Hepatitis A: From Laboratory to Clinics

**45**

**Chapter 4**

*and Dan Qiu*

**1. Introduction**

**Abstract**

Applications of Animal Models in

*Huafeng Lin, Aiping Min, Gang Li, Yan Lei Chang, Lei Shi* 

Hepatitis diseases are remaining in the list of significant threats to human health. Human hepatitis viruses are basically classified into six major hepatotropic pathogens—hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV). Among these different forms of hepatotropic viruses, HAV as the leading cause of acute viral hepatitis is characterized as a kind of tiny ribonucleic acid virus that is linked to atopic disease. As we know, animal models have been instrumental in promoting understanding of complex host-virus interactions and boosting the advancement of immune therapies. So far, animal models such as nonhuman primates (NHPs) have enabled scientists to mimic and study the pathogenicities and host immune responses for hepatitis A infection. With the exception of chimpanzees and marmosets, animals like mice, pigs, guinea pigs, and tree shrews can also be selected as alternative animal models infected with HAV under laboratory conditions. In order to gain a better insight into hepatitis A pathogenesis and relevant contents, this chapter is mainly focused on the research progress in animal models of hepatitis A, and discusses the merits and demerits of these alternative models.

**Keywords:** hepatitis A, infection experiments, animal models, virus hepatitis

Various forms of viral hepatitis represent a world health concern and challenge, generating a considerable socio-economic burden. Of these, hepatitis A as a type of food-borne hepatitis is mainly endemic in developing regions with the condition of inadequate sanitation and hygiene, such as in parts of Africa, Asia, Eastern Europe, South America, and Middle East [1, 2]. With the improvement of public health, the incidence of HAV infections in China have been gradually reduced (published data from 1990 to 2017) [3]. Up to now, 1.5 million cases of hepatitis A virus (HAV) infections are reported worldwide [2], which indicated that hepatitis A remains a primary problem in hygiene and public health. Hepatitis A has a very similar clinical symptom compared to hepatitis E. Except for the severer pathological injuries of hepatitis E than that of hepatitis A, both of two are self-limiting diseases, do not lead to liver cirrhosis and liver cancer, and transmit via orofecal route and personto-person contact [4]. Consequently, HAV-contaminated water, vegetables, fruits, blood products, and other foodstuffs, especially undercooked shellfish including clams, oysters, and mussels (**Figure 1**) [5, 6], are the major pathways of infections

Researching Hepatitis A

#### **Chapter 4**

## Applications of Animal Models in Researching Hepatitis A

*Huafeng Lin, Aiping Min, Gang Li, Yan Lei Chang, Lei Shi and Dan Qiu*

#### **Abstract**

Hepatitis diseases are remaining in the list of significant threats to human health. Human hepatitis viruses are basically classified into six major hepatotropic pathogens—hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV). Among these different forms of hepatotropic viruses, HAV as the leading cause of acute viral hepatitis is characterized as a kind of tiny ribonucleic acid virus that is linked to atopic disease. As we know, animal models have been instrumental in promoting understanding of complex host-virus interactions and boosting the advancement of immune therapies. So far, animal models such as nonhuman primates (NHPs) have enabled scientists to mimic and study the pathogenicities and host immune responses for hepatitis A infection. With the exception of chimpanzees and marmosets, animals like mice, pigs, guinea pigs, and tree shrews can also be selected as alternative animal models infected with HAV under laboratory conditions. In order to gain a better insight into hepatitis A pathogenesis and relevant contents, this chapter is mainly focused on the research progress in animal models of hepatitis A, and discusses the merits and demerits of these alternative models.

**Keywords:** hepatitis A, infection experiments, animal models, virus hepatitis

#### **1. Introduction**

Various forms of viral hepatitis represent a world health concern and challenge, generating a considerable socio-economic burden. Of these, hepatitis A as a type of food-borne hepatitis is mainly endemic in developing regions with the condition of inadequate sanitation and hygiene, such as in parts of Africa, Asia, Eastern Europe, South America, and Middle East [1, 2]. With the improvement of public health, the incidence of HAV infections in China have been gradually reduced (published data from 1990 to 2017) [3]. Up to now, 1.5 million cases of hepatitis A virus (HAV) infections are reported worldwide [2], which indicated that hepatitis A remains a primary problem in hygiene and public health. Hepatitis A has a very similar clinical symptom compared to hepatitis E. Except for the severer pathological injuries of hepatitis E than that of hepatitis A, both of two are self-limiting diseases, do not lead to liver cirrhosis and liver cancer, and transmit via orofecal route and personto-person contact [4]. Consequently, HAV-contaminated water, vegetables, fruits, blood products, and other foodstuffs, especially undercooked shellfish including clams, oysters, and mussels (**Figure 1**) [5, 6], are the major pathways of infections

**Figure 1.** *Diagram showing the possible transmission routes of HAV.*

with hepatitis A [7, 8]. Under certain circumstances, intravenous drug users with the collective use of syringes are at risk categories for HAV infections [9], and also there exist vertical transmissions of HAV from mother to child but occur very rarely (**Figure 1**) [10]. HAV as the main pathogen causing acute viral hepatitis is classified as a sole member of the genus Hepatovirus of the family *Picornaviridae*, which includes many medical and veterinary pathogens in 1991 [11–13]. HAV is a single linear positive-stranded RNA virus whose genomic full length is approximately 7500 nucleotides, which contain 5′-noncoding region (UTR), protein coding region, and 3′-noncoding region (UTR) (**Figure 2**) [13]. Researchers have found that HAV present in the form of naked, nonenveloped virions in feces aids to the viral transmissions through the environment. However, when HAV emerges in the blood of infected persons, the virion isolates itself from neutralizing antibodies by the way of producing quasi-envelope in host-derived membranes [14]. Epidemiological data showed that the most susceptible populations of HAV are the children in early childhood [2], and the disease prevalence exceeds 90% before the age of 10 [15], albeit most of infected youngers are usually mild or asymptomatic [16]. Hence, accelerating the immunological research and viral vaccine development can improve human immunity and reduce the spread of HAV. World Health Organization (WHO) recommends that vaccination combating HAV be integrated into the national immunization schedule for children aged ≥1 year on the consideration of many factors including cost-effectiveness [17]. What is noteworthiness is that, the illness infected with HAV in those people who are older than 60 will be very severe [18]. Moreover, HAV superinfections in chronic liver disease (CLD)

**47**

will be analyzed as well.

**Figure 2.**

**2. Basic biological features of HAV and beyond**

*Applications of Animal Models in Researching Hepatitis A*

sufferers (e.g., hepatitis B or C) are usually associated with raising morbidity and mortality [19, 20]. To date, animal model is one of the promising tools in the investigation of human HAV infections. Studies on HAV immunopathological mechanism and host immune response mainly used nonhuman primates such as chimpanzees and marmosets as animal models. Due to the lack of other alternative animal models that support HAV infections, the study of the HAV biology and further development of therapies for hepatitis A have been hampered. Here in this chapter, the biological features of HAV will be discussed, the animal models of hepatitis A and their characteristics will be sketched, and the merits and demerits for these models

*The genome structure, protein structure components and overall structure of HAV. Refer to [28, 35, 36].*

As early as 5000 years ago, hepatitis A-like illnesses were documented in ancient China. In Europe, similar disease known as "benign epidemic jaundice" was also described during the Hippocratic era [21]. As time goes by, in 1947, McCallum et al. termed infectious hepatitis as hepatitis A [22]. In the first half of 1967, Krugman et al. found the distinctive features between infectious hepatitis (hepatitis A) and serum hepatitis (hepatitis B) in clinical, epidemiological, and immunological aspects [23]. By 1973, Feinslone et al. firstly detected hepatitis A in feces of patients using the technology of immune electron microscopy (IEM) [24]. Morphologically and structurally, the purified HAV virion, having an outer diameter of 27–80 nm, is an icosahedral nucleocapsid protein granular which contains one linear positivestranded ribonucleic acid (RNA) genome [25]. The genome encodes a single large polyprotein of 2227 amino acids [26], which is matured and folded to produce 10 biologically active viral proteins, including four structural proteins that construct the capsid (VP4 (~2.5 kDa), VP2 (24–30 kDa), VP3 (21–28 kDa), and VP1pX) and 6 nonstructural proteins that are indispensable for replication of the RNA genome (2B, 2C, 3A, 3B [VPg], 3Cpro [a cysteine protease], and 3Dpol) (RNA-dependent

*DOI: http://dx.doi.org/10.5772/intechopen.90684*

*Applications of Animal Models in Researching Hepatitis A DOI: http://dx.doi.org/10.5772/intechopen.90684*

**Figure 2.**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

with hepatitis A [7, 8]. Under certain circumstances, intravenous drug users with the collective use of syringes are at risk categories for HAV infections [9], and also there exist vertical transmissions of HAV from mother to child but occur very rarely (**Figure 1**) [10]. HAV as the main pathogen causing acute viral hepatitis is classified as a sole member of the genus Hepatovirus of the family *Picornaviridae*, which includes many medical and veterinary pathogens in 1991 [11–13]. HAV is a single linear positive-stranded RNA virus whose genomic full length is approximately 7500 nucleotides, which contain 5′-noncoding region (UTR), protein coding region, and 3′-noncoding region (UTR) (**Figure 2**) [13]. Researchers have found that HAV present in the form of naked, nonenveloped virions in feces aids to the viral transmissions through the environment. However, when HAV emerges in the blood of infected persons, the virion isolates itself from neutralizing antibodies by the way of producing quasi-envelope in host-derived membranes [14]. Epidemiological data showed that the most susceptible populations of HAV are the children in early childhood [2], and the disease prevalence exceeds 90% before the age of 10 [15], albeit most of infected youngers are usually mild or asymptomatic [16]. Hence, accelerating the immunological research and viral vaccine development can improve human immunity and reduce the spread of HAV. World Health Organization (WHO) recommends that vaccination combating HAV be integrated into the national immunization schedule for children aged ≥1 year on the consideration of many factors including cost-effectiveness [17]. What is noteworthiness is that, the illness infected with HAV in those people who are older than 60 will be very severe [18]. Moreover, HAV superinfections in chronic liver disease (CLD)

**46**

**Figure 1.**

*Diagram showing the possible transmission routes of HAV.*

*The genome structure, protein structure components and overall structure of HAV. Refer to [28, 35, 36].*

sufferers (e.g., hepatitis B or C) are usually associated with raising morbidity and mortality [19, 20]. To date, animal model is one of the promising tools in the investigation of human HAV infections. Studies on HAV immunopathological mechanism and host immune response mainly used nonhuman primates such as chimpanzees and marmosets as animal models. Due to the lack of other alternative animal models that support HAV infections, the study of the HAV biology and further development of therapies for hepatitis A have been hampered. Here in this chapter, the biological features of HAV will be discussed, the animal models of hepatitis A and their characteristics will be sketched, and the merits and demerits for these models will be analyzed as well.

#### **2. Basic biological features of HAV and beyond**

As early as 5000 years ago, hepatitis A-like illnesses were documented in ancient China. In Europe, similar disease known as "benign epidemic jaundice" was also described during the Hippocratic era [21]. As time goes by, in 1947, McCallum et al. termed infectious hepatitis as hepatitis A [22]. In the first half of 1967, Krugman et al. found the distinctive features between infectious hepatitis (hepatitis A) and serum hepatitis (hepatitis B) in clinical, epidemiological, and immunological aspects [23]. By 1973, Feinslone et al. firstly detected hepatitis A in feces of patients using the technology of immune electron microscopy (IEM) [24]. Morphologically and structurally, the purified HAV virion, having an outer diameter of 27–80 nm, is an icosahedral nucleocapsid protein granular which contains one linear positivestranded ribonucleic acid (RNA) genome [25]. The genome encodes a single large polyprotein of 2227 amino acids [26], which is matured and folded to produce 10 biologically active viral proteins, including four structural proteins that construct the capsid (VP4 (~2.5 kDa), VP2 (24–30 kDa), VP3 (21–28 kDa), and VP1pX) and 6 nonstructural proteins that are indispensable for replication of the RNA genome (2B, 2C, 3A, 3B [VPg], 3Cpro [a cysteine protease], and 3Dpol) (RNA-dependent

RNA polymerase) (**Figure 2**) [27]. By using standard serological technique and molecular identification methods, HAV is identified to belong to merely one single serotype, and is divided into seven distinct genotypes of which three genotypes (I, II, and VII) that circulate in humans, one genotype (III) isolated from either humans or owl monkeys, and other three genotypes (V–VII) exist in nonhuman primates [28–30]. Genotypes I, II, and III are sub-classified into subtypes A and B (Genotypes IA, IB, IIA, IIB, IIIA, and IIIB) [31]. Molecular epidemiology has further revealed that HAV sub-genotype IA is responsible for the most circulations among human population [32]. For sub-genotype IB HAV strains, several reports have declared that they were associated with food such as frozen strawberries in Australia and several countries of Europe [33, 34]. Recent studies of X-ray analysis have uncovered that HAV possesses a primitive capsid architecture related to that of picorna-like viruses infecting insects, which imply a correlation of primeval evolution as well as a novel cellular entrance mechanism for viruses [35]. The structure information (especially the 3D microstructural study) of viral protein is now a robust tool for dissecting their biological functions. In 2018, Stuart et al. reviewed updated studies on the structural features of outer protein shell of HAV and proposed the future researching scopes including relevant structural elucidations of the enveloped particles, as well as the capture of intermediates in the state of assembly, attachment, and/or uncoating [36]. In terms of receptor binding mechanism, Wang et al. pointed out that using a receptor mimic mechanism for neutralization of infectivity may hold promise for the therapeutic intervention of hepatitis A [37]. With regard to the origin of human HAV, phylogenetic analyses show that, in the remote past, these ancient viruses have emerged in different host species, and ancestral state reconstructions indicate HAV is likely to have originated in rodents [38]. What's more, investigations should be fundamentally focused on therapeutic interventions and new creations of HAV vaccines as a result of hepatitis A vaccine is one of the most effective strategy for the treatment of hepatitis A [39]. To date, four inactivated monovalent HAV vaccines from different manufactures (Havrix®, Epaxal®, Avaxim®, and Vaqta®) have been commercially available to the global markets [40]. Other hepatitis A vaccines such as a Chinese live attenuated vaccine (H2 strain, Zhejiang Academy of Medical Sciences, Hangzhou, PR China) and a Vietnam one are just self-sufficient in domestic production [41]. HAV infections are still an important cause of morbidity and mortality in developed countries such as the United States [42], let alone other nations with poor sanitation. Therefore, the work of scientific research for hepatitis A vaccine is still certainly on the way.

#### **3. Applications of animal models**

According to literatures, HAV strains of wild type are quite difficult in propagating in vitro. When culturing in cell-conditioned medium, they show low growth rate characteristically, as well as have no apparent cytopathic effects [43]. Additionally, HAV has its own special life history: it primarily replicates in the hepatic tissue, is excreted in biliary system to reach the intestinal contents [44], and is mostly shed in the feces and soil [45], where the viruses may persist for an extended period of time [46, 47]. Consequently, it is significantly important for researchers to find the proper infected models that aim at the investigation of HAV. As Hirai-Yuki and his co-authors have ever pointed out that, it is essential to develop improved animal models for the deeper investigations of the molecular and cellular mechanisms associated virus-hepatocyte interactions within the distinctive environment of liver tissue of hosts [48]. Here are the examples of disease models for HAV infections showed in **Table 1**.

**49**

experiments of research [60].

**3.1 NHPs**

*Examples of disease models for HAV infections.*

**Table 1.**

Dienstag et al/1975

Amado et al/2010

LeDuc et al/1983

Hirai-Yuki et al/2016

Hornei et al/2001

Anthony et al/2015

*Applications of Animal Models in Researching Hepatitis A*

Cynomolgus monkeys

New World owl monkeys

Broadly speaking, NHPs resemble humans in anatomy, physiology, and pathology over any other animals, which make them to be considered as the principal models for diseases including HAV infections. Human HAV has successfully infected various species of NHPs, such as chimpanzees (*Pan troglodytes*) [49], common marmosets (*Callithrix jacchus*) [50, 51], Squirrel Monkeys (*Saimiri sciureus*) [52], New World owl monkeys (*Platyrrhines*) [53], African green monkeys (*Cercopithecus aethiops*) [54], owl monkey (*Aotus trivirgatus*) [55], brown macaques (*Macaca arctoides*) [56], stump-tailed monkey (*Macaca speciosa*) [57] and tamarins, etc. (**Table 1**), but the host range of this virus is still narrow [58], mainly limited to relatively few species. The most common animal models used in laboratories for interrogating HAV infection are mainly marmosets and chimpanzees, which are of scarce resources (Chimpanzees are so expensive that they are not widely available for research use) in most countries. In addition, experimental data indicated that more than 90% of wild chimpanzees carried anti-HAV antibodies [59], which made them less suitable for investigating HAV-infectious diseases, but chimpanzees reared in captivity are more susceptible to infect hepatitis A. Moreover, it is very difficult for laboratory technicians to feed and operate experimentally on these two animals in many situations. And quite importantly, ethical concerns have advocated the decreasing use of chimpanzees for invasive

pathogenesis of DHAV-3

**Authors/year Animal models Comments Refs**

monkey to HAV

Guinea pigs Useful for studying some aspects of HAV

Song et al/2016 Pigs First experimental evidence to demonstrate human

the liver

Zhan et al/1981 Tree shrews HAV can replicate in tree shrews and are potential

Liu et al/2019 Pekin ducks There are differences in the pathogenicity of

Wen et al/2019 Ducks Provided new insights into the genetics and

Chimpanzees Provided evidence for the susceptibility of chimpanzees to HAV

model to study HAV infection

HAV strains can infect pigs

Mice Provided a new paradigm for viral pathogenesis in

Cynomolgus monkey was confirmed as a suitable

Confirmed the susceptibility of New World owl

pathogenesis and for testing the safety of vaccines.

for candidate models for HAV infections

different subtypes of DHAV in ducklings

Harbor seals Describe the discovery of an HAV-like virus in seals [98]

[49]

[67]

[53]

[76]

[83]

[88]

[97]

[100]

[101]

Take chimpanzees for example, they are the candidate experimental subjects that are most closely related to humans genetically, and most probably to be simulative and predictive of human outcomes when used as disease models. In 1962, Deinhardt et al. launched the initial attemption experiment that used chimpanzees to be infected with HAV through inoculating viral materials (serum or feces), but

*DOI: http://dx.doi.org/10.5772/intechopen.90684*

*Applications of Animal Models in Researching Hepatitis A DOI: http://dx.doi.org/10.5772/intechopen.90684*


#### **Table 1.**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

RNA polymerase) (**Figure 2**) [27]. By using standard serological technique and molecular identification methods, HAV is identified to belong to merely one single serotype, and is divided into seven distinct genotypes of which three genotypes (I, II, and VII) that circulate in humans, one genotype (III) isolated from either humans or owl monkeys, and other three genotypes (V–VII) exist in nonhuman primates [28–30]. Genotypes I, II, and III are sub-classified into subtypes A and B (Genotypes IA, IB, IIA, IIB, IIIA, and IIIB) [31]. Molecular epidemiology has further revealed that HAV sub-genotype IA is responsible for the most circulations among human population [32]. For sub-genotype IB HAV strains, several reports have declared that they were associated with food such as frozen strawberries in Australia and several countries of Europe [33, 34]. Recent studies of X-ray analysis have uncovered that HAV possesses a primitive capsid architecture related to that of picorna-like viruses infecting insects, which imply a correlation of primeval evolution as well as a novel cellular entrance mechanism for viruses [35]. The structure information (especially the 3D microstructural study) of viral protein is now a robust tool for dissecting their biological functions. In 2018, Stuart et al. reviewed updated studies on the structural features of outer protein shell of HAV and proposed the future researching scopes including relevant structural elucidations of the enveloped particles, as well as the capture of intermediates in the state of assembly, attachment, and/or uncoating [36]. In terms of receptor binding mechanism, Wang et al. pointed out that using a receptor mimic mechanism for neutralization of infectivity may hold promise for the therapeutic intervention of hepatitis A [37]. With regard to the origin of human HAV, phylogenetic analyses show that, in the remote past, these ancient viruses have emerged in different host species, and ancestral state reconstructions indicate HAV is likely to have originated in rodents [38]. What's more, investigations should be fundamentally focused on therapeutic interventions and new creations of HAV vaccines as a result of hepatitis A vaccine is one of the most effective strategy for the treatment of hepatitis A [39]. To date, four inactivated monovalent HAV vaccines from different manufactures (Havrix®, Epaxal®, Avaxim®, and Vaqta®) have been commercially available to the global markets [40]. Other hepatitis A vaccines such as a Chinese live attenuated vaccine (H2 strain, Zhejiang Academy of Medical Sciences, Hangzhou, PR China) and a Vietnam one are just self-sufficient in domestic production [41]. HAV infections are still an important cause of morbidity and mortality in developed countries such as the United States [42], let alone other nations with poor sanitation. Therefore, the work of scientific research for hepatitis A vaccine is still certainly on the way.

**48**

**3. Applications of animal models**

for HAV infections showed in **Table 1**.

According to literatures, HAV strains of wild type are quite difficult in propa-

gating in vitro. When culturing in cell-conditioned medium, they show low growth rate characteristically, as well as have no apparent cytopathic effects [43]. Additionally, HAV has its own special life history: it primarily replicates in the hepatic tissue, is excreted in biliary system to reach the intestinal contents [44], and is mostly shed in the feces and soil [45], where the viruses may persist for an extended period of time [46, 47]. Consequently, it is significantly important for researchers to find the proper infected models that aim at the investigation of HAV. As Hirai-Yuki and his co-authors have ever pointed out that, it is essential to develop improved animal models for the deeper investigations of the molecular and cellular mechanisms associated virus-hepatocyte interactions within the distinctive environment of liver tissue of hosts [48]. Here are the examples of disease models

*Examples of disease models for HAV infections.*

#### **3.1 NHPs**

Broadly speaking, NHPs resemble humans in anatomy, physiology, and pathology over any other animals, which make them to be considered as the principal models for diseases including HAV infections. Human HAV has successfully infected various species of NHPs, such as chimpanzees (*Pan troglodytes*) [49], common marmosets (*Callithrix jacchus*) [50, 51], Squirrel Monkeys (*Saimiri sciureus*) [52], New World owl monkeys (*Platyrrhines*) [53], African green monkeys (*Cercopithecus aethiops*) [54], owl monkey (*Aotus trivirgatus*) [55], brown macaques (*Macaca arctoides*) [56], stump-tailed monkey (*Macaca speciosa*) [57] and tamarins, etc. (**Table 1**), but the host range of this virus is still narrow [58], mainly limited to relatively few species. The most common animal models used in laboratories for interrogating HAV infection are mainly marmosets and chimpanzees, which are of scarce resources (Chimpanzees are so expensive that they are not widely available for research use) in most countries. In addition, experimental data indicated that more than 90% of wild chimpanzees carried anti-HAV antibodies [59], which made them less suitable for investigating HAV-infectious diseases, but chimpanzees reared in captivity are more susceptible to infect hepatitis A. Moreover, it is very difficult for laboratory technicians to feed and operate experimentally on these two animals in many situations. And quite importantly, ethical concerns have advocated the decreasing use of chimpanzees for invasive experiments of research [60].

Take chimpanzees for example, they are the candidate experimental subjects that are most closely related to humans genetically, and most probably to be simulative and predictive of human outcomes when used as disease models. In 1962, Deinhardt et al. launched the initial attemption experiment that used chimpanzees to be infected with HAV through inoculating viral materials (serum or feces), but

the gained results could not provide conclusive evidences for the transmission of infective hepatitis from humans to chimpanzees [61]. Intriguingly, in 1963, Hillis presented biochemical and histologic evidences that promisingly proved chimpanzees as useful as experimental hosts for human hepatitis viruses [62, 63]. In the mid-1970s, results of most of numerous publishment, which attempt to spread hepatitis A to chimpanzees yielded negative or equivocal results [64]. By 1984, Tsiquaye et al. carried out a study on acute hepatitis A infection occurred in hepatitis B chimpanzee carriers, which showed that superinfection can significantly alter the parameters of HBV chronicity in chimpanzees [65]. The authors pointed out that further observations were needed to establish the degree of severity of concurrent infection of HBV carriers with HAV, since such changes may have implications in some countries where the proportion of HBV carriers is high plus hepatitis A is highly prevalent [65]. For the purpose to locate where does the HAV might duplicate in the body, in 1989, Cohen and his colleagues conducted a study of single chimpanzee involvement in experiment, and found a possible oropharyngeal site for viral replication due to the emergence of HAV in saliva and throat swabs [66]. Similarly, Amado and co-authors acquired an experimental result that salivary gland was an extrahepatic site for early HAV replication in cynomolgus monkeys [67]. In the following two decades, the investigators shifted the focus of animal models to other NHPs instead of chimpanzees either because of the high cost of chimpanzee research or because of the poor contribution of chimpanzee experiments for biomedical applications [68]. Until 2011, Lanford et al. utilized three chimpanzee models to study the early innate immune responses to HAV infections. They found that HAV has a better property of keeping itself latent compared to HCV during early stage of acute resolving infection, and HAV infections represent a distinctly different paradigm in the course of intrahepatic interactions of virus-host [69].

The chimpanzees have been demonstrated to be an invaluable model tools for the investigation on HBV-induced disease pathogenesis and the discovery of novel prophylactic drugs and anti-viral therapies [70]. Optimistically, with the advancement of biotechnology, utilizing chimpanzee and other NHPs as disease models for HAV infection will surely play significant roles in HAV-associated studies in the future.

#### **3.2 Pigs**

Compared with NHPs, pigs have several advantages such as easy breeding and rearing, convenient handling and fewer ethical concerns, which make them be widely used in biomedical research [71]. Under natural conditions, it had been reported that HAV infections are being restricted to humans and nonhuman primates [72], and the appropriate models used for HAV infection have been restricted to nonhuman primates [73]. Due to several limitations of such animal models, other surrogate models need to be developed for further study. According to literatures, the immune system in pigs shares many similarities with humans for over 80% of analytical parameters, which made swine as a more suitable and common animal model for humans [74, 75]. Moreover, pigs have been used preclinically as disease models for preclinical studies usually. Until 2016, Song et al. firstly found the experiment evidence to prove human HAV strains can also infect swine [76], which took the first step to approach swine models for HAV infections. In this experiment, Song and colleagues observed that HAV can survive and replicate in pigs, which have replaced NHPs. However, there were no significant changes in the clinical manifestations and serum markers for pigs infected with HAV. Finally, they further suggested that pigs might be a suitable animal model for future studies related to HAV pathogenesis [76].

**51**

**3.4 Guinea pigs**

*Applications of Animal Models in Researching Hepatitis A*

Over the last two decades, mouse models have been successfully used in tackling various biological questions associated with intrahepatic immune response mechanisms for disease pathogenesis and clearing of HBV [77]. And also, such types of models can be applied to study the adaptive immune response to hepacivirus infection and will play roles in vaccine development. However, HAV is not capable to replicate in mice due to incompatibilities in the interaction of the virus and the innate immune system of mice. Therefore, scientists tackle this difficulty by utilizing chimeric mice, which facilitated the successful replication of HAV in the body through bypassing the cytosolic pattern recognition recep-

Generally speaking, certain cellular receptors are the key molecules that medi-

Previously, Yang et al. reported that, by using cell culture method, HAV ablates type 1 IFN responses thereby disrupting activation of IRF3 through the MDA5 pathway [85]. In 2013, Pang used HAV to infect SCID-beige/Alb-uPA mice with chimeric human/mouse livers for the purpose to test the susceptibility of mice to HAV. The result shows that these chimeric mice are permissive to HAV infection and represent valuable small animal models for future studies [86]. In 2016, Hirai-Yuki et al. applied the murine models with genetic defects in the induction of type I interferon (IFN) responses for HAV infection to reveal a previously undefined link between innate immune responses to virus infection and acute liver injury, which furnishes a novel paradigm for viral etiopathogenesis in the liver [83]. In 2018, a research team of Hirai-Yuki wrote a review of the study on "Murine models of hepatitis A virus infection" in which they provided an extensive and in-depth perspective into the development and application of mice models for HAV [48]. Additionally, in this chapter, it emphatically introduced the mechanism of degrading MAVS via viral proteases, in which it facilitates long-term survival of virus and spread through escaping from IFN-mediated restriction of virus replication and limiting pathogen-

Till now, mouse models have been applied to support infections with HBV, HCV, and even HAV successfully. This probably has to do with building infections in the mouse liver, which is a key point in the development of viral hepatitis. Predictably, it has a promising future for utilizing mice as effective models for the investigation of HAV infection with the technological development of biomedical models.

The guinea pig models are more similar to humans than other small animal models in physiology and immunology. Specifically, the guinea pigs have the

ate viruses of entry into special kinds of cells in the body. Human membrane protein TIM-1 (T cell immunoglobulin and mucin domain protein-1) is a type of phosphatidylserine receptor that was firstly described as HAVCR1 [79], which helps cellular entry and infection with innumerable conventional enveloped viruses that bind phosphatidylserine on their surface [80]. And specially, TIM-3 receptor facilitates HAV for its entrance into target cells in humans [81]. However, recent research showed that TIM-1 is not an essential hepatovirus factor although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver damage in mice [82]. For most of mouse models, wild-type mice are naturally resistant to HAV infection [83], and murine cell lines still exist defects in viral entry processes functionally [84]. For these reasons, multiple approaches have been developed by investigators to generate "humanize" mice at a genetic level to aid

*DOI: http://dx.doi.org/10.5772/intechopen.90684*

**3.3 Chimeric mice/gene knock-out mice**

them susceptible to infection with HAV.

esis and hepatic damage [48].

tor, MAVS [78].

#### **3.3 Chimeric mice/gene knock-out mice**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

interactions of virus-host [69].

the future.

**3.2 Pigs**

the gained results could not provide conclusive evidences for the transmission of infective hepatitis from humans to chimpanzees [61]. Intriguingly, in 1963, Hillis presented biochemical and histologic evidences that promisingly proved chimpanzees as useful as experimental hosts for human hepatitis viruses [62, 63]. In the mid-1970s, results of most of numerous publishment, which attempt to spread hepatitis A to chimpanzees yielded negative or equivocal results [64]. By 1984, Tsiquaye et al. carried out a study on acute hepatitis A infection occurred in hepatitis B chimpanzee carriers, which showed that superinfection can significantly alter the parameters of HBV chronicity in chimpanzees [65]. The authors pointed out that further observations were needed to establish the degree of severity of concurrent infection of HBV carriers with HAV, since such changes may have implications in some countries where the proportion of HBV carriers is high plus hepatitis A is highly prevalent [65]. For the purpose to locate where does the HAV might duplicate in the body, in 1989, Cohen and his colleagues conducted a study of single chimpanzee involvement in experiment, and found a possible oropharyngeal site for viral replication due to the emergence of HAV in saliva and throat swabs [66]. Similarly, Amado and co-authors acquired an experimental result that salivary gland was an extrahepatic site for early HAV replication in cynomolgus monkeys [67]. In the following two decades, the investigators shifted the focus of animal models to other NHPs instead of chimpanzees either because of the high cost of chimpanzee research or because of the poor contribution of chimpanzee experiments for biomedical applications [68]. Until 2011, Lanford et al. utilized three chimpanzee models to study the early innate immune responses to HAV infections. They found that HAV has a better property of keeping itself latent compared to HCV during early stage of acute resolving infection, and HAV infections represent a distinctly different paradigm in the course of intrahepatic

The chimpanzees have been demonstrated to be an invaluable model tools for the investigation on HBV-induced disease pathogenesis and the discovery of novel prophylactic drugs and anti-viral therapies [70]. Optimistically, with the advancement of biotechnology, utilizing chimpanzee and other NHPs as disease models for HAV infection will surely play significant roles in HAV-associated studies in

Compared with NHPs, pigs have several advantages such as easy breeding and rearing, convenient handling and fewer ethical concerns, which make them be widely used in biomedical research [71]. Under natural conditions, it had been reported that HAV infections are being restricted to humans and nonhuman primates [72], and the appropriate models used for HAV infection have been restricted to nonhuman primates [73]. Due to several limitations of such animal models, other surrogate models need to be developed for further study. According to literatures, the immune system in pigs shares many similarities with humans for over 80% of analytical parameters, which made swine as a more suitable and common animal model for humans [74, 75]. Moreover, pigs have been used preclinically as disease models for preclinical studies usually. Until 2016, Song et al. firstly found the experiment evidence to prove human HAV strains can also infect swine [76], which took the first step to approach swine models for HAV infections. In this experiment, Song and colleagues observed that HAV can survive and replicate in pigs, which have replaced NHPs. However, there were no significant changes in the clinical manifestations and serum markers for pigs infected with HAV. Finally, they further suggested that pigs might be a suitable

animal model for future studies related to HAV pathogenesis [76].

**50**

Over the last two decades, mouse models have been successfully used in tackling various biological questions associated with intrahepatic immune response mechanisms for disease pathogenesis and clearing of HBV [77]. And also, such types of models can be applied to study the adaptive immune response to hepacivirus infection and will play roles in vaccine development. However, HAV is not capable to replicate in mice due to incompatibilities in the interaction of the virus and the innate immune system of mice. Therefore, scientists tackle this difficulty by utilizing chimeric mice, which facilitated the successful replication of HAV in the body through bypassing the cytosolic pattern recognition receptor, MAVS [78].

Generally speaking, certain cellular receptors are the key molecules that mediate viruses of entry into special kinds of cells in the body. Human membrane protein TIM-1 (T cell immunoglobulin and mucin domain protein-1) is a type of phosphatidylserine receptor that was firstly described as HAVCR1 [79], which helps cellular entry and infection with innumerable conventional enveloped viruses that bind phosphatidylserine on their surface [80]. And specially, TIM-3 receptor facilitates HAV for its entrance into target cells in humans [81]. However, recent research showed that TIM-1 is not an essential hepatovirus factor although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver damage in mice [82]. For most of mouse models, wild-type mice are naturally resistant to HAV infection [83], and murine cell lines still exist defects in viral entry processes functionally [84]. For these reasons, multiple approaches have been developed by investigators to generate "humanize" mice at a genetic level to aid them susceptible to infection with HAV.

Previously, Yang et al. reported that, by using cell culture method, HAV ablates type 1 IFN responses thereby disrupting activation of IRF3 through the MDA5 pathway [85]. In 2013, Pang used HAV to infect SCID-beige/Alb-uPA mice with chimeric human/mouse livers for the purpose to test the susceptibility of mice to HAV. The result shows that these chimeric mice are permissive to HAV infection and represent valuable small animal models for future studies [86]. In 2016, Hirai-Yuki et al. applied the murine models with genetic defects in the induction of type I interferon (IFN) responses for HAV infection to reveal a previously undefined link between innate immune responses to virus infection and acute liver injury, which furnishes a novel paradigm for viral etiopathogenesis in the liver [83]. In 2018, a research team of Hirai-Yuki wrote a review of the study on "Murine models of hepatitis A virus infection" in which they provided an extensive and in-depth perspective into the development and application of mice models for HAV [48]. Additionally, in this chapter, it emphatically introduced the mechanism of degrading MAVS via viral proteases, in which it facilitates long-term survival of virus and spread through escaping from IFN-mediated restriction of virus replication and limiting pathogenesis and hepatic damage [48].

Till now, mouse models have been applied to support infections with HBV, HCV, and even HAV successfully. This probably has to do with building infections in the mouse liver, which is a key point in the development of viral hepatitis. Predictably, it has a promising future for utilizing mice as effective models for the investigation of HAV infection with the technological development of biomedical models.

#### **3.4 Guinea pigs**

The guinea pig models are more similar to humans than other small animal models in physiology and immunology. Specifically, the guinea pigs have the

property of being analogous to humans in reproductive physiology and estrous cycle [87], etc. Guinea pigs have been used as an HAV infection model, but their use is limited because of the lack of development of anti-HAV antibodies in inoculated guinea pigs. In 2001, Hornei et al. conducted a study to determine whether HAV is capable to infect guinea pigs and whether they can be valid as a disease model for replicating HAV pathogenesis in humans and for the evaluation of vaccines [88]. The authors found that very low levels of HAV were detected in the livers of guinea pigs, which inoculated with human HAV [88]. Furthermore, they also described that the experimental guinea pigs shared similar response pattern with a New World nonhuman primate (*Callithrix jacchus*) after being challenged with HAV materials [88, 89]. The method of using guinea pigs to establish models for HAV infection is still under controversy. In 2010, de Castro Araujo and colleagues designed a research project to respond to the question "Whether HAV is capable to infect guinea pigs?". However, they failed to establish a guinea pig as model for HAV [90].

#### **3.5 Tree shrews**

Chinese tree shrew (*Tupaia belangeri chinensis*), mainly distributing in Yunnan and Guangxi provinces in China, is a class of small animals having a closer evolutionary relationship with humans compared to rodents [91]. Tree shrews have emerged in the vision of scientists for more than 30 years as a result of having many valuable features that are suitable in animals utilized as experimental models in biomedical studies [92], particularly in the fields of toxicology and virology [93]. To date, there are many attempts to employ tree shrews as models for human disorders such as hepatitis B [94] and hepatitis C infections [95, 96]. In 1981, Zhan et al. used fecal suspension of hepatitis A patients (concentration: 5%) to infect nine tree shrews through oral route, and eventually no apparent clinical symptoms of acute hepatitis were found. About 7–13 days after the viral infections, seven tree shrews were detected HAV that lies in their stools in 12–27 days, which indicated that HAV could reproduce in the body of tree shrews. The experimental results indicated that HAV can replicate in tree shrews and are potential for candidate models for HAV infections [97]. Additionally, they also found disease symptoms including increased alanine transaminase (ALT), hepatic hyperemia, hepatic edema, steatosis, and hyperplasia of Kupffer cells in the infected tree shrews, which further manifested that HAV can propagate in tree shrews [97].

#### **3.6 Other animal models**

Early studies suggested that HAV was unable to lead to infections of any common small laboratory animals successfully except NHPs. However, this "prejudice" has already been challenged and overturned by animal model engineering as well as by new scientific discoveries. In 2015, several strains of human HAV have been found in seals, which may indicate that the first natural nonprimate HAV to be discovered, and provide further understanding for the evolutionary history and pathogenicity of HAV [98]. Moreover, in recent years, HAV-associated hepatoviruses have been found in bats, rodents, hedgehogs [38], duck [99–101], and woodchucks [102, 103], which suggested that there may be more candidate animals potentially used as animal models of HAV. On the contrary, some scholars believed that these new viruses are substantially more divergent from each other and from human HAV (including simian HAV), which is in accordance with them being assigned to several additional species in taxonomy [78].

**53**

*Applications of Animal Models in Researching Hepatitis A*

The animal experiments definitely play an important role in the development of life sciences and medical sciences. Therefore, ethical analysis concerning animal experiments is essential because it cannot completely avoid the use of animals [104] in the process of biomedicine and preclinical medicine research. Specially, NHPs act as the particularly valuable models for testing interventions against the Ebola and Marburg viruses in the field of studying of current infectious diseases, which can effectively objectively simulate human diseases via infections in these animals [105], and further contribute to the development of new protective and therapeutic vaccines. At a certain level, ethical issues become more important than scientific interest for this type of animal test [104] because such infections are often lethal to the experimental animals, which are commonly viewed as unethical. Similarly, experiments with HAV infection also expose animals (mainly NHPs) to injury or disease. Consequently, how to balance the contradiction between ethical challenges

and NHPs infectious experiments becomes a vitally important subject.

Animal model research is entering a new and exciting stage along with the technologies of computational information and molecular biochemistry. For example, it is now possible for us to employ genome-edited techniques (e.g., ZFNs, TALENs, and CRISPR/Cas) to knockout specific genes, to knock in new genes, or to introduce specific mutations, and then to produce valuable animal models that benefited for our investigations. However, as we know, "no model is perfect, but many are useful" [106]. Therefore, establishing susceptible animal models is one of the methods in the research fields of HAV. By using appropriate and reliable animal models, virologist can perform a series of studies associated with hepatitis A including epidemiologic features, viral infectivity, humoral and cellular immunity, cytokine responses, virus pathogenesis, as well as the research and development of

For the development of hepatitis A vaccines, it is worth mentioning that a highly effective vaccine against HAV was manufactured by classical inactivation of the whole virus generated from cell culture [107], which commendably avoids the ethical controversy of using NHPs models. Moreover, there is a need to provide more support for the studies of long-term protection vaccines against hepatitis A

Last but not least, it is very likely that a much wider host range of HAVassociated viruses will be discovered in other mammalian species in the future [38].

is known to do good" [109]. However, as we all know, "viruses are not omnipotent." For hepatitis viruses, the narrow hepatic tissue tropism maybe is the cause

The Nobel laureate Peter Medawar have ever succinctly concluded that "No virus

*DOI: http://dx.doi.org/10.5772/intechopen.90684*

**4. Ethical aspects**

**5. Future directions**

antiviral vaccinations.

infection [108].

**6. Conclusion**

**5.2 Cell culture methods**

**5.1 Animal model methods**

### **4. Ethical aspects**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

to establish a guinea pig as model for HAV [90].

that HAV can propagate in tree shrews [97].

assigned to several additional species in taxonomy [78].

**3.6 Other animal models**

**3.5 Tree shrews**

property of being analogous to humans in reproductive physiology and estrous cycle [87], etc. Guinea pigs have been used as an HAV infection model, but their use is limited because of the lack of development of anti-HAV antibodies in inoculated guinea pigs. In 2001, Hornei et al. conducted a study to determine whether HAV is capable to infect guinea pigs and whether they can be valid as a disease model for replicating HAV pathogenesis in humans and for the evaluation of vaccines [88]. The authors found that very low levels of HAV were detected in the livers of guinea pigs, which inoculated with human HAV [88]. Furthermore, they also described that the experimental guinea pigs shared similar response pattern with a New World nonhuman primate (*Callithrix jacchus*) after being challenged with HAV materials [88, 89]. The method of using guinea pigs to establish models for HAV infection is still under controversy. In 2010, de Castro Araujo and colleagues designed a research project to respond to the question "Whether HAV is capable to infect guinea pigs?". However, they failed

Chinese tree shrew (*Tupaia belangeri chinensis*), mainly distributing in Yunnan and Guangxi provinces in China, is a class of small animals having a closer evolutionary relationship with humans compared to rodents [91]. Tree shrews have emerged in the vision of scientists for more than 30 years as a result of having many valuable features that are suitable in animals utilized as experimental models in biomedical studies [92], particularly in the fields of toxicology and virology [93]. To date, there are many attempts to employ tree shrews as models for human disorders such as hepatitis B [94] and hepatitis C infections [95, 96]. In 1981, Zhan et al. used fecal suspension of hepatitis A patients (concentration: 5%) to infect nine tree shrews through oral route, and eventually no apparent clinical symptoms of acute hepatitis were found. About 7–13 days after the viral infections, seven tree shrews were detected HAV that lies in their stools in 12–27 days, which indicated that HAV could reproduce in the body of tree shrews. The experimental results indicated that HAV can replicate in tree shrews and are potential for candidate models for HAV infections [97]. Additionally, they also found disease symptoms including increased alanine transaminase (ALT), hepatic hyperemia, hepatic edema, steatosis, and hyperplasia of Kupffer cells in the infected tree shrews, which further manifested

Early studies suggested that HAV was unable to lead to infections of any common small laboratory animals successfully except NHPs. However, this "prejudice" has already been challenged and overturned by animal model engineering as well as by new scientific discoveries. In 2015, several strains of human HAV have been found in seals, which may indicate that the first natural nonprimate HAV to be discovered, and provide further understanding for the evolutionary history and pathogenicity of HAV [98]. Moreover, in recent years, HAV-associated hepatoviruses have been found in bats, rodents, hedgehogs [38], duck [99–101], and woodchucks [102, 103], which suggested that there may be more candidate animals potentially used as animal models of HAV. On the contrary, some scholars believed that these new viruses are substantially more divergent from each other and from human HAV (including simian HAV), which is in accordance with them being

**52**

The animal experiments definitely play an important role in the development of life sciences and medical sciences. Therefore, ethical analysis concerning animal experiments is essential because it cannot completely avoid the use of animals [104] in the process of biomedicine and preclinical medicine research. Specially, NHPs act as the particularly valuable models for testing interventions against the Ebola and Marburg viruses in the field of studying of current infectious diseases, which can effectively objectively simulate human diseases via infections in these animals [105], and further contribute to the development of new protective and therapeutic vaccines. At a certain level, ethical issues become more important than scientific interest for this type of animal test [104] because such infections are often lethal to the experimental animals, which are commonly viewed as unethical. Similarly, experiments with HAV infection also expose animals (mainly NHPs) to injury or disease. Consequently, how to balance the contradiction between ethical challenges and NHPs infectious experiments becomes a vitally important subject.

#### **5. Future directions**

#### **5.1 Animal model methods**

Animal model research is entering a new and exciting stage along with the technologies of computational information and molecular biochemistry. For example, it is now possible for us to employ genome-edited techniques (e.g., ZFNs, TALENs, and CRISPR/Cas) to knockout specific genes, to knock in new genes, or to introduce specific mutations, and then to produce valuable animal models that benefited for our investigations. However, as we know, "no model is perfect, but many are useful" [106]. Therefore, establishing susceptible animal models is one of the methods in the research fields of HAV. By using appropriate and reliable animal models, virologist can perform a series of studies associated with hepatitis A including epidemiologic features, viral infectivity, humoral and cellular immunity, cytokine responses, virus pathogenesis, as well as the research and development of antiviral vaccinations.

#### **5.2 Cell culture methods**

For the development of hepatitis A vaccines, it is worth mentioning that a highly effective vaccine against HAV was manufactured by classical inactivation of the whole virus generated from cell culture [107], which commendably avoids the ethical controversy of using NHPs models. Moreover, there is a need to provide more support for the studies of long-term protection vaccines against hepatitis A infection [108].

Last but not least, it is very likely that a much wider host range of HAVassociated viruses will be discovered in other mammalian species in the future [38].

#### **6. Conclusion**

The Nobel laureate Peter Medawar have ever succinctly concluded that "No virus is known to do good" [109]. However, as we all know, "viruses are not omnipotent." For hepatitis viruses, the narrow hepatic tissue tropism maybe is the cause

of constraining the host ranges of hepatitis viruses to relatively few special host species. As previously reported, only one serotype of HAV had been found globally [110]. However, according to Bosch et al., there exists the possibility of emergence of a novel serotype originated from zoonotic reservoirs [18]. In summary, it is necessary to further develop candidate animal models for hepatitis A infection although HAV is easily capable of adapting growth in the condition of conventional mammalian cell cultures [92].

In recent decades, HAV has been ignored by viral research circles to a certain extent due to the research spending and interest have shifted to other hepatotropic pathogens. Finally, animal model research, as a preclinical study aiming to hepatitis A, can offer a scientific platform to accelerate the pace for drugs screening and vaccines development.

#### **Acknowledgements**

We would like to express our sincere thanks to Professor Costin Teodor Streba, Professor Cristin Constantin Vere and Professor Ion Rogoveanu for initiating this book project.

#### **Conflict of interest**

No conflicts of interest were reported.

#### **Financial support**

This work was supported in part by grants from the National Key R&D Program Projects (2017YFF0104904), National Key Research and Development Plan (2016YFD0500600), Guangdong Provincial Science and Technology Plan Project (2017B020207004), Maternal and Child Health Molecular Genetic Medicine Research Project of Maternal and Child Health Center in Chinese CDCP (FY-ZX-ZD-0285, MHBD-0833-001), Youth Innovation Project of Sichuan Medical Research (Q16036), Key Science & Technology Project of Leshan in 2018 (18SZD150).

**55**

authors.

**Author details**

Huafeng Lin1,2†, Aiping Min3

Leshan, Sichuan, P.R. China

Guangzhou, Guangdong, P.R. China

University, Guangzhou, P.R. China

provided the original work is properly cited.

\* †

University, Guangzhou, Guangdong, P.R. China

\*Address all correspondence to: map5198@163.com

, Gang Li4†, Yan Lei Chang2

1 Department of Biotechnology, College of Life Science and Technology, Jinan

2 Institute of Food Safety and Nutrition, Jinan University, Guangzhou, P.R. China

3 Department of Gynecology and Obstetrics, The People's Hospital of Leshan,

4 Institute of Biomedicine and Department of Cell Biology, Jinan University,

5 Combined TCM and Western Medicine Clinics, College of Basic Medicine, Jinan

† These authors contributed equally to this work and should be considered co-first

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Lei Shi2

and Dan Qiu5

*Applications of Animal Models in Researching Hepatitis A*

TALENs transcription activator-like effector nucleases

*DOI: http://dx.doi.org/10.5772/intechopen.90684*

WHO World Health Organization ZFNs zinc finger nucleases

#### **Acronyms and abbreviations**


*Applications of Animal Models in Researching Hepatitis A DOI: http://dx.doi.org/10.5772/intechopen.90684*

TALENs transcription activator-like effector nucleases WHO World Health Organization ZFNs zinc finger nucleases

#### **Author details**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

mammalian cell cultures [92].

vaccines development.

**Acknowledgements**

**Conflict of interest**

**Financial support**

(18SZD150).

**Acronyms and abbreviations**

IFN type I interferon

NHPs nonhuman primates

ALT alanine transaminase CLD chronic liver disease DHAV duck hepatitis A virus HAV hepatitis A virus HBV hepatitis B virus HCV hepatitis C virus HDV hepatitis D virus HEV hepatitis E virus HGV hepatitis G virus

IEM immune electron microscopy

IRES internal ribosomal entry site

No conflicts of interest were reported.

book project.

of constraining the host ranges of hepatitis viruses to relatively few special host species. As previously reported, only one serotype of HAV had been found globally [110]. However, according to Bosch et al., there exists the possibility of emergence of a novel serotype originated from zoonotic reservoirs [18]. In summary, it is necessary to further develop candidate animal models for hepatitis A infection although HAV is easily capable of adapting growth in the condition of conventional

In recent decades, HAV has been ignored by viral research circles to a certain extent due to the research spending and interest have shifted to other hepatotropic pathogens. Finally, animal model research, as a preclinical study aiming to hepatitis A, can offer a scientific platform to accelerate the pace for drugs screening and

We would like to express our sincere thanks to Professor Costin Teodor Streba, Professor Cristin Constantin Vere and Professor Ion Rogoveanu for initiating this

This work was supported in part by grants from the National Key R&D Program Projects (2017YFF0104904), National Key Research and Development Plan (2016YFD0500600), Guangdong Provincial Science and Technology Plan Project (2017B020207004), Maternal and Child Health Molecular Genetic Medicine Research Project of Maternal and Child Health Center in Chinese CDCP (FY-ZX-ZD-0285, MHBD-0833-001), Youth Innovation Project of Sichuan Medical Research (Q16036), Key Science & Technology Project of Leshan in 2018

**54**

Huafeng Lin1,2†, Aiping Min3 \* † , Gang Li4†, Yan Lei Chang2 , Lei Shi2 and Dan Qiu5

1 Department of Biotechnology, College of Life Science and Technology, Jinan University, Guangzhou, Guangdong, P.R. China

2 Institute of Food Safety and Nutrition, Jinan University, Guangzhou, P.R. China

3 Department of Gynecology and Obstetrics, The People's Hospital of Leshan, Leshan, Sichuan, P.R. China

4 Institute of Biomedicine and Department of Cell Biology, Jinan University, Guangzhou, Guangdong, P.R. China

5 Combined TCM and Western Medicine Clinics, College of Basic Medicine, Jinan University, Guangzhou, P.R. China

\*Address all correspondence to: map5198@163.com

† These authors contributed equally to this work and should be considered co-first authors.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[82] Das A, Hirai-Yuki A, González-López O, Rhein B, Moller-Tank S, Brouillette R, et al. TIM1 (HAVCR1) is not essential for cellular entry of either quasi-enveloped or naked hepatitis A virions. MBio. 2017;**8**(5):e00969-e00917. DOI: 10.1128/mBio.00969-17

[83] Hirai-Yuki A, Hensley L, McGivern DR, González-López O, Das A, Feng H, et al. MAVS-dependent host species range and pathogenicity of human hepatitis A virus. Science. 2016;**353**(6307):1541-1545. DOI: 10.1126/science.aaf8325

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[85] Yang Y, Liang Y, Qu L, Chen Z, Yi M, Li K, et al. Disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor. Proceedings of the National Academy of Sciences. 2007;**104**(17):7253-7258. DOI: 10.1073/pnas.0611506104

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[92] Fan Y, Huang ZY, Cao CC, Chen CS, Chen YX, Fan DD, et al. Genome of the Chinese tree shrew. Nature Communications. 2013;**4**:1426. DOI: 10.1038/ncomms2416

[93] Fuchs E, Flügge G. Social stress in tree shrews: Effects on physiology, brain function, and behavior of subordinate individuals. Pharmacology Biochemistry and Behavior. 2002;**73**(1):247-258. DOI: 10.1016/s0091-3057(02)00795-5

[94] Yan RQ, Su JJ, Huang DR, Gan YC, Yang C, Huang GH. Human hepatitis B virus and hepatocellular carcinoma II. Experimental induction of hepatocellular carcinoma in tree shrews exposed to hepatitis B virus and aflatoxin B1. Journal of Cancer Research and Clinical Oncology. 1996;**122**(5):289- 295. DOI: 10.1007/BF01261405

[95] Zhao X, Tang ZY, Klumpp B, Wolff-Vorbeck G, Barth H, Levy S, et al. Primary hepatocytes of Tupaia belangeri as a potential model for hepatitis C virus infection. The Journal of Clinical Investigation. 2002;**109**(2):221-232. DOI: 10.1172/JCI13011

[96] Feng Y, Feng YM, Lu C, Han Y, Liu L, Sun X, et al. Tree shrew, a potential animal model for hepatitis C, supports the infection and replication of HCV in vitro and in vivo. The Journal of General Virology. 2017;**98**(8):2069. DOI: 10.1099/jgv.0.000869

[97] Zhan MY, Liu CB, Li CM, Zhang WY, Zhu C, Pang QF, et al. A preliminary study of hepatitis a virus in Chinese Tupaia (author's transl). Acta Academiae Medicinae Sinicae. 1981;**3**(3):148-152

[98] Anthony SJ, Leger JS, Liang E, Hicks AL, Sanchez-Leon MD, Jain K, et al. Discovery of a novel hepatovirus (phopivirus of seals) related to human hepatitis A virus. MBio. 2015;**6**(4):e01180-e01115. DOI: 10.1128/ mBio.01180-15

[99] Fu Y, Pan M, Wang X, Xu Y, Yang H, Zhang D. Molecular detection and typing of duck hepatitis A virus directly from clinical specimens. Veterinary Microbiology. 2008;**131**(3-4):247-257. DOI: 10.1016/j.vetmic.2008.03.011

[100] Liu R, Shi S, Huang Y, Chen Z, Chen C, Cheng L, et al. Comparative pathogenicity of different subtypes of duck hepatitis A virus in Pekin ducklings. Veterinary Microbiology.

2019;**228**:181-187. DOI: 10.1016/j. vetmic.2018.11.030

[101] Wen X, Guo J, Sun D, Wang M, Cao D, Cheng A, et al. Mutations in VP0 and 2C proteins of duck hepatitis A virus type 3 attenuate viral infection and virulence. Vaccine. 2019;**7**(3):111. DOI: 10.3390/vaccines7030111

[102] Yu JM, Li LL, Zhang CY, Lu S, Ao YY, Gao HC, et al. A novel hepatovirus identified in wild woodchuck Marmota himalayana. Scientific Reports. 2016;**6**:22361

[103] Sander AL, Corman VM, Lukashev AN, Drexler JF. Evolutionary origins of enteric hepatitis viruses. Cold Spring Harbor Perspectives in Medicine. 2018;**8**(12):a031690. DOI: 10.1101/ cshperspect.a031690

[104] Gross D, Tolba RH. Ethics in animal-based research. European Surgical Research. 2015;**55**(1-2):43-57. DOI: 10.1159/000377721

[105] Barnhill A, Joffe S, Miller FG. The ethics of infection challenges in primates. Hastings Center Report. 2016;**46**(4):20-26. DOI: 10.1002/ hast.580

[106] Baxter VK, Griffin DE. Animal models: No model is perfect, but many are useful. In: Viral Pathogenesis. New York: Academic Press; 2016. pp. 125-138. DOI:10.1016/ B978-0-12-800964-2.00010-0

[107] Plotkin SA, Plotkin SL. The development of vaccines: How the past led to the future. Nature Reviews Microbiology. 2011;**9**(12):889-893. DOI: 10.1038/nrmicro2668

[108] Ott JJ, Irving G, Wiersma ST. Longterm protective effects of hepatitis A vaccines. A systematic review. Vaccine. 2012;**31**(1):3-11. DOI: 10.1016/j. vaccine.2012.04.104

**63**

*Applications of Animal Models in Researching Hepatitis A*

*DOI: http://dx.doi.org/10.5772/intechopen.90684*

[109] Cuthbert JA. Hepatitis A: Old and new. Clinical Microbiology Reviews. 2001;**14**(1):38-58. DOI: 10.1128/

Foodborne viruses and fresh produce. Journal of Applied Microbiology. 2001;**91**(5):759-773. DOI: 10.1046/

CMR.14.1.38-58.2001

[110] Seymour IJ, Appleton H.

j.1365-2672.2001. 01427.x

*Applications of Animal Models in Researching Hepatitis A DOI: http://dx.doi.org/10.5772/intechopen.90684*

[109] Cuthbert JA. Hepatitis A: Old and new. Clinical Microbiology Reviews. 2001;**14**(1):38-58. DOI: 10.1128/ CMR.14.1.38-58.2001

*Hepatitis A and Other Associated Hepatobiliary Diseases*

2019;**228**:181-187. DOI: 10.1016/j.

DOI: 10.3390/vaccines7030111

[103] Sander AL, Corman VM,

[104] Gross D, Tolba RH. Ethics in animal-based research. European Surgical Research. 2015;**55**(1-2):43-57.

[105] Barnhill A, Joffe S, Miller FG. The ethics of infection challenges in primates. Hastings Center Report. 2016;**46**(4):20-26. DOI: 10.1002/

[106] Baxter VK, Griffin DE. Animal

models: No model is perfect, but many are useful. In: Viral Pathogenesis. New York: Academic Press; 2016. pp. 125-138. DOI:10.1016/

B978-0-12-800964-2.00010-0

10.1038/nrmicro2668

vaccine.2012.04.104

[107] Plotkin SA, Plotkin SL. The development of vaccines: How the past led to the future. Nature Reviews Microbiology. 2011;**9**(12):889-893. DOI:

2012;**31**(1):3-11. DOI: 10.1016/j.

[108] Ott JJ, Irving G, Wiersma ST. Longterm protective effects of hepatitis A vaccines. A systematic review. Vaccine.

cshperspect.a031690

DOI: 10.1159/000377721

hast.580

Lukashev AN, Drexler JF. Evolutionary origins of enteric hepatitis viruses. Cold Spring Harbor Perspectives in Medicine. 2018;**8**(12):a031690. DOI: 10.1101/

[102] Yu JM, Li LL, Zhang CY, Lu S, Ao YY, Gao HC, et al. A novel hepatovirus identified in wild woodchuck Marmota himalayana. Scientific Reports. 2016;**6**:22361

[101] Wen X, Guo J, Sun D, Wang M, Cao D, Cheng A, et al. Mutations in VP0 and 2C proteins of duck hepatitis A virus type 3 attenuate viral infection and virulence. Vaccine. 2019;**7**(3):111.

vetmic.2018.11.030

[94] Yan RQ, Su JJ, Huang DR, Gan YC, Yang C, Huang GH. Human hepatitis B virus and hepatocellular carcinoma II. Experimental induction of hepatocellular carcinoma in tree shrews exposed to hepatitis B virus and aflatoxin B1. Journal of Cancer Research and Clinical Oncology. 1996;**122**(5):289-

295. DOI: 10.1007/BF01261405

DOI: 10.1172/JCI13011

10.1099/jgv.0.000869

1981;**3**(3):148-152

mBio.01180-15

[97] Zhan MY, Liu CB, Li CM, Zhang WY, Zhu C, Pang QF, et al. A preliminary study of hepatitis a virus in Chinese Tupaia (author's transl). Acta Academiae Medicinae Sinicae.

[98] Anthony SJ, Leger JS, Liang E, Hicks AL, Sanchez-Leon MD, Jain K, et al. Discovery of a novel hepatovirus (phopivirus of seals) related to human hepatitis A virus. MBio.

2015;**6**(4):e01180-e01115. DOI: 10.1128/

[99] Fu Y, Pan M, Wang X, Xu Y, Yang H, Zhang D. Molecular detection and typing of duck hepatitis A virus directly from clinical specimens. Veterinary Microbiology. 2008;**131**(3-4):247-257. DOI: 10.1016/j.vetmic.2008.03.011

[100] Liu R, Shi S, Huang Y, Chen Z, Chen C, Cheng L, et al. Comparative pathogenicity of different subtypes of duck hepatitis A virus in Pekin ducklings. Veterinary Microbiology.

[95] Zhao X, Tang ZY, Klumpp B, Wolff-Vorbeck G, Barth H, Levy S, et al. Primary hepatocytes of Tupaia belangeri as a potential model for hepatitis C virus infection. The Journal of Clinical Investigation. 2002;**109**(2):221-232.

[96] Feng Y, Feng YM, Lu C, Han Y, Liu L, Sun X, et al. Tree shrew, a potential animal model for hepatitis C, supports the infection and replication of HCV in vitro and in vivo. The Journal of General Virology. 2017;**98**(8):2069. DOI:

**62**

[110] Seymour IJ, Appleton H. Foodborne viruses and fresh produce. Journal of Applied Microbiology. 2001;**91**(5):759-773. DOI: 10.1046/ j.1365-2672.2001. 01427.x

**65**

**Chapter 5**

**Abstract**

developed countries.

**Key points**

against hepatitis A.

basic sanitation conditions and vaccination.

12 months of age in a single-dose regimen.

Hepatitis A: How We Are after the

Hepatitis A is a disease known for a long time. It has a universal distribution, although it has a higher prevalence in places with poor sanitary conditions due to its main form of transmission: fecal-oral. The local health conditions also influence the age of acquisition of the disease and, therefore, its clinical presentation, because the disease in young children is usually asymptomatic. It is a viral disease whose prevention is possible through improvements in the population's basic sanitation conditions and vaccination. Since the introduction of vaccines, it has been possible to see a reduction in its incidence, especially in places where universal vaccination of children has been instituted. In recent years immunoglobulin therapy is being replaced by vaccination in pre- and postexposure prophylaxis (PEP), except in specific situations. Its incidence, even in developing countries, has decreased after introduction of hepatitis A vaccine. The vaccine is recommended in two doses for children, starting at the age of 1. Argentina and, more recently, Brazil have adopted the universal vaccination of all children upon completion of 12 months of age in a single-dose regimen. Despite this breakthrough isolated outbreaks in homeless and drug users are still described in

*Julia Teixeira Rodrigues, Priscila Menezes Ferri Liu and* 

**Keywords:** hepatitis viruses, classification, diagnosis, prevention, control

• The vaccine is recommended in two doses for children, starting at the age of 1.

because although it is a rare complication, it may require hepatic transplantation.

• Hepatitis A is a viral disease whose prevention is possible through improvements in the population's

• The incidence, even in developing countries, has been reduced since the introduction of vaccines

• Argentina and, more recently, Brazil have adopted the universal vaccination of all children upon

While dealing with hepatitis A diagnosis, be aware of the signs of acute hepatic encephalopathy,

Introduction of Vaccines

*Adriana Teixeira Rodrigues*

#### **Chapter 5**

## Hepatitis A: How We Are after the Introduction of Vaccines

*Julia Teixeira Rodrigues, Priscila Menezes Ferri Liu and Adriana Teixeira Rodrigues*

#### **Abstract**

Hepatitis A is a disease known for a long time. It has a universal distribution, although it has a higher prevalence in places with poor sanitary conditions due to its main form of transmission: fecal-oral. The local health conditions also influence the age of acquisition of the disease and, therefore, its clinical presentation, because the disease in young children is usually asymptomatic. It is a viral disease whose prevention is possible through improvements in the population's basic sanitation conditions and vaccination. Since the introduction of vaccines, it has been possible to see a reduction in its incidence, especially in places where universal vaccination of children has been instituted. In recent years immunoglobulin therapy is being replaced by vaccination in pre- and postexposure prophylaxis (PEP), except in specific situations. Its incidence, even in developing countries, has decreased after introduction of hepatitis A vaccine. The vaccine is recommended in two doses for children, starting at the age of 1. Argentina and, more recently, Brazil have adopted the universal vaccination of all children upon completion of 12 months of age in a single-dose regimen. Despite this breakthrough isolated outbreaks in homeless and drug users are still described in developed countries.

**Keywords:** hepatitis viruses, classification, diagnosis, prevention, control

#### **Key points**


While dealing with hepatitis A diagnosis, be aware of the signs of acute hepatic encephalopathy, because although it is a rare complication, it may require hepatic transplantation.

#### **1. Introduction**

Hepatitis A is an acute viral disease caused by the hepatitis A virus (HAV), a picornavirus that infects only primates and has a low mutation rate compared to the other viruses capable of causing acute viral hepatitis [1–3].

The hepatitis A virus is transmitted by the fecal-oral route, either through ingestion of contaminated food [1] and water [4] or person-to-person contact. It has the ability to survive on surfaces for up to 60 days, and it is relatively resistant to alcohol and ether. As a result, the hygiene of bathrooms and toys in day care facilities must be meticulous [2, 5, 6].

The disease endemic character is related to poor sanitation conditions. That explains why its prevalence is higher in developing countries, where children generally become infected during the first years of life. That justifies the predominance of the asymptomatic form of the disease in such places [2].

Although it is related to inadequate sanitation conditions, there are records of the disease even in developed countries where the major concern is the people, mainly adults, who travel to exotic locations or developing countries. Besides that, some outbreaks due to food contamination are also described. Adult's disease, unlike the one that occurs in children, is symptomatic in 80% of cases [2]. Fortunately, since the introduction of vaccines, there has been a progressive decrease in the number of the cases of hepatitis A [7].

#### **2. The virus**

Hepatitis A virus (HAV) is one of the five etiological agents of viral hepatic inflammation (HAV, HBV, HCV, HDV, and HEV), whose incidence, according to the WHO, is sporadic and occurs in the form of epidemics around the whole world, which tend to present cyclical recurrences [8]. It belongs to the genus *Hepatovirus* that belongs to the family *Picornaviridae* [9].

#### **2.1 Structure of the virus**

HAV is described as being a naked virus; however, there is evidence that it can be released from a preinfected cell, through a non-lytic path, inside a small extracellular vesicle whose membrane surrounds the entire capsid and provides protection against the mechanisms of the host immune system [10].

The HAV presents as a genetic material a single-strand RNA with positive polarity, which confers the ability to act as messenger RNA (mRNA) and to interact directly with the ribosome to initiate the synthesis of the viral proteins. Its genome is divided in three parts [11]:


**67**

capsid [17].

*Hepatitis A: How We Are after the Introduction of Vaccines*

another individual who had been previously infected.

The HAV is transmitted through fecal-oral route; the individual becomes infected from the ingestion of water and food contaminated by fecal material from

After ingestion, the virus falls into the blood vessels and, through the portal circulation, reaches the hepatocytes. The first contact occurs through the basolateral membrane of the hepatocytes in the space of Disse [9]. After the processes of adsorption, penetration, denudation, and synthesis of viral genome and viral proteins, the virus is assembled and released from the host cell through a non-cytolytic process and undergoes cell exocytosis [12]. This release can occur through the apical membrane of the hepatocyte, which will cause the new virus to go to the bile canaliculi and, consequently, be sent to the intestine along with the bile. In addition, the release can also occur by the basolateral membrane, which causes the virus to return to the

After being sent to the intestine, the virus will be sent to the external environment through the feces. During excretion, a large amount of virus is eliminated. This starts about 10 days before the onset of clinical manifestations [13]. The period of greatest transmissibility occurs between the previous 15 days and 7 days after the

The immune response built by the host leads to the destruction of the hepatocytes infected by the virus and causes the appearance of the symptoms and signs of the disease. The HAV's slow replication does not appear to cause cytopathic

It is possible that several mechanisms are involved in the development of signs of the disease. In one study fibroblasts and peripheral blood lymphocytes from patients with acute hepatitis A were used to demonstrate that the IFN-γ produced by HAV-specific cytotoxic T lymphocytes might play an important role in the

Another analysis evidenced the presence of IFN-γ, TNF, IL-2, and IL-21 produced by polyfunctional CD4 + T cells. These results place the immune response modulated by CD4 + T lymphocytes as being more crucial in the control of HAV

In contrast to studies that propose that adaptive immunity is more important in the pathogenesis and resolution of hepatitis A, it has been proposed that cells of the innate immune system, especially natural killer and lymphokine-activated killer (LAK) cells, play a crucial role in hepatic cell damage, which is inflicted prior to that

In addition, there is an antibody-mediated response. HAV-specific immunoglobulin M (IgM) antibody and IgA antibodies can be detected, from the onset of the first clinical signs, in the patient's serum or plasma during the acute phase of the disease. IgG antibodies appear 1 week after the onset of the disease and can be detected for years even after healing. IgG can also be detected in vaccinated individuals. IgM and IgG immunoglobulins can neutralize the virus by recognizing epitopes of the HAV's structural proteins, VP1, VP2, and VP3, located in the

*DOI: http://dx.doi.org/10.5772/intechopen.88851*

**2.2 Biological cycle**

bloodstream [10, 12].

onset of symptoms [4].

pathogenesis of the disease [14].

performed by cytotoxic T lymphocytes [16].

**3. Pathogenesis**

effects [13].

replication [15].

3.Noncoding region 3′UTR which has a poly-A tail [11]

#### **2.2 Biological cycle**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

other viruses capable of causing acute viral hepatitis [1–3].

the asymptomatic form of the disease in such places [2].

that belongs to the family *Picornaviridae* [9].

**2.1 Structure of the virus**

is divided in three parts [11]:

Hepatitis A is an acute viral disease caused by the hepatitis A virus (HAV), a picornavirus that infects only primates and has a low mutation rate compared to the

The disease endemic character is related to poor sanitation conditions. That explains why its prevalence is higher in developing countries, where children generally become infected during the first years of life. That justifies the predominance of

Hepatitis A virus (HAV) is one of the five etiological agents of viral hepatic inflammation (HAV, HBV, HCV, HDV, and HEV), whose incidence, according to the WHO, is sporadic and occurs in the form of epidemics around the whole world, which tend to present cyclical recurrences [8]. It belongs to the genus *Hepatovirus*

HAV is described as being a naked virus; however, there is evidence that it can be released from a preinfected cell, through a non-lytic path, inside a small extracellular vesicle whose membrane surrounds the entire capsid and provides protection against the mechanisms of the host immune

The HAV presents as a genetic material a single-strand RNA with positive polarity, which confers the ability to act as messenger RNA (mRNA) and to interact directly with the ribosome to initiate the synthesis of the viral proteins. Its genome

1.Noncoding region 5′UTR covalently linked to viral protein VPg [11]

nonstructural proteins that act during viral multiplication [11]

3.Noncoding region 3′UTR which has a poly-A tail [11]

2.A single open reading frame (ORF) subdivided into P1, which encodes the viral capsid proteins (VP1, VP2, VP3, and VP4), and P2 and P3, which encode

Although it is related to inadequate sanitation conditions, there are records of the disease even in developed countries where the major concern is the people, mainly adults, who travel to exotic locations or developing countries. Besides that, some outbreaks due to food contamination are also described. Adult's disease, unlike the one that occurs in children, is symptomatic in 80% of cases [2]. Fortunately, since the introduction of vaccines, there has been a progressive decrease in the number of the cases of

The hepatitis A virus is transmitted by the fecal-oral route, either through ingestion of contaminated food [1] and water [4] or person-to-person contact. It has the ability to survive on surfaces for up to 60 days, and it is relatively resistant to alcohol and ether. As a result, the hygiene of bathrooms and toys in day care facilities must

**1. Introduction**

be meticulous [2, 5, 6].

hepatitis A [7].

**2. The virus**

system [10].

**66**

The HAV is transmitted through fecal-oral route; the individual becomes infected from the ingestion of water and food contaminated by fecal material from another individual who had been previously infected.

After ingestion, the virus falls into the blood vessels and, through the portal circulation, reaches the hepatocytes. The first contact occurs through the basolateral membrane of the hepatocytes in the space of Disse [9]. After the processes of adsorption, penetration, denudation, and synthesis of viral genome and viral proteins, the virus is assembled and released from the host cell through a non-cytolytic process and undergoes cell exocytosis [12]. This release can occur through the apical membrane of the hepatocyte, which will cause the new virus to go to the bile canaliculi and, consequently, be sent to the intestine along with the bile. In addition, the release can also occur by the basolateral membrane, which causes the virus to return to the bloodstream [10, 12].

After being sent to the intestine, the virus will be sent to the external environment through the feces. During excretion, a large amount of virus is eliminated. This starts about 10 days before the onset of clinical manifestations [13]. The period of greatest transmissibility occurs between the previous 15 days and 7 days after the onset of symptoms [4].

#### **3. Pathogenesis**

The immune response built by the host leads to the destruction of the hepatocytes infected by the virus and causes the appearance of the symptoms and signs of the disease. The HAV's slow replication does not appear to cause cytopathic effects [13].

It is possible that several mechanisms are involved in the development of signs of the disease. In one study fibroblasts and peripheral blood lymphocytes from patients with acute hepatitis A were used to demonstrate that the IFN-γ produced by HAV-specific cytotoxic T lymphocytes might play an important role in the pathogenesis of the disease [14].

Another analysis evidenced the presence of IFN-γ, TNF, IL-2, and IL-21 produced by polyfunctional CD4 + T cells. These results place the immune response modulated by CD4 + T lymphocytes as being more crucial in the control of HAV replication [15].

In contrast to studies that propose that adaptive immunity is more important in the pathogenesis and resolution of hepatitis A, it has been proposed that cells of the innate immune system, especially natural killer and lymphokine-activated killer (LAK) cells, play a crucial role in hepatic cell damage, which is inflicted prior to that performed by cytotoxic T lymphocytes [16].

In addition, there is an antibody-mediated response. HAV-specific immunoglobulin M (IgM) antibody and IgA antibodies can be detected, from the onset of the first clinical signs, in the patient's serum or plasma during the acute phase of the disease. IgG antibodies appear 1 week after the onset of the disease and can be detected for years even after healing. IgG can also be detected in vaccinated individuals. IgM and IgG immunoglobulins can neutralize the virus by recognizing epitopes of the HAV's structural proteins, VP1, VP2, and VP3, located in the capsid [17].

#### **4. Epidemiology**

The hepatitis A virus is distributed worldwide. However, the highest incidence of hepatitis A occurs in developing countries and in the ones with poor health conditions. In developed countries the disease acquired by traveler accounts for almost half of the cases reported.

The introduction of universal vaccination of children can change this general picture. The implementation of this program may reduce the rate of seropositive children against hepatitis A virus [18].

Luxemburger and Dutta show Brazil as a country with high endemicity of hepatitis A, meaning that more than 90% of children between 5 and 14 years old were seropositive for hepatitis A [19].

After that date there have been modifications in epidemiology of this disease. Checking the Epidemiological Bulletin of the Ministry of Health of Brazil in 2018, it's possible to notice that after 2007 the incidence rate of hepatitis A has shown a progressive tendency of falling, going from 7.1 cases to 1.0 per 100,000 inhabitants in 2017.

Between 1997 and 2017, most cases of hepatitis A occurred in children under 10 years of age (53.8%). In the last 2 years, there has been not only an increase in the incidence of the disease among people from 20 to 39 years of age but also a modification in the route of contamination. There was a significant reduction in cases related to food contamination and an increase in those related to sexual transmission. The incidence reduction preceded the universal vaccine. Universal vaccination of children from 12 months onwards was introduced in Brazil's vaccination calendar only in 2014 [20].

In relation to hepatitis A mortality in Brazil, between 2000 and 2016, there were 1125 deaths associated with viral hepatitis A. There is no data yet to compare mortality after the onset of the universal vaccine of Brazilian children [20]. Considering the distribution of deaths associated with all viral hepatitis in Brazil between 2000 and 2016 (66,196 obits), the proportion of obits was 1.7% associated with viral hepatitis A; 21.4% to hepatitis B; 75.8% to hepatitis C; and 1.1% to hepatitis D. In the document there is no report of viral hepatitis E [20].

Recently, the person-to-person HAV outbreaks involving people who use drugs or people experiencing homelessness are ongoing in United States, and this could signal a shift in HAV infection epidemiology in the United States [21].

#### **5. Clinical condition**

The virus's incubation period is long (15–50 days), and the first symptoms are non-specific and reminiscent of common viral disease. The following may be present: fever, malaise, headache, and abdominal pain. Eventually, jaundice, hepatomegaly, splenomegaly, and bradycardia can appear. The icteric phase has a variable duration, on average from 4 to 30 days, and is associated with dark urine and acholic stools. Laboratory elevation of aminotransferases and direct hyperbilirubinemia is observed [2].

Hepatitis A can have different forms of evolution, although most patients progress to healing within 2 months. Approximately 10% of hepatitis A patients present a biphasic form, in which relapses are observed in the first 6 months of evolution. Other forms considered atypical [6] may also be observed as the prolonged form, in which the symptoms persist for up to 120 days [22], and the cholestatic form presents the following alterations: elevation of the direct fraction of bilirubin, presence of significant pruritus, malabsorption of nutrients, and weight loss. Fortunately, all these forms present evolution for healing. Only a small fraction of patients will develop acute liver failure that is associated with increased morbidity and mortality [22].

**69**

*Hepatitis A: How We Are after the Introduction of Vaccines*

According to CDC [23], suspected hepatitis A occurs in the presence of a suggestive clinical picture, characterized by the presence of fever, headache, malaise, anorexia, nausea, vomiting, diarrhea, abdominal pain, or dark urine, associated with suggestive laboratory alterations of the direct bilirubin fraction or ALT > 200 IU/L, in the absence of another diagnosis that explains such alterations.

The presence of immunoglobulin M (IgM) antibody against hepatitis A virus or

Hepatitis A has no specific treatment, and usually only support and monitoring measures are adopted. Although the disease is self-limiting in the vast majority of cases, some patients develop severe hepatitis, which can lead to fulminant hepatic

The fulminant hepatic insufficiency diagnosis should be considered in those patients with viral hepatitis who present any alteration of consciousness accompanied by some coagulation disorder [23] characterized by INR ≥ 1.5. Those findings indicate hospitalization of the patient in an intensive care unit with possibility of

It is important to be aware of the fact that the symptoms of fulminant hepatic insufficiency become noticeable late when most of the liver functions are already compromised [26]. And so, the patient need to be transferred to a transplant center as soon as possible if he or she presents metabolic acidosis with arterial pH < 7.3, arterial lactate > 3 mmol/L (27 mg/dL), INR > 6.5, creatinine > 3.4 mg/ dL, and presence of grade 3 or 4 hepatic encephalopathy all within the 24-h

Special attention should also be given to the child who evolves with the cholestatic form due to the nutritional risk secondary to malabsorption of fat-soluble nutrients and vitamins. During disease, until the cholestasis is fully treated, it may be necessary to provide increased caloric intake in addition to vitamins A, D, and

The Brazilian Ministry of Health advices that the prevention of the disease is best achieved by improving basic sanitation and personal hygiene conditions as

1.Wash the hands after going to the bathroom and before eating or preparing

E. Nutritional care should be maintained until cholestasis improves [27].

2.Wash, with treated water, foods that are consumed raw [28].

4.Wash dishes, glasses, cutlery, and bottles properly [28].

The presence of sensorineural alterations should raise the suspicion of acute liver failure, and the diagnosis is confirmed if there is an association of sensory alterations with a high prothrombin time in more than 4–6 s (INR ≥ 1.5) [24, 25].

viral RNA detection provides laboratory evidence for the diagnosis [23].

*DOI: http://dx.doi.org/10.5772/intechopen.88851*

insufficiency, and need liver transplantation.

transfer to a liver transplant center.

**6. Diagnosis**

**7. Treatment**

period [25].

**8. Prevention**

follows [28]:

food [28].

3.Cook the food before eating it [28].

#### **6. Diagnosis**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

The hepatitis A virus is distributed worldwide. However, the highest incidence of hepatitis A occurs in developing countries and in the ones with poor health conditions. In developed countries the disease acquired by traveler accounts for almost

The introduction of universal vaccination of children can change this general picture. The implementation of this program may reduce the rate of seropositive

Luxemburger and Dutta show Brazil as a country with high endemicity of hepatitis A, meaning that more than 90% of children between 5 and 14 years old

After that date there have been modifications in epidemiology of this disease. Checking the Epidemiological Bulletin of the Ministry of Health of Brazil in 2018, it's possible to notice that after 2007 the incidence rate of hepatitis A has shown a progressive tendency of falling, going from 7.1 cases to 1.0 per 100,000 inhabitants in 2017. Between 1997 and 2017, most cases of hepatitis A occurred in children under 10 years of age (53.8%). In the last 2 years, there has been not only an increase in the incidence of the disease among people from 20 to 39 years of age but also a modification in the route of contamination. There was a significant reduction in cases related to food contamination and an increase in those related to sexual transmission. The incidence reduction preceded the universal vaccine. Universal vaccination of children from 12 months onwards was introduced in Brazil's vaccination calendar only in 2014 [20]. In relation to hepatitis A mortality in Brazil, between 2000 and 2016, there were 1125 deaths associated with viral hepatitis A. There is no data yet to compare mortality after the onset of the universal vaccine of Brazilian children [20]. Considering the distribution of deaths associated with all viral hepatitis in Brazil between 2000 and 2016 (66,196 obits), the proportion of obits was 1.7% associated with viral hepatitis A; 21.4% to hepatitis B; 75.8% to hepatitis C; and 1.1% to hepatitis D. In the

Recently, the person-to-person HAV outbreaks involving people who use drugs or people experiencing homelessness are ongoing in United States, and this could

The virus's incubation period is long (15–50 days), and the first symptoms are non-specific and reminiscent of common viral disease. The following may be present: fever, malaise, headache, and abdominal pain. Eventually, jaundice, hepatomegaly, splenomegaly, and bradycardia can appear. The icteric phase has a variable duration, on average from 4 to 30 days, and is associated with dark urine and acholic stools. Laboratory elevation of aminotransferases and direct hyperbili-

Hepatitis A can have different forms of evolution, although most patients progress

to healing within 2 months. Approximately 10% of hepatitis A patients present a biphasic form, in which relapses are observed in the first 6 months of evolution. Other forms considered atypical [6] may also be observed as the prolonged form, in which the symptoms persist for up to 120 days [22], and the cholestatic form presents the following alterations: elevation of the direct fraction of bilirubin, presence of significant pruritus, malabsorption of nutrients, and weight loss. Fortunately, all these forms present evolution for healing. Only a small fraction of patients will develop acute liver

failure that is associated with increased morbidity and mortality [22].

signal a shift in HAV infection epidemiology in the United States [21].

**4. Epidemiology**

half of the cases reported.

children against hepatitis A virus [18].

were seropositive for hepatitis A [19].

document there is no report of viral hepatitis E [20].

**5. Clinical condition**

rubinemia is observed [2].

**68**

According to CDC [23], suspected hepatitis A occurs in the presence of a suggestive clinical picture, characterized by the presence of fever, headache, malaise, anorexia, nausea, vomiting, diarrhea, abdominal pain, or dark urine, associated with suggestive laboratory alterations of the direct bilirubin fraction or ALT > 200 IU/L, in the absence of another diagnosis that explains such alterations.

The presence of immunoglobulin M (IgM) antibody against hepatitis A virus or viral RNA detection provides laboratory evidence for the diagnosis [23].

The presence of sensorineural alterations should raise the suspicion of acute liver failure, and the diagnosis is confirmed if there is an association of sensory alterations with a high prothrombin time in more than 4–6 s (INR ≥ 1.5) [24, 25].

#### **7. Treatment**

Hepatitis A has no specific treatment, and usually only support and monitoring measures are adopted. Although the disease is self-limiting in the vast majority of cases, some patients develop severe hepatitis, which can lead to fulminant hepatic insufficiency, and need liver transplantation.

The fulminant hepatic insufficiency diagnosis should be considered in those patients with viral hepatitis who present any alteration of consciousness accompanied by some coagulation disorder [23] characterized by INR ≥ 1.5. Those findings indicate hospitalization of the patient in an intensive care unit with possibility of transfer to a liver transplant center.

It is important to be aware of the fact that the symptoms of fulminant hepatic insufficiency become noticeable late when most of the liver functions are already compromised [26]. And so, the patient need to be transferred to a transplant center as soon as possible if he or she presents metabolic acidosis with arterial pH < 7.3, arterial lactate > 3 mmol/L (27 mg/dL), INR > 6.5, creatinine > 3.4 mg/ dL, and presence of grade 3 or 4 hepatic encephalopathy all within the 24-h period [25].

Special attention should also be given to the child who evolves with the cholestatic form due to the nutritional risk secondary to malabsorption of fat-soluble nutrients and vitamins. During disease, until the cholestasis is fully treated, it may be necessary to provide increased caloric intake in addition to vitamins A, D, and E. Nutritional care should be maintained until cholestasis improves [27].

#### **8. Prevention**

The Brazilian Ministry of Health advices that the prevention of the disease is best achieved by improving basic sanitation and personal hygiene conditions as follows [28]:


As a prophylactic measure for travelers, the vaccine has replaced immunoglobulin (Ig). The protection achieved by Ig when used before exposure is approximately 80–90% [20], whereas a single dose of hepatitis A vaccine provides protection of 85% of cases in the first 6 weeks and up to 95–00% after this time period [29]. Postexposure prophylaxis (PEP) with hepatitis A vaccine prevents hepatitis A virus infection when administered within 2 weeks of exposure [25, 30]. The use of Ig for PEP is indicated in the following situations: children aged <12 months; immunocompromised people; patients with chronic liver disease; and those for whom the vaccine is contraindicated. For people over 40, immunoglobulin is preferred, but the vaccine can be used on a non-distant immunoglobulin obtained [25, 28]. The recommended dose of IG was modified to 0.1 mL/kg [30].

Pre-exposure prevention for travelers aged 12 months to 40 years should be given by vaccination as soon as traveling to endemic sites is considered, and the second dose should be administered at the regular interval recommended by the vaccine manufacturer. The vaccine can be administered between 6 and 11 months for pre-exposure prophylaxis, but this dose should not be considered when the child initiates the usual vaccination schedule at 12 months of age. In the case of infants <6 months of age, Ig should be given. The dose in these cases depends on the duration of the trip: for trips with a duration of up to 1 month 0.1 mL/kg is recommended; and with longer duration, the dose will be 0.2 mL/kg repeated every 2 months until the end of the trip. Travelers aged >40 years, immunocompromised or with chronic liver disease, are recommended to be vaccinated against hepatitis A and use Ig at the dose of 0.1 mL/kg at the same time but applied at separate sites [30].

Since the introduction of vaccines, there has been a reduction in the prevalence of the disease. The vaccine is effective, even if given as a single dose, although it is usually recommended in two doses. The initial dose should be administered at 1 year of age, especially in endemic areas where contact with the virus is early. The booster dose may occur at varying intervals, usually 6–18 months after the first dose.

The effectiveness of the single-dose vaccine was initially described among people vaccinated for travel to exotic locations or with inadequate sanitary conditions [31, 32]. In 2003 a randomized, double blind study in Nicaragua showed that one dose of the vaccine had good efficacy, reaching up to 100% of children after 6 weeks (95% CI: 79.8–100%) [29].

Young children who present hepatitis A are asymptomatic and therefore able to spread the virus in the community. That is why universal vaccination of all children between 1 and 5 years of age is recommended in populations where the incidence of the disease is >20 cases/100,000 inhabitants. The monovalent vaccine (Havrix®, Vaqta®) should be administered via intramuscular injection in two doses at a 6- to 12-month interval between doses [21, 25].

In 2005, Argentina adopted a universal vaccination schedule for children aged 12 months in a single dose, and since then the incidence of hepatitis A has decreased by >80% in all age groups [33, 34].

**71**

*Hepatitis A: How We Are after the Introduction of Vaccines*

in the long run whether the booster dose will be needed [34].

For developing countries, this may be a cheaper and simpler strategy than twodose schedules; however, it is necessary to deploy a surveillance system to determine

The United Nations (UN) reports that viral hepatitis is a serious threat to global health, mainly related to hepatitis B and C viruses that cause chronic liver disease. The UN estimates suggest that 325 million people are infected worldwide, with 70 million on the African continent alone. Although the reports focus on hepatitis B and C because of their chronicity, the UN and WHO are committed to reducing hepatitis A-related deaths by 10 percent by the year 2030. According to the WHO, viral hepatitis A is a viral infection of the disease. It can be eliminated from Africa with vaccination and improved sanitation and access to safe drinking water. This

latter measure may also reduce the incidence of viral hepatitis E [36, 37].

it's possible that this goal may be achieved within the following decades.

other countries where hepatitis A is also endemic.

facilities, especially in endemic regions.

using microwire electrodes [39].

DNA and cut it [40, 41].

**9. Future perspectives**

In order to make vaccination against HAV feasible for developing countries, it is necessary to evaluate effective and cost-effective strategies. Vizzotti et al. [38] evaluated the impact of single-dose vaccination in Argentina and found an impressive decline in hepatitis A cases accompanied by a decrease in medical and nonmedical costs in the first 5 years. The authors then suggested that this could be a simpler and less costly strategy thus becoming an economically viable alternative to

Since both the world population and the life expectancy are increasing, it's imperative that new techniques, fast, accessible, and sensitive ones, are developed in order to guarantee accurate diagnosis and proper treatment to anyone who is suffering from a disease. With new technologies being released in a daily basis and several researches being done in fields like molecular diagnostics, immunodiagnostics, and gene therapy,

So as to improve the diagnosis of hepatitis and several other diseases, either through the detection of pathogens or elements present due to the host's immune response, it's essential that new, highly sensitive tests become available in healthcare

One possibility is to use new techniques that are being developed and allow the detection of antibodies. One example is the capacitive immunosensor developed to detect anti-Zika virus and anti-chikungunya virus antibodies in low concentrations

Another possibility is to use the CRISPR-Cas technology to detect the pathogen's genetic material. This technique was developed based on the analysis of a specific defense mechanism of bacteria and archaea, organisms in which clusters of regularly interspaced short palindromic repeats (CRISPR), a specific region of the DNA, are transcribed into CRISPR RNA (crRNA) when they are infected by viruses [40, 41]. When the crRNA and the trans-activating crRNA (tracrRNA) associate with Cas9, an enzyme, the crRNA-Cas9 complex will then target a foreign

Studies have shown that, through modification, the CRISPR-Cas complex is capable of targeting RNA [42] and adapting to different intracellular environments,

In Brazil, the hepatitis A vaccine was added to the national immunization program (NIP) only in 2014. Universal vaccination of children with a single dose of the inactivated vaccine was adopted at 12 months of age. In an official document, the NIP undertook to monitor the epidemiological situation of hepatitis A, aiming at the definition of whether or not to include a second dose in the child's immuniza-

*DOI: http://dx.doi.org/10.5772/intechopen.88851*

tion schedule [35].

*Hepatitis A and Other Associated Hepatobiliary Diseases*

bleach when washing the restroom [28].

using 2.5% sodium hypochlorite or bleach [28].

recommended dose of IG was modified to 0.1 mL/kg [30].

at the same time but applied at separate sites [30].

6 weeks (95% CI: 79.8–100%) [29].

by >80% in all age groups [33, 34].

12-month interval between doses [21, 25].

compromise the water table that feeds the well [28].

5.Avoid the construction of ditches near wells and river springs, so as not to

6.If there is a patient with hepatitis A at home, use 2.5% sodium hypochlorite, or

7.In nurseries, preschools, cafeterias, restaurants, and closed institutions, adopt strict hygiene measures, such as disinfection of objects, benches, and floors

As a prophylactic measure for travelers, the vaccine has replaced immunoglobulin (Ig). The protection achieved by Ig when used before exposure is approximately 80–90% [20], whereas a single dose of hepatitis A vaccine provides protection of 85% of cases in the first 6 weeks and up to 95–00% after this time period [29]. Postexposure prophylaxis (PEP) with hepatitis A vaccine prevents hepatitis A virus infection when administered within 2 weeks of exposure [25, 30]. The use of Ig for PEP is indicated in the following situations: children aged <12 months; immunocompromised people; patients with chronic liver disease; and those for whom the vaccine is contraindicated. For people over 40, immunoglobulin is preferred, but the vaccine can be used on a non-distant immunoglobulin obtained [25, 28]. The

Pre-exposure prevention for travelers aged 12 months to 40 years should be given by vaccination as soon as traveling to endemic sites is considered, and the second dose should be administered at the regular interval recommended by the vaccine manufacturer. The vaccine can be administered between 6 and 11 months for pre-exposure prophylaxis, but this dose should not be considered when the child initiates the usual vaccination schedule at 12 months of age. In the case of infants <6 months of age, Ig should be given. The dose in these cases depends on the duration of the trip: for trips with a duration of up to 1 month 0.1 mL/kg is recommended; and with longer duration, the dose will be 0.2 mL/kg repeated every 2 months until the end of the trip. Travelers aged >40 years, immunocompromised or with chronic liver disease, are recommended to be vaccinated against hepatitis A and use Ig at the dose of 0.1 mL/kg

Since the introduction of vaccines, there has been a reduction in the prevalence of the disease. The vaccine is effective, even if given as a single dose, although it is usually recommended in two doses. The initial dose should be administered at 1 year of age, especially in endemic areas where contact with the virus is early. The

booster dose may occur at varying intervals, usually 6–18 months after the

The effectiveness of the single-dose vaccine was initially described among people vaccinated for travel to exotic locations or with inadequate sanitary conditions [31, 32]. In 2003 a randomized, double blind study in Nicaragua showed that one dose of the vaccine had good efficacy, reaching up to 100% of children after

Young children who present hepatitis A are asymptomatic and therefore able to spread the virus in the community. That is why universal vaccination of all children between 1 and 5 years of age is recommended in populations where the incidence of the disease is >20 cases/100,000 inhabitants. The monovalent vaccine (Havrix®, Vaqta®) should be administered via intramuscular injection in two doses at a 6- to

In 2005, Argentina adopted a universal vaccination schedule for children aged 12 months in a single dose, and since then the incidence of hepatitis A has decreased

**70**

first dose.

For developing countries, this may be a cheaper and simpler strategy than twodose schedules; however, it is necessary to deploy a surveillance system to determine in the long run whether the booster dose will be needed [34].

In Brazil, the hepatitis A vaccine was added to the national immunization program (NIP) only in 2014. Universal vaccination of children with a single dose of the inactivated vaccine was adopted at 12 months of age. In an official document, the NIP undertook to monitor the epidemiological situation of hepatitis A, aiming at the definition of whether or not to include a second dose in the child's immunization schedule [35].

The United Nations (UN) reports that viral hepatitis is a serious threat to global health, mainly related to hepatitis B and C viruses that cause chronic liver disease. The UN estimates suggest that 325 million people are infected worldwide, with 70 million on the African continent alone. Although the reports focus on hepatitis B and C because of their chronicity, the UN and WHO are committed to reducing hepatitis A-related deaths by 10 percent by the year 2030. According to the WHO, viral hepatitis A is a viral infection of the disease. It can be eliminated from Africa with vaccination and improved sanitation and access to safe drinking water. This latter measure may also reduce the incidence of viral hepatitis E [36, 37].

In order to make vaccination against HAV feasible for developing countries, it is necessary to evaluate effective and cost-effective strategies. Vizzotti et al. [38] evaluated the impact of single-dose vaccination in Argentina and found an impressive decline in hepatitis A cases accompanied by a decrease in medical and nonmedical costs in the first 5 years. The authors then suggested that this could be a simpler and less costly strategy thus becoming an economically viable alternative to other countries where hepatitis A is also endemic.

#### **9. Future perspectives**

Since both the world population and the life expectancy are increasing, it's imperative that new techniques, fast, accessible, and sensitive ones, are developed in order to guarantee accurate diagnosis and proper treatment to anyone who is suffering from a disease. With new technologies being released in a daily basis and several researches being done in fields like molecular diagnostics, immunodiagnostics, and gene therapy, it's possible that this goal may be achieved within the following decades.

So as to improve the diagnosis of hepatitis and several other diseases, either through the detection of pathogens or elements present due to the host's immune response, it's essential that new, highly sensitive tests become available in healthcare facilities, especially in endemic regions.

One possibility is to use new techniques that are being developed and allow the detection of antibodies. One example is the capacitive immunosensor developed to detect anti-Zika virus and anti-chikungunya virus antibodies in low concentrations using microwire electrodes [39].

Another possibility is to use the CRISPR-Cas technology to detect the pathogen's genetic material. This technique was developed based on the analysis of a specific defense mechanism of bacteria and archaea, organisms in which clusters of regularly interspaced short palindromic repeats (CRISPR), a specific region of the DNA, are transcribed into CRISPR RNA (crRNA) when they are infected by viruses [40, 41]. When the crRNA and the trans-activating crRNA (tracrRNA) associate with Cas9, an enzyme, the crRNA-Cas9 complex will then target a foreign DNA and cut it [40, 41].

Studies have shown that, through modification, the CRISPR-Cas complex is capable of targeting RNA [42] and adapting to different intracellular environments, such as the eukaryotic one [43]. Other experiments with CRISPR-Cas demonstrate that it can detect both Zika virus and dengue virus, RNA viruses [44]. Besides, this last analysis has also shown that a test based on this technique would be fast and sensitive and the costs would be low [44].

Regarding the treatment, some studies have shown that genome editing using the CRISPR-Cas system might also allow the development of effective antiviral therapies. Experiments done in vitro using human cells demonstrate that this system can target herpesvirus and provide either clearance of some strains of this virus or cause decay in other strains' replication [45]. Another work, by demonstrating that the CRISPR-Cas system was able to inhibit the accumulation of hepatitis B virus (HBV) DNA in human cells, has shown that this system has the capacity to be developed into an effective therapy for viral diseases [46].

Nevertheless, to ensure a decrease in the number of people infected with hepatitis, it's also important to develop strategies to prevent the spread of the virus. Since hepatitis A is transmitted through a fecal-oral pathway, to achieve this goal, it's essential that the water used by the population receives proper treatment in order to guarantee that all the pathogenic organisms are eliminated. In this context, water and sanitation projects developed by humanitarian organizations can contribute to the decrease in the number of people infected with fecal-oral transmitted diseases.

Some examples are the water safety plans (WSPs), created by the World Health Organization, and the Sustainable Development Goal 6, created by the United Nations. The former focusses on assembling a team that will develop a WSP considering all the hazardous events that can affect the safety of a water supply so as to determine and validate control measures that will be used to develop and implement improvements, which ensure that the drinking water supply is safe and accepted by the population [47]. The latter sets the goals for the following decades and analyzes indicators in order to monitor and promote the implementation of plans of action made to ensure universal and equitable access to safe and affordable drinking water for all, access to adequate and equitable sanitation, hygiene for all, end open defecation [48], and several other measures that can help control fecaloral transmitted diseases.

#### **10. Conclusion**

Hepatitis A is a viral disease whose prevention is possible through improvements in basic sanitation and vaccination of the population. The vaccine provides good protection and is recommended in two doses for children, starting at the age of 1 year. The efficacy of the single-dose vaccine has been reported between people traveling to exotic locations and then by developing countries that have adopted this single-dose schedule. In those places where the single-dose schedule has been adopted, a surveillance system should be in place to determine whether the booster dose will be necessary over the long term. Patients with hepatitis A present evolution to the healing in the majority of the cases, but it is necessary to be aware that, in rare occasions, they can develop acute hepatic insufficiency, which is associated to greater morbimortality.

**73**

**Author details**

Julia Teixeira Rodrigues, Priscila Menezes Ferri Liu and Adriana Teixeira Rodrigues\*

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil

\*Address all correspondence to: adrianatr92@gmail.com

provided the original work is properly cited.

*Hepatitis A: How We Are after the Introduction of Vaccines*

*DOI: http://dx.doi.org/10.5772/intechopen.88851*

#### **Conflict of interest**

The authors declare no conflict of interest.

*Hepatitis A: How We Are after the Introduction of Vaccines DOI: http://dx.doi.org/10.5772/intechopen.88851*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

sensitive and the costs would be low [44].

oral transmitted diseases.

greater morbimortality.

**Conflict of interest**

The authors declare no conflict of interest.

**10. Conclusion**

developed into an effective therapy for viral diseases [46].

such as the eukaryotic one [43]. Other experiments with CRISPR-Cas demonstrate that it can detect both Zika virus and dengue virus, RNA viruses [44]. Besides, this last analysis has also shown that a test based on this technique would be fast and

Regarding the treatment, some studies have shown that genome editing using the CRISPR-Cas system might also allow the development of effective antiviral therapies. Experiments done in vitro using human cells demonstrate that this system can target herpesvirus and provide either clearance of some strains of this virus or cause decay in other strains' replication [45]. Another work, by demonstrating that the CRISPR-Cas system was able to inhibit the accumulation of hepatitis B virus (HBV) DNA in human cells, has shown that this system has the capacity to be

Nevertheless, to ensure a decrease in the number of people infected with hepatitis, it's also important to develop strategies to prevent the spread of the virus. Since hepatitis A is transmitted through a fecal-oral pathway, to achieve this goal, it's essential that the water used by the population receives proper treatment in order to guarantee that all the pathogenic organisms are eliminated. In this context, water and sanitation projects developed by humanitarian organizations can contribute to the decrease in the number of people infected with fecal-oral transmitted diseases. Some examples are the water safety plans (WSPs), created by the World Health

Organization, and the Sustainable Development Goal 6, created by the United Nations. The former focusses on assembling a team that will develop a WSP considering all the hazardous events that can affect the safety of a water supply so as to determine and validate control measures that will be used to develop and implement improvements, which ensure that the drinking water supply is safe and accepted by the population [47]. The latter sets the goals for the following decades and analyzes indicators in order to monitor and promote the implementation of plans of action made to ensure universal and equitable access to safe and affordable drinking water for all, access to adequate and equitable sanitation, hygiene for all, end open defecation [48], and several other measures that can help control fecal-

Hepatitis A is a viral disease whose prevention is possible through improvements in basic sanitation and vaccination of the population. The vaccine provides good protection and is recommended in two doses for children, starting at the age of 1 year. The efficacy of the single-dose vaccine has been reported between people traveling to exotic locations and then by developing countries that have adopted this single-dose schedule. In those places where the single-dose schedule has been adopted, a surveillance system should be in place to determine whether the booster dose will be necessary over the long term. Patients with hepatitis A present evolution to the healing in the majority of the cases, but it is necessary to be aware that, in rare occasions, they can develop acute hepatic insufficiency, which is associated to

**72**

#### **Author details**

Julia Teixeira Rodrigues, Priscila Menezes Ferri Liu and Adriana Teixeira Rodrigues\* Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil

\*Address all correspondence to: adrianatr92@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[1] Matheny SC, Kingery JE. University of Kentucky College of Medicine, Lexington, Kentucky. American Family Physician. 2012;**86**(11):1027-1034

[2] Brundage SC, Fitzpatrick AN. Hepatitis A. American Family Physician. 2006;**73**(12):2162-2168

[3] Cristina J, Costa-Matiioli M. Genetic variability and molecular evolution of Hepatitis A virus. Virus Research. 2007;**127**(2):151-157. DOI: 10.1016/j. virusres.2007.01.005

[4] Acheson D, Fiore AE. Hepatitis A transmitted by food. Clinical Infectious Diseases. 2004;**38**(5):705-715. DOI: 10.1086/381671

[5] Kramer A, Schwebke I, Kampf G. How long do nosocomial pathogens persist on inanimate surfaces? A systematic review. BMC Infectious Diseases. 2006;**6**:130

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[7] Mutsch M et al. Hepatitis A virus infections in travelers, 1988- 2004. Clinical Infectious Diseases. 2006;**42**(4):490-497

[8] WHO (World Health Organization). Hepatitis A. 2018. Available from: https://www.who.int/news-room/factsheets/detail/hepatitis-a [Accessed: 20 June 2019]

[9] Lemon SM et al. Type A viral hepatitis: A summary and update on the molecular virology, epidemiology, pathogenesis and prevention. Journal of Hepatology. 2018;**68**:167-184

[10] Efrain E et al. Cellular entry and uncoating of naked and

quasi-enveloped human hepatoviruses. eLife. 2019;**8**:e43983. DOI: 10.7554/ eLife.43983

[11] Tanton BN, editor. Hepatitis viruses: Virology and epidemiology. In: Tropical Gastroenterology. New Delhi: New Elsevier; 2008. 261 p

[12] Cuthbert JA. Hepatitis A: Old and new. Clinical Microbiology Reviews. 2001;**14**(1):38-58

[13] Murray PR, Rosenthal KS, Pfaller MA, editors. Virus da hepatite. In: Microbiologia Médica. 7th ed. Vol. 2014. Rio de Janeiro: Elsevier. 1115-1121 p

[14] Maier K et al. Human gamma interferon production by cytotoxic T lymphocytes sensitized during hepatitis A virus infection. Journal of Virology. 1988;**62**:3756-3763

[15] Zhou Y et al. Dominance of the CD4<sup>+</sup> T helper cell response during acute resolving hepatitis A virus infection. Journal of Experimental Medicine. 2012;**209**(8):1481-1492

[16] Baba M et al. Cytolytic activity of natural killer cells and lymphokine activated killer cells against hepatitis A virus infected fibroblasts. Journal of Clinical & Laboratory Immunology. 1993;**40**(2):47-60

[17] Pereira FEL, Gonçalves CS. Hepatite A. Revista da Sociedade Brasileira de Medicina Tropical. 2003;**36**(3):387-400

[18] Dagan R et al. Incidence of hepatitis A in Israel following universal immunization of toddlers. Journal of the American Medical Association. 2005;**294**(2):202-210. DOI: 10.1001/ jama.294.2.202

[19] Luxemburger C, Dutta AK. Overlapping epidemiologies of hepatitis

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vaccination. Journal of Viral Hepatitis.

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Ministério da Saúde do Brasil. Available from: http://www.aids.gov.br/pt-br/ publico-geral/o-que-sao-hepatites/ hepatite [Accessed: 24 July 2019]

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A and typhoid fever: The needs of the traveler. Journal of Travel Medicine.

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of the advisory committee on immunization practices for use of hepatitis A vaccine for persons experiencing homelessness. MMWR. Morbidity and Mortality Weekly Report. 2019;**68**:153-156

[22] Pereira FEL, Gonçalves CS. Hepatitis A. Revistada Sociedade Brasileirade Medicina Tropical.

[23] CDC (Center of Disease Control). Hepatitis A, Acute. Case Definition. 2019. Available from https://wwwn.cdc. gov/nndss/conditions/hepatitis-a-acute/ case-definition/2019/ [Accessed: 30 May

[24] Jayakumar S et al. Fulminant viral hepatitis. Critical Care Clinics. 2013;**29**:677-697. DOI: 10.1016/j.

[25] DynaMed [Internet]. Ipswich (MA): EBSCO Information Services. 1995. Record No. 900055, Acute liver failure; [updated 2018 Nov 30]. Available from: http://www.dynamed.com/login.aspx ?direct=true&site=DynaMed&id=90 0055 Registration and login required

[26] Leikin JB. Current and prospective therapies for acute liver failure. Diseasea-Month. 2018;**64**(12):492. DOI: 10.1016/j.disamonth.2018.10.001

assessment and support of children with

[27] Barbosa PSH et al. Nutrition

2003;**36**(3):387-400

[20] Secretaria de Vigilância em Saúde e Ministério da Saúde. Boletim Epidemiológico. 2018;**49**(31). Available from: http://portalarquivos2.saude.gov. br/images/pdf/2018/julho/05/Boletim-Hepatites-2018.pdf [Accessed: 23 June

2005;**12**(1):S12

2019]

2019]

ccc.2013.03.013

[Accessed: 20 June 2019]

*Hepatitis A: How We Are after the Introduction of Vaccines DOI: http://dx.doi.org/10.5772/intechopen.88851*

A and typhoid fever: The needs of the traveler. Journal of Travel Medicine. 2005;**12**(1):S12

[20] Secretaria de Vigilância em Saúde e Ministério da Saúde. Boletim Epidemiológico. 2018;**49**(31). Available from: http://portalarquivos2.saude.gov. br/images/pdf/2018/julho/05/Boletim-Hepatites-2018.pdf [Accessed: 23 June 2019]

[21] Doshani M et al. Recommendations of the advisory committee on immunization practices for use of hepatitis A vaccine for persons experiencing homelessness. MMWR. Morbidity and Mortality Weekly Report. 2019;**68**:153-156

[22] Pereira FEL, Gonçalves CS. Hepatitis A. Revistada Sociedade Brasileirade Medicina Tropical. 2003;**36**(3):387-400

[23] CDC (Center of Disease Control). Hepatitis A, Acute. Case Definition. 2019. Available from https://wwwn.cdc. gov/nndss/conditions/hepatitis-a-acute/ case-definition/2019/ [Accessed: 30 May 2019]

[24] Jayakumar S et al. Fulminant viral hepatitis. Critical Care Clinics. 2013;**29**:677-697. DOI: 10.1016/j. ccc.2013.03.013

[25] DynaMed [Internet]. Ipswich (MA): EBSCO Information Services. 1995. Record No. 900055, Acute liver failure; [updated 2018 Nov 30]. Available from: http://www.dynamed.com/login.aspx ?direct=true&site=DynaMed&id=90 0055 Registration and login required [Accessed: 20 June 2019]

[26] Leikin JB. Current and prospective therapies for acute liver failure. Diseasea-Month. 2018;**64**(12):492. DOI: 10.1016/j.disamonth.2018.10.001

[27] Barbosa PSH et al. Nutrition assessment and support of children with cholestasis. Revista Médica de Minas Gerais. 2013;**23**(Supl 2):S34-S40. DOI: 10.5935/2238-3182.2013S006

[28] Departamento de Doenças de Condições Crônicas e Infecções Sexualmente Transmissíveis do Ministério da Saúde do Brasil. Available from: http://www.aids.gov.br/pt-br/ publico-geral/o-que-sao-hepatites/ hepatite [Accessed: 24 July 2019]

[29] Mayorga PO et al. Efficacy of virosome hepatitis A vaccine in young children in Nicaragua: Randomized placebo-controlled trial. The Journal of Infectious Diseases. 2003;**188**:671-677

[30] Nelson NP, Link-Gelles R, Hofmeister MG, et al. Update: Recommendations of the advisory committee on immunization practices for use of hepatitis A vaccine for postexposure prophylaxis and for preexposure prophylaxis for international travel. MMWR. Morbidity and Mortality Weekly Report. 2018;**67**:1216-1220

[31] Iwarson S et al. Excellent booster response 4 to 8 years after a single primary dose of an inactivated hepatitis A vaccine. Journal of Travel Medicine. 2004;**11**:120-121

[32] Hatz C et al. Successful memory response following a booster dose with a virosome-formulated hepatitis a vaccine delayed up to 11 years. Clinical and Vaccine Immunology. 2011;**18**:885-887

[33] Vacchino MN. Incidence of hepatitis A in Argentina after vaccination. Journal of Viral Hepatitis. 2008;**15**(2):47-50

[34] Contents 261 WHO position paper on hepatitis A vaccines—June 2012. Weekly Epidemiological Record. 2012;**87**:261-276. Available from: http:// www.who.int/wer [Accessed: 13 June 2019]

**74**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

quasi-enveloped human hepatoviruses. eLife. 2019;**8**:e43983. DOI: 10.7554/

[11] Tanton BN, editor. Hepatitis viruses: Virology and epidemiology. In: Tropical Gastroenterology. New Delhi: New

[12] Cuthbert JA. Hepatitis A: Old and new. Clinical Microbiology Reviews.

Pfaller MA, editors. Virus da hepatite. In: Microbiologia Médica. 7th ed. Vol. 2014. Rio de Janeiro: Elsevier. 1115-1121 p

[13] Murray PR, Rosenthal KS,

[14] Maier K et al. Human gamma interferon production by cytotoxic T lymphocytes sensitized during hepatitis A virus infection. Journal of Virology.

[15] Zhou Y et al. Dominance of the

acute resolving hepatitis A virus infection. Journal of Experimental Medicine. 2012;**209**(8):1481-1492

[17] Pereira FEL, Gonçalves CS. Hepatite A. Revista da Sociedade Brasileira de Medicina Tropical.

[18] Dagan R et al. Incidence of

[19] Luxemburger C, Dutta AK.

hepatitis A in Israel following universal immunization of toddlers. Journal of the American Medical Association. 2005;**294**(2):202-210. DOI: 10.1001/

Overlapping epidemiologies of hepatitis

T helper cell response during

[16] Baba M et al. Cytolytic activity of natural killer cells and lymphokine activated killer cells against hepatitis A virus infected fibroblasts. Journal of Clinical & Laboratory Immunology.

eLife.43983

Elsevier; 2008. 261 p

2001;**14**(1):38-58

1988;**62**:3756-3763

1993;**40**(2):47-60

2003;**36**(3):387-400

jama.294.2.202

CD4<sup>+</sup>

[1] Matheny SC, Kingery JE. University of Kentucky College of Medicine, Lexington, Kentucky. American Family Physician. 2012;**86**(11):1027-1034

Hepatitis A. American Family Physician.

[3] Cristina J, Costa-Matiioli M. Genetic variability and molecular evolution of Hepatitis A virus. Virus Research. 2007;**127**(2):151-157. DOI: 10.1016/j.

[4] Acheson D, Fiore AE. Hepatitis A transmitted by food. Clinical Infectious Diseases. 2004;**38**(5):705-715. DOI:

[5] Kramer A, Schwebke I, Kampf G. How long do nosocomial pathogens persist on inanimate surfaces? A systematic review. BMC Infectious

[6] Ferreira CT, Silveira TR. Hepatites virais: Aspectos da epidemiologia e da prevenção. Revista Brasileira de Epidemiologia. 2004;*7*(4):473-487. DOI: 10.1590/S1415-790X2004000400010

[8] WHO (World Health Organization). Hepatitis A. 2018. Available from: https://www.who.int/news-room/factsheets/detail/hepatitis-a [Accessed: 20

[7] Mutsch M et al. Hepatitis A virus infections in travelers, 1988- 2004. Clinical Infectious Diseases.

[9] Lemon SM et al. Type A viral hepatitis: A summary and update on the molecular virology, epidemiology, pathogenesis and prevention. Journal of

Hepatology. 2018;**68**:167-184

[10] Efrain E et al. Cellular entry and uncoating of naked and

[2] Brundage SC, Fitzpatrick AN.

2006;**73**(12):2162-2168

**References**

virusres.2007.01.005

10.1086/381671

Diseases. 2006;**6**:130

2006;**42**(4):490-497

June 2019]

[35] Ministério da Saúde e Secretaria de Vigilância em Saúde Departamento de Vigilância Epidemiológica Coordenação Geral do Programa Nacional de Imunizações. Informe Técnico da Introdução da Vacina Adsorvida Hepatite A (inativada). Brasilia. 2014. Available from: http://portalarquivos2.saude.gov. br/images/pdf/2015/junho/26/Informet--cnico-vacina-hepatite-A-junho-2014. pdf [Accessed: 18 June 2019]

[36] ONU News. Meta da África é eliminar a hepatite viral até 2030. Available from: https://news.un.org/pt/ story/2016/08/1560571-meta-da-africae-eliminar-hepatite-viral-ate-2030 [Accessed: 30 June 2019]

[37] WHO. Mensagem da Directora Regional da OMS para a África, Dr.ª Matshidiso Moeti, por ocasião do Dia Mundial contra a Hepatite. 2017. Available from: https://afro.who.int/pt/ regional-director/speeches-messages/ mensagem-da-directora-regional-daoms-para-africa-dra-0 [Accessed: 30 June 2019]

[38] Vizzotti C et al. Economic analysis of the single-dose immunization strategy against hepatitis A in Argentina. Vaccine. 2015;**33S**:A227-A232. DOI: 10.1016/j. vaccine.2014.12.077

[39] Wang L et al. An ultrasensitive capacitive microwire sensor for pathogen-specific serum antibody responses. Biosensors and Bioelectronics. 2019;**131**:46-52

[40] Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;**346**(6213): 12580961-12580969. DOI: 10.1126/ science.1258096. Available from https:// innovativegenomics.org/wp-content/ uploads/2016/06/Doudna-Charpentier-Science-2014.pdf [Accessed: 23 July 2019]

[41] Barrangou R, Doudna JA. Applications of CRISPR technologies in research and beyond. Nature Biotechnology. 2016;**34**:933-941

[42] O'Connell MR et al. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature. 2014;**516**(7530):263-266

[43] Price AA et al. Cas9-mediated targeting of viral RNA in eukaryotic cells. Proceedings of the National Academy of Sciences of the United States of America. 2015;**112**(19):6164-6169

[44] Gootenberg JS et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;**356**(6336):438-442

[45] van Diemen FR, Kruse EM, Hooykaas MJG, Bruggeling CE, Schürch AC, van Ham PM, et al. CRISPR/Cas9-mediated genome editing of herpesviruses limits productive and latent infections. PLoS Pathogens. 2016;**12**(6):e1005701. DOI: 10.1371/ journal.ppat.1005701

[46] Kennedy EM et al. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology. 2015;**476**:196-205

[47] Bartram J et al. Water Safety Plan Manual: Step by Step Risk Management for Drinking-water Supplies. Geneva: World Health Organization; 2009. Available from: https://apps.who.int/iris/ handle/10665/75141 [Accessed: 23 July 2019]

[48] United Nations. Economic and Social Council. Agenda items 5(a) and 6, May 2019. Available from: https:// sustainabledevelopment.un.org/ hlpf/2019/ [Accessed: 23 July 2019]

**77**

**Chapter 6**

**Abstract**

interactions.

**1. Introduction**

Hepatitis-A Virus

*Damian Chukwu Odimegwu* 

*and Uzochukwu Gospel Ukachukwu*

**Keywords:** hepatitis A, antiviral, natural products, infections

and their application were adequately discussed.

**2. Therapeutic anti-HAV natural products**

Antiviral Natural Products against

The review on antiviral anti-hepatitis A virus agents is warranted given the importance of hepatitis A virus (HAV) as a human pathogen. Novel antiviral drugs have been sourced from natural agents and developed into products for management of viral infections. The role of purified natural products in treatment and as adjunctives in the management of HAV infections is clearly plausible. Treatments against Hepatitis A virus infection is currently limited. In this chapter, the antiviral natural products against hepatitis-A virus (HAV), their sources as well as their treatment approach and their application have been discussed. The antiviral natural products could be sourced generally from plants, herbs and animals. These natural agents have been shown to demonstrate substantial antiviral activity against HAV and could target various stages of the viral life cycle, replication, assemblage, release, as well as targeting virus-host specific

The role of purified natural products in prophylaxis, palliative and curative treatment of myriad diseases of bacterial, fungal and viral origin cannot be overemphasized. Novel antiviral drugs have been sourced from natural agents and developed into products for prophylactic and therapeutic purposes [1]. These natural agents have been shown to demonstrate antiviral activity by interfering with viral life cycle, replication, assemblage, release, as well as targeting virus-host specific interactions [1]. Antiviral natural products can be sourced generally from plants or herbs, microbes, animals and humans. In this chapter, the antiviral natural products against hepatitis-A virus (HAV), their sources as well as their treatment approach

Hepatitis A virus is among the pathogens that find their way into the human system through ingestion of food contaminated with them, and most of these food-borne viruses lack licensed antivirals. Vaccine development and immunization

#### **Chapter 6**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

in research and beyond. Nature Biotechnology. 2016;**34**:933-941

Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature.

[43] Price AA et al. Cas9-mediated

[44] Gootenberg JS et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;**356**(6336):438-442

CRISPR/Cas9-mediated genome editing of herpesviruses limits productive and latent infections. PLoS Pathogens. 2016;**12**(6):e1005701. DOI: 10.1371/

[46] Kennedy EM et al. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology.

[47] Bartram J et al. Water Safety Plan Manual: Step by Step Risk Management for Drinking-water Supplies. Geneva: World Health Organization; 2009. Available from: https://apps.who.int/iris/ handle/10665/75141 [Accessed: 23 July

[48] United Nations. Economic and Social Council. Agenda items 5(a) and 6, May 2019. Available from: https:// sustainabledevelopment.un.org/ hlpf/2019/ [Accessed: 23 July 2019]

[45] van Diemen FR, Kruse EM, Hooykaas MJG, Bruggeling CE, Schürch AC, van Ham PM, et al.

[42] O'Connell MR et al.

2014;**516**(7530):263-266

targeting of viral RNA in eukaryotic cells. Proceedings of the National Academy of Sciences of the United States of America.

2015;**112**(19):6164-6169

journal.ppat.1005701

2015;**476**:196-205

2019]

[35] Ministério da Saúde e Secretaria de Vigilância em Saúde Departamento de Vigilância Epidemiológica Coordenação

Introdução da Vacina Adsorvida Hepatite A (inativada). Brasilia. 2014. Available from: http://portalarquivos2.saude.gov. br/images/pdf/2015/junho/26/Informet--cnico-vacina-hepatite-A-junho-2014.

Geral do Programa Nacional de Imunizações. Informe Técnico da

pdf [Accessed: 18 June 2019]

[Accessed: 30 June 2019]

2019]

[36] ONU News. Meta da África é eliminar a hepatite viral até 2030. Available from: https://news.un.org/pt/ story/2016/08/1560571-meta-da-africae-eliminar-hepatite-viral-ate-2030

[37] WHO. Mensagem da Directora Regional da OMS para a África, Dr.ª Matshidiso Moeti, por ocasião do Dia Mundial contra a Hepatite. 2017. Available from: https://afro.who.int/pt/ regional-director/speeches-messages/ mensagem-da-directora-regional-daoms-para-africa-dra-0 [Accessed: 30 June

[38] Vizzotti C et al. Economic analysis of the single-dose immunization strategy against hepatitis A in Argentina. Vaccine. 2015;**33S**:A227-A232. DOI: 10.1016/j.

vaccine.2014.12.077

[39] Wang L et al. An ultrasensitive capacitive microwire sensor for pathogen-specific serum antibody responses. Biosensors and Bioelectronics. 2019;**131**:46-52

[41] Barrangou R, Doudna JA.

Applications of CRISPR technologies

[40] Doudna JA, Charpentier E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;**346**(6213): 12580961-12580969. DOI: 10.1126/ science.1258096. Available from https:// innovativegenomics.org/wp-content/ uploads/2016/06/Doudna-Charpentier-Science-2014.pdf [Accessed: 23 July 2019]

**76**

## Antiviral Natural Products against Hepatitis-A Virus

*Damian Chukwu Odimegwu and Uzochukwu Gospel Ukachukwu*

#### **Abstract**

The review on antiviral anti-hepatitis A virus agents is warranted given the importance of hepatitis A virus (HAV) as a human pathogen. Novel antiviral drugs have been sourced from natural agents and developed into products for management of viral infections. The role of purified natural products in treatment and as adjunctives in the management of HAV infections is clearly plausible. Treatments against Hepatitis A virus infection is currently limited. In this chapter, the antiviral natural products against hepatitis-A virus (HAV), their sources as well as their treatment approach and their application have been discussed. The antiviral natural products could be sourced generally from plants, herbs and animals. These natural agents have been shown to demonstrate substantial antiviral activity against HAV and could target various stages of the viral life cycle, replication, assemblage, release, as well as targeting virus-host specific interactions.

**Keywords:** hepatitis A, antiviral, natural products, infections

#### **1. Introduction**

The role of purified natural products in prophylaxis, palliative and curative treatment of myriad diseases of bacterial, fungal and viral origin cannot be overemphasized. Novel antiviral drugs have been sourced from natural agents and developed into products for prophylactic and therapeutic purposes [1]. These natural agents have been shown to demonstrate antiviral activity by interfering with viral life cycle, replication, assemblage, release, as well as targeting virus-host specific interactions [1]. Antiviral natural products can be sourced generally from plants or herbs, microbes, animals and humans. In this chapter, the antiviral natural products against hepatitis-A virus (HAV), their sources as well as their treatment approach and their application were adequately discussed.

#### **2. Therapeutic anti-HAV natural products**

Hepatitis A virus is among the pathogens that find their way into the human system through ingestion of food contaminated with them, and most of these food-borne viruses lack licensed antivirals. Vaccine development and immunization against several viruses including hepatitis A virus lack preventive and efficient antiviral therapies, as they are often challenged by counter-production of viral escape mutants that evade the immune system [1]. Also, the development of efficient and low-cost vaccines for economically unprivileged countries will be difficult, including countries with low prevalence where vaccine is recommended only for high-risk individuals [2]. Post-exposure of the human system to viral infections requires an efficient therapeutic approach to clear infections off the human system. It is imperative to develop effective antiviral therapeutic agents against these viruses, and interest in the employment of natural products as effective antiviral therapeutic agents has widely increased.

Flavonoids, polyphenols, saponin, proanthocyanins, polysaccharides, organic acids, proteins, polypeptides, and essential oils obtained from plant, animals or microorganisms can control and eradicate food-borne viral infections including hepatitis A [3, 4]. Over the past two decades, much effort has been aimed at identifying natural products, mostly of plant origin, to control food-borne viruses. Extracts from natural plants potentially have several applications, not limited to increasing the safety of food products and enhancing their quality, but also to serve as natural antiviral agents. For instance, these extracts possess several natural compounds that have been reported to demonstrate virucidal activity against surrogates of the human novovirus, a known food-borne virus [5]. In this section, we will discuss the antiviral therapeutic activities of several natural products and herbal medicines against hepatitis A viral infection.

#### **2.1 Plant-based**

#### *2.1.1 Green tea extract*

Green tea extract (GTE) is produced from the leaves of cultivated evergreen tea plant, *Camellia sinensis* L., of the family Theaceae [6]. It is rich in polyphenols and proanthocyanidins, and has been widely used to nutritionally enrich various food and beverages due to reports about its diverse health benefits such as possessing antioxidant, anti-inflammatory, and anti-carcinogenic properties [7–9]. Studies have revealed that GTE exhibits inhibitory properties against a wide variety of food-borne pathogens [10, 11]. Chemical composition of GTE includes mainly catechins, a group of flavonoids [12] that possess antimicrobial properties on a wide spectrum of Gram-positive and Gram-negative bacteria [11]. In a study, catechins such as epigallocatechin-3-gallate (EGCG) and epicatechin gallate (ECG), contained in GTE demonstrated the strongest antiviral properties [13], and also exhibited significant antiviral properties when encapsulated within chitosan electrosprayed microcapsules [14].

Recent *in vitro* study revealed that GTE demonstrated excellent antiviral activity against hepatitis A virus under controlled conditions of concentration, pH, temperature and also time exposure. It was shown that 5 mg/ml GTE incubated with the viral suspension for 2 h at 37°C and pH of 7.2 observed that there was complete inactivation of the virus in the suspension [6]. Findings suggested that GTE antiviral activity thrived better under increasing alkaline conditions. GTE has also been evaluated as a natural sanitizer of farm produce, demonstrating that HAV titers in lettuce and spinach were drastically reduced after 30 min treatment with 10 mg/ ml GTE. Hence GTE holds promise for food-borne viral infection control through disinfection of food produce before consumption. Although the antiviral mechanisms of GTE have not yet been elucidated, some extrapolations could be drawn from the action of EGCG on viruses as it is the chief constituent compound in GTE [14, 15]. EGCG has high affinity for viral surface proteins but binds nonspecifically to them.

**79**

infection.

food produce.

*Antiviral Natural Products against Hepatitis-A Virus DOI: http://dx.doi.org/10.5772/intechopen.91869*

undetectable levels in intestinal fluid after 6 h.

similar mechanism.

*2.1.2 Grape seed extract*

Therefore it exhibits its antiviral activity against a wide variety of enveloped and non-enveloped viruses by interfering with viral attachment to cell membrane receptors upon binding to them; thus, HAV infection could be curbed by GTE via

Grape seed extract (GSE), *Vitis vinifera*, is generally obtained as a by-product of the grape juice and wine industry during processing of grapes [16]. It is reported to possess diverse bioactive principles including anthocyanins, flavonoids, proanthocyanidins, polyphenols, procyanidins and resveratrol, a derivative of stilbene [17]. The antioxidative, anti-inflammatory, cardioprotective, hepatoprotective, neuroprotective, and antimicrobial properties of these compounds make the extract

GSE demonstrates antimicrobial activity against many food-borne bacterial pathogens including *Listeria monocytogenes*, *Staphylococcus aureus*, methicillinresistant *S. aureus* (MRSA), *Escherichia coli* O157:H7, *Salmonella enterica* serovar Enteritidis, and *S. typhimurium* [19–21]. Moreover, studies have reported the antiviral activities of GSE against some food-borne viruses including hepatitis A virus (HAV), human norovirus surrogates (feline calicivirus (FCV-F9)) and murine norovirus (MNV-1) [22, 23]. Under simulated gastrointestinal conditions, GSE reduced the HAV titer to undetectable levels in a dose-dependent fashion at varied temperatures (room temperature, 37°C) and time not exceeding 24 h. Emphatically, 2 mg/ml GSE drastically reduced HAV titer among other food-borne viruses to

However, this success may not be reproducible in the human system as the HAV strain, HM175, used during the study was a lab-adapted strain that was not sensitive to low pH as observed in the wild type strain. Again, some studies showed that GSE anti-HAV activity decreased in the presence of increasing concentrations of 0.02 and 0.2% dried milk or lettuce extract, where a higher dose is required to inactivate viral replication [24]. This implies that proteins could interfere with GSE antiviral activity and consequently decreases its effectiveness for treatments. Also, at concentrations ranging from 0.25 to 1 mg/ml GSE was said to diminish food-borne viral contamination levels on food produce (lettuce and peppers) without causing notable color changes on them. Therefore, GSE could be considered as a control measure for hepatitis A virus contamination on food produce before consumption, though may require a synergistic approach to combat persistent contamination of

The antiviral mechanisms of GSE are not yet well expounded. However, some studies suggest that resveratrol (RV), a nonflavonoid polyphenol found in grapes modulate some intracellular signaling pathways of the influenza virus [25]. In a study evaluating the effect of GSE on the adsorption and replicativity of HAV, it was revealed that treatment of the host cells with GSE prior to viral infection caused significant decline in HAV titer [26]. Post-viral infection of the host cells showed that HAV titers decreased insignificantly. This implies that GSE may have a moderate antiviral effect on adsorption of HAV on the host cells but with less effect on its replication [26]. Likewise, GSE was reported to down-regulate the expression of HIV entry coreceptors, implying that GSE may interrupt the binding of the virus to the cell receptors and in turn prevent HIV entry into normal lymphocytes [27]. Presently, GSE appears not to cause any structural damage to the viral capsid of HAV, rather it is more likely to exert greater antiviral activity by potentially blocking the host cell receptors and consequently prevents viral entry, replication, and

to exhibit impressive pharmacological and therapeutic benefits [18].

Therefore it exhibits its antiviral activity against a wide variety of enveloped and non-enveloped viruses by interfering with viral attachment to cell membrane receptors upon binding to them; thus, HAV infection could be curbed by GTE via similar mechanism.

#### *2.1.2 Grape seed extract*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

medicines against hepatitis A viral infection.

agents has widely increased.

**2.1 Plant-based**

*2.1.1 Green tea extract*

electrosprayed microcapsules [14].

against several viruses including hepatitis A virus lack preventive and efficient antiviral therapies, as they are often challenged by counter-production of viral escape mutants that evade the immune system [1]. Also, the development of efficient and low-cost vaccines for economically unprivileged countries will be difficult, including countries with low prevalence where vaccine is recommended only for high-risk individuals [2]. Post-exposure of the human system to viral infections requires an efficient therapeutic approach to clear infections off the human system. It is imperative to develop effective antiviral therapeutic agents against these viruses, and interest in the employment of natural products as effective antiviral therapeutic

Flavonoids, polyphenols, saponin, proanthocyanins, polysaccharides, organic acids, proteins, polypeptides, and essential oils obtained from plant, animals or microorganisms can control and eradicate food-borne viral infections including hepatitis A [3, 4]. Over the past two decades, much effort has been aimed at identifying natural products, mostly of plant origin, to control food-borne viruses. Extracts from natural plants potentially have several applications, not limited to increasing the safety of food products and enhancing their quality, but also to serve as natural antiviral agents. For instance, these extracts possess several natural compounds that have been reported to demonstrate virucidal activity against surrogates of the human novovirus, a known food-borne virus [5]. In this section, we will discuss the antiviral therapeutic activities of several natural products and herbal

Green tea extract (GTE) is produced from the leaves of cultivated evergreen tea plant, *Camellia sinensis* L., of the family Theaceae [6]. It is rich in polyphenols and proanthocyanidins, and has been widely used to nutritionally enrich various food and beverages due to reports about its diverse health benefits such as possessing antioxidant, anti-inflammatory, and anti-carcinogenic properties [7–9]. Studies have revealed that GTE exhibits inhibitory properties against a wide variety of food-borne pathogens [10, 11]. Chemical composition of GTE includes mainly catechins, a group of flavonoids [12] that possess antimicrobial properties on a wide spectrum of Gram-positive and Gram-negative bacteria [11]. In a study, catechins such as epigallocatechin-3-gallate (EGCG) and epicatechin gallate (ECG), contained in GTE demonstrated the strongest antiviral properties [13], and also exhibited significant antiviral properties when encapsulated within chitosan

Recent *in vitro* study revealed that GTE demonstrated excellent antiviral activity against hepatitis A virus under controlled conditions of concentration, pH, temperature and also time exposure. It was shown that 5 mg/ml GTE incubated with the viral suspension for 2 h at 37°C and pH of 7.2 observed that there was complete inactivation of the virus in the suspension [6]. Findings suggested that GTE antiviral activity thrived better under increasing alkaline conditions. GTE has also been evaluated as a natural sanitizer of farm produce, demonstrating that HAV titers in lettuce and spinach were drastically reduced after 30 min treatment with 10 mg/ ml GTE. Hence GTE holds promise for food-borne viral infection control through disinfection of food produce before consumption. Although the antiviral mechanisms of GTE have not yet been elucidated, some extrapolations could be drawn from the action of EGCG on viruses as it is the chief constituent compound in GTE [14, 15]. EGCG has high affinity for viral surface proteins but binds nonspecifically to them.

**78**

Grape seed extract (GSE), *Vitis vinifera*, is generally obtained as a by-product of the grape juice and wine industry during processing of grapes [16]. It is reported to possess diverse bioactive principles including anthocyanins, flavonoids, proanthocyanidins, polyphenols, procyanidins and resveratrol, a derivative of stilbene [17]. The antioxidative, anti-inflammatory, cardioprotective, hepatoprotective, neuroprotective, and antimicrobial properties of these compounds make the extract to exhibit impressive pharmacological and therapeutic benefits [18].

GSE demonstrates antimicrobial activity against many food-borne bacterial pathogens including *Listeria monocytogenes*, *Staphylococcus aureus*, methicillinresistant *S. aureus* (MRSA), *Escherichia coli* O157:H7, *Salmonella enterica* serovar Enteritidis, and *S. typhimurium* [19–21]. Moreover, studies have reported the antiviral activities of GSE against some food-borne viruses including hepatitis A virus (HAV), human norovirus surrogates (feline calicivirus (FCV-F9)) and murine norovirus (MNV-1) [22, 23]. Under simulated gastrointestinal conditions, GSE reduced the HAV titer to undetectable levels in a dose-dependent fashion at varied temperatures (room temperature, 37°C) and time not exceeding 24 h. Emphatically, 2 mg/ml GSE drastically reduced HAV titer among other food-borne viruses to undetectable levels in intestinal fluid after 6 h.

However, this success may not be reproducible in the human system as the HAV strain, HM175, used during the study was a lab-adapted strain that was not sensitive to low pH as observed in the wild type strain. Again, some studies showed that GSE anti-HAV activity decreased in the presence of increasing concentrations of 0.02 and 0.2% dried milk or lettuce extract, where a higher dose is required to inactivate viral replication [24]. This implies that proteins could interfere with GSE antiviral activity and consequently decreases its effectiveness for treatments. Also, at concentrations ranging from 0.25 to 1 mg/ml GSE was said to diminish food-borne viral contamination levels on food produce (lettuce and peppers) without causing notable color changes on them. Therefore, GSE could be considered as a control measure for hepatitis A virus contamination on food produce before consumption, though may require a synergistic approach to combat persistent contamination of food produce.

The antiviral mechanisms of GSE are not yet well expounded. However, some studies suggest that resveratrol (RV), a nonflavonoid polyphenol found in grapes modulate some intracellular signaling pathways of the influenza virus [25]. In a study evaluating the effect of GSE on the adsorption and replicativity of HAV, it was revealed that treatment of the host cells with GSE prior to viral infection caused significant decline in HAV titer [26]. Post-viral infection of the host cells showed that HAV titers decreased insignificantly. This implies that GSE may have a moderate antiviral effect on adsorption of HAV on the host cells but with less effect on its replication [26]. Likewise, GSE was reported to down-regulate the expression of HIV entry coreceptors, implying that GSE may interrupt the binding of the virus to the cell receptors and in turn prevent HIV entry into normal lymphocytes [27]. Presently, GSE appears not to cause any structural damage to the viral capsid of HAV, rather it is more likely to exert greater antiviral activity by potentially blocking the host cell receptors and consequently prevents viral entry, replication, and infection.

#### *2.1.3 Egyptian red sea seagrass extract*

Seagrass is a critical part of the marine ecosystem and is generally distributed along the tropical and temperate coastal zones of the world [28]. It was said to be the only marine flowering plant that completes its lifecycle in sea water and often lives entirely submerged [29]. It is of ecological importance and is employed in folklore medicine for therapeutic purposes [30, 31]. The Egyptian Red Sea seagrass, *Thalassodendron ciliatum*, is said to be one of the longest and most common sea grasses along the Egyptian Red Sea. Its leaves are characterized by many 'tannin cells' more than in any other sea grass [32], which infers that it possesses a high phenolic content.

Compounds isolated from the sea grass crude extract have been shown to exhibit antioxidant and cytotoxic activities [28]. The crude extract demonstrated 100% inhibition of hepatitis A (HAV) and Herpes Simplex (HSV-1) viruses at 20 μg/ mL. The antiviral activity of the crude extract against HAV was lost by fractionation, which could be explained by the synergistic action of several compounds in the crude extract [28]. Moreover, knowledge about the mechanism of anti-HAV activity of *T. ciliatum* has not yet been elucidated. Further studies are required to evaluate the toxicity of *T. ciliatum* on humans after consumption as food supplement or on formulation as a therapeutic drug against HAV.

#### *2.1.4 Essential oils*

Essential oils (EOs) are aromatic oily liquids derived from plant materials such as flowers, buds, seeds, leaves, branches, bark, grass, wood, fruit, and roots. Production of essential oils is majorly by steam distillation or by other methods such as solvent-heat extraction, pressing, fermentation or enfleurage [33]. Chemical components contained in these essential oils have been shown to be effective in combating pathogens [34, 35]. Few essential oils have been tested for their antiviral activities against food-borne viruses, particularly for HAV [36].

The anti-HAV activity of essential oils obtained from lemon (*Citrus limon*), sweet orange (*Citrus sinensis*), grapefruit (*Citrus paradisi*), and rosemary cineole (*Rosmarinus officinalis*) have been reported [33]. Essential oils belonging to the genus *Citrus* contain 85–99% of volatile compounds such as sesquiterpenes, monoterpene (limonene), and hydrocarbons, with their oxygenated products including aldehydes (citral), acids, ketones, alcohols (linalool), and esters [37]. *Rosmarinus officinalis* of the family, *Lamiaceae*, is generally applied during the preparation of some European cuisine and is also used as a medicinal plant, because of the strong antiseptic properties, antibacterial and antioxidant activities of it's essential oil [38]; rosemary oil is also used as a natural food preservative [39, 40].

Essential oil treatment of ATCC/HM-175 strain of HAV propagated in Frp3 cells revealed that after an hour incubation at room temperature, the greatest reduction in cell infectivity was observed for rosemary cineole EO, followed by grapefruit and lemon EOs, while orange EO, although reducing HAV infectivity was not statistically significant [33]. Orange and grapefruit EOs were found to be cytotoxic for Frp3 cells at concentrations that exceeded 0.1%, while lemon and rosemary cineole EOs were cytotoxic at concentrations exceeding 0.5% and 0.05%, respectively. Studies have also revealed that treatment of contaminated berries with all four EOs from lemon, orange, grapefruit and rosemary cineole reduced the viral titer of HAV at room temperature. Essential oil from rosemary cineole was shown to be the most effective, as it significantly reduced the HAV titer on the berries followed by essential oils from grapefruit and lemon respectively [33]. Anti-HAV activity of essential oil from orange was not significant though there was a reduction in the

**81**

*Antiviral Natural Products against Hepatitis-A Virus DOI: http://dx.doi.org/10.5772/intechopen.91869*

activity of EOs have not yet been elucidated.

*2.1.5 Korean red ginseng extract and ginsenosides*

be evaluated in further study using *in vivo* models.

*2.1.6 Blueberry juice and blueberry proanthocyanidins*

induced by this pathway also contribute to the antiviral response.

HAV titer on the berries. However, application of these essential oils alone may not be sufficient to decontaminate soft fruits (berries) laden with higher viral (HAV) loads [33]. Therefore, it is imperative that the essential oils be considered for use in food sterilization in combination with other treatments. It is also necessary to evaluate the minimum time it takes for EOs to reduce the maximum HAV loads on food produce so that adequate awareness is made to individuals to achieve food product safety before consumption [33]. Moreover, the mechanisms of anti-HAV

Ginseng (*Panax ginseng* Meyer) is a famous medicinal herb that has been used for over 5000 years in Korea and China [41]. Ginseng contains myriad bioactive components including, ginsenosides, phytosterols, polysaccharides, polyacetylenes, polyacetylenic alcohols, fatty acids and peptides [42]. There exists already documentations on the anti-stress, anti-carcinogenic, anti-inflammatory, antioxidant, anti-bacterial, anti-viral and anti-fungal activities of ginseng [42–44]. Furthermore, ginseng demonstrates useful activity on endocrine diseases, cardiovascular diseases and the immune system [45]. During processing, Red ginseng is usually steamed and fermented with skinned ginseng and this alters the composition saponin contained in it when done repeatedly [46]. Red ginseng has been shown to possess anti-cancer, anti-diabetic, anti-obesity and immunomodulatory properties [3, 4]. Likewise zidovudine, red ginseng has also been applied as a therapeutic supplement

for the treatment of patients with human immunodeficiency virus [47].

Studies have shown that red ginseng extract and its ginsenosides inactivate food borne viruses such as the human norovirus (huNoV) surrogates (feline calicivirus and murine norovirus) [43]. A plaque assay performed on FRhK-4 cell lines pretreated and co-treated with varied concentrations of Korean red ginseng (KRG) extract and purified ginsenosides (Rg1 and Rb1) showed that after inoculation of HAV HM-175 strain on the cell lines, KRG and the ginsenosides reduced significantly the HAV concentration [3, 4]. Korean red ginseng's extract demonstrated cytotoxicity at concentration above 10 μg/mL, while the purified ginsenosides showed no cytotoxic activity even up to 40 μg/mL. Although co-treatment of cell lines with KRG and the ginsenosides exhibited significant reduction of HAV concentration in the study, anti-HAV activity of the pretreated cell lines was quite higher [3, 4]. Hence, pretreatment with ginseng may be effective in preventing HAV infection. Also co-treatment of cell lines with KRG and the ginsenosides may

The anti-HAV mechanisms of KRG extract and its ginsenosides are not clearly defined. However, reports from studies have shown that HAV-infected FRhK-4 cells activate the 2′-5′ oligoadenylate synthetase/RNaseL pathway [48]. Activation of RNase L degrades viral RNA and cellular single-stranded RNA; hence, KRG extract and its ginsenosides may tour a similar path. In addition, previous studies have reported that ginseng polysaccharides and ginsenosides have the capacity to boost the production of cytokines via stimulation of immune cells [3, 4]. Interferons

Blueberries are said to contain about 88–261 mg of proanthocyanidin/100 g of edible portion according to the USDA database for flavonoid content (USDA Database for the proanthocyanidin Content of Selected Foods, August 2004). Again, blue berries possess some other structurally related polyphenols such as

#### *Antiviral Natural Products against Hepatitis-A Virus DOI: http://dx.doi.org/10.5772/intechopen.91869*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

ment or on formulation as a therapeutic drug against HAV.

activities against food-borne viruses, particularly for HAV [36].

[38]; rosemary oil is also used as a natural food preservative [39, 40].

Seagrass is a critical part of the marine ecosystem and is generally distributed along the tropical and temperate coastal zones of the world [28]. It was said to be the only marine flowering plant that completes its lifecycle in sea water and often lives entirely submerged [29]. It is of ecological importance and is employed in folklore medicine for therapeutic purposes [30, 31]. The Egyptian Red Sea seagrass, *Thalassodendron ciliatum*, is said to be one of the longest and most common sea grasses along the Egyptian Red Sea. Its leaves are characterized by many 'tannin cells' more than in any other sea grass [32], which infers that it possesses a high

Compounds isolated from the sea grass crude extract have been shown to exhibit antioxidant and cytotoxic activities [28]. The crude extract demonstrated 100% inhibition of hepatitis A (HAV) and Herpes Simplex (HSV-1) viruses at 20 μg/ mL. The antiviral activity of the crude extract against HAV was lost by fractionation, which could be explained by the synergistic action of several compounds in the crude extract [28]. Moreover, knowledge about the mechanism of anti-HAV activity of *T. ciliatum* has not yet been elucidated. Further studies are required to evaluate the toxicity of *T. ciliatum* on humans after consumption as food supple-

Essential oils (EOs) are aromatic oily liquids derived from plant materials such as flowers, buds, seeds, leaves, branches, bark, grass, wood, fruit, and roots. Production of essential oils is majorly by steam distillation or by other methods such as solvent-heat extraction, pressing, fermentation or enfleurage [33]. Chemical components contained in these essential oils have been shown to be effective in combating pathogens [34, 35]. Few essential oils have been tested for their antiviral

The anti-HAV activity of essential oils obtained from lemon (*Citrus limon*), sweet orange (*Citrus sinensis*), grapefruit (*Citrus paradisi*), and rosemary cineole (*Rosmarinus officinalis*) have been reported [33]. Essential oils belonging to the genus *Citrus* contain 85–99% of volatile compounds such as sesquiterpenes, monoterpene (limonene), and hydrocarbons, with their oxygenated products including aldehydes (citral), acids, ketones, alcohols (linalool), and esters [37]. *Rosmarinus officinalis* of the family, *Lamiaceae*, is generally applied during the preparation of some European cuisine and is also used as a medicinal plant, because of the strong antiseptic properties, antibacterial and antioxidant activities of it's essential oil

Essential oil treatment of ATCC/HM-175 strain of HAV propagated in Frp3 cells revealed that after an hour incubation at room temperature, the greatest reduction in cell infectivity was observed for rosemary cineole EO, followed by grapefruit and lemon EOs, while orange EO, although reducing HAV infectivity was not statistically significant [33]. Orange and grapefruit EOs were found to be cytotoxic for Frp3 cells at concentrations that exceeded 0.1%, while lemon and rosemary cineole EOs were cytotoxic at concentrations exceeding 0.5% and 0.05%, respectively. Studies have also revealed that treatment of contaminated berries with all four EOs from lemon, orange, grapefruit and rosemary cineole reduced the viral titer of HAV at room temperature. Essential oil from rosemary cineole was shown to be the most effective, as it significantly reduced the HAV titer on the berries followed by essential oils from grapefruit and lemon respectively [33]. Anti-HAV activity of essential oil from orange was not significant though there was a reduction in the

*2.1.3 Egyptian red sea seagrass extract*

phenolic content.

*2.1.4 Essential oils*

**80**

HAV titer on the berries. However, application of these essential oils alone may not be sufficient to decontaminate soft fruits (berries) laden with higher viral (HAV) loads [33]. Therefore, it is imperative that the essential oils be considered for use in food sterilization in combination with other treatments. It is also necessary to evaluate the minimum time it takes for EOs to reduce the maximum HAV loads on food produce so that adequate awareness is made to individuals to achieve food product safety before consumption [33]. Moreover, the mechanisms of anti-HAV activity of EOs have not yet been elucidated.

#### *2.1.5 Korean red ginseng extract and ginsenosides*

Ginseng (*Panax ginseng* Meyer) is a famous medicinal herb that has been used for over 5000 years in Korea and China [41]. Ginseng contains myriad bioactive components including, ginsenosides, phytosterols, polysaccharides, polyacetylenes, polyacetylenic alcohols, fatty acids and peptides [42]. There exists already documentations on the anti-stress, anti-carcinogenic, anti-inflammatory, antioxidant, anti-bacterial, anti-viral and anti-fungal activities of ginseng [42–44]. Furthermore, ginseng demonstrates useful activity on endocrine diseases, cardiovascular diseases and the immune system [45]. During processing, Red ginseng is usually steamed and fermented with skinned ginseng and this alters the composition saponin contained in it when done repeatedly [46]. Red ginseng has been shown to possess anti-cancer, anti-diabetic, anti-obesity and immunomodulatory properties [3, 4]. Likewise zidovudine, red ginseng has also been applied as a therapeutic supplement for the treatment of patients with human immunodeficiency virus [47].

Studies have shown that red ginseng extract and its ginsenosides inactivate food borne viruses such as the human norovirus (huNoV) surrogates (feline calicivirus and murine norovirus) [43]. A plaque assay performed on FRhK-4 cell lines pretreated and co-treated with varied concentrations of Korean red ginseng (KRG) extract and purified ginsenosides (Rg1 and Rb1) showed that after inoculation of HAV HM-175 strain on the cell lines, KRG and the ginsenosides reduced significantly the HAV concentration [3, 4]. Korean red ginseng's extract demonstrated cytotoxicity at concentration above 10 μg/mL, while the purified ginsenosides showed no cytotoxic activity even up to 40 μg/mL. Although co-treatment of cell lines with KRG and the ginsenosides exhibited significant reduction of HAV concentration in the study, anti-HAV activity of the pretreated cell lines was quite higher [3, 4]. Hence, pretreatment with ginseng may be effective in preventing HAV infection. Also co-treatment of cell lines with KRG and the ginsenosides may be evaluated in further study using *in vivo* models.

The anti-HAV mechanisms of KRG extract and its ginsenosides are not clearly defined. However, reports from studies have shown that HAV-infected FRhK-4 cells activate the 2′-5′ oligoadenylate synthetase/RNaseL pathway [48]. Activation of RNase L degrades viral RNA and cellular single-stranded RNA; hence, KRG extract and its ginsenosides may tour a similar path. In addition, previous studies have reported that ginseng polysaccharides and ginsenosides have the capacity to boost the production of cytokines via stimulation of immune cells [3, 4]. Interferons induced by this pathway also contribute to the antiviral response.

#### *2.1.6 Blueberry juice and blueberry proanthocyanidins*

Blueberries are said to contain about 88–261 mg of proanthocyanidin/100 g of edible portion according to the USDA database for flavonoid content (USDA Database for the proanthocyanidin Content of Selected Foods, August 2004). Again, blue berries possess some other structurally related polyphenols such as anthocyanins and flavonoids [49]. Blueberry juice and its polyphenols have been found to have promising health benefits which include their cardioprotective, neuroprotective, anticarcinogenic, antibacterial, and antiviral properties [50]. Ethanol and water extracts of blueberries were reported to decrease *Listeria monocytogenes* by 5.90 log CFU/ml at 24 ppm and 37°C after 24 h *in-vitro* [51]. Also, 0.4 g/L gallic acid from blueberries caused a reduction in of *E. coli* O157:H7 titer in addition to the disruption of its cell-membrane after 24 h at 37°C *in-vitro* [52]. In addition, in a hepatitis C virus replicon cell system, methanol extract fraction of blueberry leaves (0.112–2200 lg/ml) was shown to suppress hepatitis C virus (HCV) subgenomic expression at 37°C after 72 h [53].

Recent study evaluated the antiviral activities of Blueberry juice and its proanthocyanidins (B-type) against HAV and some of human norovirus surrogates [50]. It was shown that in suspension, HAV titers were reduced by proanthocyanidins (2 and 5 mg/ml) to undetectable levels after 30 min, and after 3 h by 1 mg/ml proanthocyanidins. HAV titer was only reduced to by 2 log PFU/ml with Blue berry juice at pH 2.8 and 37°C after 24 h [50]. FRhK4 cells pre-infected and post-infected with HAV (strain; HM175) were also investigated for viral adsorption and replication upon treatment with the Blueberry juice and isolated proanthocyanidins [50]. The Blue berry proanthocyanidins showed promising preventive capacity as it moderately reduced HAV infectivity in the pre-infected cells but did not affect the replication of HAV in the post-infected cells. Hence, the Blue berry proanthocyanidins interrupt HAV binding and entry much more than it can limit its replication in the host cells; suggesting that it's antiviral efficacy is more preventive than therapeutic.

#### *2.1.7 Aqueous extracts of* Hibiscus sabdariffa *calyces*

*Hibiscus sabdariffa*, belonging to the family, Malvaceae, is an annual tropical or subtropical shrub species found in countries including Mexico, Sudan, India, and Thailand [54]. It is commonly called 'roselle' and is used for ornamental purposes, and the red calyces of *H. sabdariffa* are often used in the preparation of cold or hot beverages [55]. The calyces are said to be rich in bioactive compounds like anthocyanins, saponins, phenolic acids, organic acids and alkaloids [56]. Presence of organic acids like malic and tartaric acids identified in the calyces, possess a low pH of approximately 2–2.5 [54]. Aqueous extracts of the calyces are considered generally as safe and are approved for use as food additives by the U.S. Food and Drug Administration (21 CFR 172.510) in the flavoring of beverages [22, 23]. The calyces of *H. sabdariffa* are reported to possess a wide range of health benefits including antioxidant, anticancer, cardioprotective, anti-diabetic, and antimicrobial effects [57–59]. Protocatechuic acid (PCA), an essential component of *H. sabdariffa* has been shown to be the component responsible for its antimicrobial activity [60]. Another chemical component of the genus Hibiscus, known as Ferulic acid (FA) has also been reported to exhibit antimicrobial properties and antifilarial activity against *Setaria cervi* [61, 62].

Recent study evaluated the antiviral activity of *H. sabdariffa* against human novovirus surrogates and HAV. Findings revealed that aqueous extracts of calyces of *H. sabdariffa* (100 and 40 mg/ml) reduced HAV titer in suspension to undetectable levels at 37°C after 24 h [22, 23]. However, PCA demonstrated a moderate antiviral effect on HAV as it significantly reduced the HAV titer in suspension but not to undetectable levels. Pre- and post-infection assays with the aqueous extract of the calyces of *H. sabdariffa* (5 mg/ml) demonstrated no notable change in titres observed for HAV [22, 23]. Higher concentrations (40 and 100 mg/ml) of the aqueous extract was found to be cytotoxic to the host cell lines when added; observation for visual cytopathic effect under the light microscope showed that cells were

**83**

*2.1.9 Protamine, taxifolin and atropine*

*Antiviral Natural Products against Hepatitis-A Virus DOI: http://dx.doi.org/10.5772/intechopen.91869*

*2.1.8 4-phenylcoumarin derivatives*

of the extracts under *in vivo* conditions are required.

target structure for the discovery of new antiviral agents [66, 67].

peeling off [22, 23]. It is likely the aqueous extract is effective for alleviating viral burden; however this has not yet been substantiated as more studies into model food systems and simulation of gastrointestinal tract conditions to test the efficacy

Coumarin was first isolated from tonka beans, *Dipteryx odoranta*, also called Coumarou and biological activities of thousands of natural coumarins from plants, bacteria and fungi and chemical synthesis have been reported [63]. Coumarin and its derivatives have been used to manufacture drugs serving as anticoagulants including warfarin, acenocoumarin and phenprocoumon, and also for production of novobiocin, a potent inhibitor of bacterial DNA gyrase [63]. Coumarins (2H-chromen-2-ones) are recognized as a privileged bioactive scaffold for designing new agents with high affinity and specificity to various molecular targets [64], especially as antiviral agents [65]. In recent years, 4-Phenylcoumarins (neoflavones) which are bio-isosteres of flavonoids, have been of much interest as lead

A more recent study demonstrated that some coumarin derivatives possess anti-HAV activity. Newly modified 4-phenylcoumarin-based compounds were developed and evaluated for inhibition of 3C proteases [63]. Similar to other picornaviruses, HAV genome encodes a key processing protease, known as HAV 3C protease (HAV 3Cpro), which is a nonstructural cysteine protein responsible for the cleavage process within the viral polyprotein (250 kDa) that is critical for the replication process [63]. These proteases are responsible for processing the polyprotein precursor and also cleaving specific cellular factors needed for transcription and translation processes as well as nucleo-cytoplasmic trafficking in order to alter cell physiology to enhance viral replication; thus 3Cpro is vital to viral life cycle, making the viral 3C proteases choice targets for antiviral therapy [63]. Evaluation of the target compounds for their antiviral activity against hepatitis A virus revealed that the derivative, 1-(2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)acetyl) 4-ethylthiosemicarbazide had the most potent virucidal activity (IC50 = 3.1 μg/ml, TI = 83). The derivatives, 2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N′-(1-(4-chlorophenyl) ethylidene)acetohydrazide and 2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N′-(1- (4-bromophenyl)ethylidene)acetohydrazide demonstrated the strongest virustatic effects against HAV adsorption and replication, respectively (IC50 = 8.5 μg/ml, TI =88; IC50 = 10.7 μg/ml, TI = 91). Furthermore, studies reported that the three newly derived compounds were tested against HAV 3C protease and they exhibited remarkable inhibition effects (Ki = 1.903, 0.104 and 0.217 μM, respectively) indicating strong binding to HAV 3Cpro [63]. Also, the three compounds were docked within the pocket site of HAV 3C protease (PDB code: 2HAL) which illustrated that they had strong H-profiles with the amino acids Gly170 and Cys172. Findings suggested that the target compounds inhibited virus infection through the interrupting virus adsorption to the cell surface. This may have occurred via blocking of the cellular surface receptors by the target compounds which consequently led to an anti-HAV effect. Deduction from the post-treatment assay suggested that the target compounds inhibited the activities of some viral enzymes needed to complete the replication cycle or that they interfered with one or more steps in the viral life cycle.

Protamine, a cationic peptide, is generally obtained from fish milt (spermatic cells) and is applied medically as a heparin antagonist, an injectable insulin-carrier, peeling off [22, 23]. It is likely the aqueous extract is effective for alleviating viral burden; however this has not yet been substantiated as more studies into model food systems and simulation of gastrointestinal tract conditions to test the efficacy of the extracts under *in vivo* conditions are required.

#### *2.1.8 4-phenylcoumarin derivatives*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

*2.1.7 Aqueous extracts of* Hibiscus sabdariffa *calyces*

expression at 37°C after 72 h [53].

anthocyanins and flavonoids [49]. Blueberry juice and its polyphenols have been found to have promising health benefits which include their cardioprotective, neuroprotective, anticarcinogenic, antibacterial, and antiviral properties [50]. Ethanol and water extracts of blueberries were reported to decrease *Listeria monocytogenes* by 5.90 log CFU/ml at 24 ppm and 37°C after 24 h *in-vitro* [51]. Also, 0.4 g/L gallic acid from blueberries caused a reduction in of *E. coli* O157:H7 titer in addition to the disruption of its cell-membrane after 24 h at 37°C *in-vitro* [52]. In addition, in a hepatitis C virus replicon cell system, methanol extract fraction of blueberry leaves (0.112–2200 lg/ml) was shown to suppress hepatitis C virus (HCV) subgenomic

Recent study evaluated the antiviral activities of Blueberry juice and its proanthocyanidins (B-type) against HAV and some of human norovirus surrogates [50]. It was shown that in suspension, HAV titers were reduced by proanthocyanidins (2 and 5 mg/ml) to undetectable levels after 30 min, and after 3 h by 1 mg/ml proanthocyanidins. HAV titer was only reduced to by 2 log PFU/ml with Blue berry juice at pH 2.8 and 37°C after 24 h [50]. FRhK4 cells pre-infected and post-infected with HAV (strain; HM175) were also investigated for viral adsorption and replication upon treatment with the Blueberry juice and isolated proanthocyanidins [50]. The Blue berry proanthocyanidins showed promising preventive capacity as it moderately reduced HAV infectivity in the pre-infected cells but did not affect the replication of HAV in the post-infected cells. Hence, the Blue berry proanthocyanidins interrupt HAV binding and entry much more than it can limit its replication in the host cells; suggesting that it's antiviral efficacy is more preventive than therapeutic.

*Hibiscus sabdariffa*, belonging to the family, Malvaceae, is an annual tropical or subtropical shrub species found in countries including Mexico, Sudan, India, and Thailand [54]. It is commonly called 'roselle' and is used for ornamental purposes, and the red calyces of *H. sabdariffa* are often used in the preparation of cold or hot beverages [55]. The calyces are said to be rich in bioactive compounds like anthocyanins, saponins, phenolic acids, organic acids and alkaloids [56]. Presence of organic acids like malic and tartaric acids identified in the calyces, possess a low pH of approximately 2–2.5 [54]. Aqueous extracts of the calyces are considered generally as safe and are approved for use as food additives by the U.S. Food and Drug Administration (21 CFR 172.510) in the flavoring of beverages [22, 23]. The calyces of *H. sabdariffa* are reported to possess a wide range of health benefits including antioxidant, anticancer, cardioprotective, anti-diabetic, and antimicrobial effects [57–59]. Protocatechuic acid (PCA), an essential component of *H. sabdariffa* has been shown to be the component responsible for its antimicrobial activity [60]. Another chemical component of the genus Hibiscus, known as Ferulic acid (FA) has also been reported to exhibit antimicrobial properties and antifilarial activity

Recent study evaluated the antiviral activity of *H. sabdariffa* against human novovirus surrogates and HAV. Findings revealed that aqueous extracts of calyces of *H. sabdariffa* (100 and 40 mg/ml) reduced HAV titer in suspension to undetectable levels at 37°C after 24 h [22, 23]. However, PCA demonstrated a moderate antiviral effect on HAV as it significantly reduced the HAV titer in suspension but not to undetectable levels. Pre- and post-infection assays with the aqueous extract of the calyces of *H. sabdariffa* (5 mg/ml) demonstrated no notable change in titres observed for HAV [22, 23]. Higher concentrations (40 and 100 mg/ml) of the aqueous extract was found to be cytotoxic to the host cell lines when added; observation for visual cytopathic effect under the light microscope showed that cells were

**82**

against *Setaria cervi* [61, 62].

Coumarin was first isolated from tonka beans, *Dipteryx odoranta*, also called Coumarou and biological activities of thousands of natural coumarins from plants, bacteria and fungi and chemical synthesis have been reported [63]. Coumarin and its derivatives have been used to manufacture drugs serving as anticoagulants including warfarin, acenocoumarin and phenprocoumon, and also for production of novobiocin, a potent inhibitor of bacterial DNA gyrase [63]. Coumarins (2H-chromen-2-ones) are recognized as a privileged bioactive scaffold for designing new agents with high affinity and specificity to various molecular targets [64], especially as antiviral agents [65]. In recent years, 4-Phenylcoumarins (neoflavones) which are bio-isosteres of flavonoids, have been of much interest as lead target structure for the discovery of new antiviral agents [66, 67].

A more recent study demonstrated that some coumarin derivatives possess anti-HAV activity. Newly modified 4-phenylcoumarin-based compounds were developed and evaluated for inhibition of 3C proteases [63]. Similar to other picornaviruses, HAV genome encodes a key processing protease, known as HAV 3C protease (HAV 3Cpro), which is a nonstructural cysteine protein responsible for the cleavage process within the viral polyprotein (250 kDa) that is critical for the replication process [63]. These proteases are responsible for processing the polyprotein precursor and also cleaving specific cellular factors needed for transcription and translation processes as well as nucleo-cytoplasmic trafficking in order to alter cell physiology to enhance viral replication; thus 3Cpro is vital to viral life cycle, making the viral 3C proteases choice targets for antiviral therapy [63]. Evaluation of the target compounds for their antiviral activity against hepatitis A virus revealed that the derivative, 1-(2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)acetyl) 4-ethylthiosemicarbazide had the most potent virucidal activity (IC50 = 3.1 μg/ml, TI = 83). The derivatives, 2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N′-(1-(4-chlorophenyl) ethylidene)acetohydrazide and 2-(2-Oxo-4-phenyl-2H-chromen-7-yloxy)-N′-(1- (4-bromophenyl)ethylidene)acetohydrazide demonstrated the strongest virustatic effects against HAV adsorption and replication, respectively (IC50 = 8.5 μg/ml, TI =88; IC50 = 10.7 μg/ml, TI = 91). Furthermore, studies reported that the three newly derived compounds were tested against HAV 3C protease and they exhibited remarkable inhibition effects (Ki = 1.903, 0.104 and 0.217 μM, respectively) indicating strong binding to HAV 3Cpro [63]. Also, the three compounds were docked within the pocket site of HAV 3C protease (PDB code: 2HAL) which illustrated that they had strong H-profiles with the amino acids Gly170 and Cys172. Findings suggested that the target compounds inhibited virus infection through the interrupting virus adsorption to the cell surface. This may have occurred via blocking of the cellular surface receptors by the target compounds which consequently led to an anti-HAV effect. Deduction from the post-treatment assay suggested that the target compounds inhibited the activities of some viral enzymes needed to complete the replication cycle or that they interfered with one or more steps in the viral life cycle.

#### *2.1.9 Protamine, taxifolin and atropine*

Protamine, a cationic peptide, is generally obtained from fish milt (spermatic cells) and is applied medically as a heparin antagonist, an injectable insulin-carrier, and recently as an antibacterial ingredient in some food products [68]. Taxifolin (dihydroquercetin) is a flavononol amply found in grapes, olive oil, citrus fruits and onions [69]. It has been shown to possess strong pharmacological activities, including antioxidative, hepatoprotective, cardioprotective, anti-diabetic, anti-inflammatory, antitumor, neuroprotective effects, and had played a remarkable role in the preclusion of Alzheimer's disease [69]. Atropine is naturally occurring compound (alkaloid) majorly found in belladonna (Solanaceae) plants. It is a muscuranic receptor antagonist and is used medically to modulate muscular contractions and dilations which consequently regulate blood flow to cells and tissues [70].

A previous study investigated the inhibitory potential of protamine, atropine and taxifolin against HAV replication in PLC/PRF/5 cells, and found out that the trio exhibited some significant but not drastic effects on HAV replication [2]. Atropine demonstrated a concentration–dependent reduction in the infectivity of HAV but the antigenicity of the virus was not affected. HAV titer was reduced at the maximum concentration of 50, 59 and 50 μg/ml of protamine, taxifolin and atropine, respectively. It was suggested that further studies be done to determine the effect of these compounds on several multiplicities of HAV infection and also investigate possible synergistic effects of these compounds with other substances that have potential for clinical use against HAV infection [2]. The mechanisms of HAV titer reduction by the compounds are not yet clearly elucidated.

#### **3. Adjunctive anti-HAV natural products**

#### **3.1 Japanese rice-koji miso extracts**

Koji, also known as *Aspergillus oryzae*, is a filamentous fungus employed by the Japanese to ferment certain kinds of food like soybeans, potatoes, rice and some other grains [71]. Miso is one of the by-products of the fermentation of Japanese rice by Koji. Miso is conventional Japanese seasoning used for preparing miso soup, a staple Japanese cuisine [71]. Previous studies showed that Japanese miso extract increases the expression of a heat-shock protein known as glucose-regulated protein 78 (GRP78) and suppresses ultraviolet C mutagenesis [72]. Some researchers observed that HAV replication was retarded upon expression of GRP78 [71]; hence GRP78 has become a potential host antiviral against HAV infection [73]. Recent post-infection assay examined miso extracts obtained from Japanese rice-koji for antiviral activity against HAV, and it was shown that the miso extracts inhibited HAV replication by enhancing the expression of GRP78 in human hepatocytes (Huh7 and PXB cells) [71]. These findings suggested that Japanese miso extracts may synergistically work as antivirals against HAV infection by partially modulating GRP78 expression [71]. Miso extracts may also serve as effective dietary supplements for the control of acute hepatitis A infection.

#### **3.2 Korean soy sauce**

Conventional Korean soy sauce is generally made with germinated soybean, salt and water [74]. The soy sauce is fermented after cooking and crushing soybean, then mold it into a block form (Meju) with concurrent addition of salt (NaCl) and water before exposing it to natural conditions [3, 4]. The percentage salt content of traditional Korean soy sauce is around 16.3–20.8% NaCl [75]. Studies have shown that soy sauce possesses diverse biological activities such as angiotensin inhibitory, anti-platelet, anticarcinogenic, and anti-oxidant activities [74]. Also, there is a report about the antibacterial activity of soy sauce against *Escherichia coli* O157:H7 [76]. The

**85**

*Antiviral Natural Products against Hepatitis-A Virus DOI: http://dx.doi.org/10.5772/intechopen.91869*

preservation and storage before consumption (**Table 1**).

at 37°C and pH of 7.2

2 mg/ml for 6 h at 37°C

0.1% (EO from grapefruit); 0.5% (EO from lemon); 0.05% (EO from rosemary cineole)

5–10 μg/mL For 24 h at 37°C

for 24 h

2 and 5 mg/ml for 30 min at 37°C

Blueberry Juice pH 2.8 at 37°C

tives [74].

**Evaluated natural products**

Grape Seed Extract

Egyptian Red Sea Seagrass Crude Extract

Essential Oils (EO) from lemon, grapefruit and rosemary cineole

Korean Red Ginseng Extract and Ginsenosides

Blueberry Proanthocyanidins

Green Tea Extract 5 mg/ml for 2 h

antimicrobial effects of soy sauce were attributed to the presence of a combination of ingredients and properties including NaCl, ethanol, pH, organic acids, and preserva-

A study that evaluated the antiviral activity of the Korean soy sauce on HAV inoculated in raw fresh crabs (*Portunus trituberculantus*) to simulate storage conditions for homemade Ganjanggejang (a salted preserved raw seafood in Korean cuisine) revealed that there was an over 90% reduction of the HAV titer in the Ganjanggejang marinated in soy sauce containing 20% NaCl for at least 3 days [74]. Hence, the soy sauce was synergistically more effective at increasing salt concentrations. The antiviral activity of soy sauce is majorly due to the salt (NaCl) concentrations and partially attributable to its other constituents, such as ethanol, organic acids, and preservatives, and the pH of 5.11–6.98 [77]. Inhibition of HAV in crabs by NaCl in soy sauce might be due to changes in water activity which may affect virus survival [77]. In addition, antiviral mechanisms associated with NaCl may include altering the molecular structure of the viral RNA and inhibiting the viral enzymes' activity [74]. However, it's not likely that Korean soy sauce will be of relevance in clinical practice rather it may be instrumental for immediate food

**Concentration Result Proposed** 

Complete inactivation of HAV in suspension

20 μg/mL 100% inhibition

assay

Significant reduction in cell infectivity in the order; rosemary cineole > grapefruit > lemon.

Significant reduction of HAV titer with dosedependent manner in pretreated FRhk-4 cells

Reduced HAV titer by 2 log PFU/ml

Reduced HAV titer to undetectable levels in suspension

Reduced HAV titer to undetectable levels under simulated gastrointestinal conditions

of HAV in a plaque

**mechanism of action**

them

Interfers with viral attachment to cell membrane receptors upon binding to

Interrupt the binding of HAV to the cell receptors, preventing adsorption.

— [28]

— [33]

(1) Activation of the 2′-5′oligoadenylate synthetase/RNaseL pathway; (2) boost the production of cytokines

Interfers with HAV binding to host cells

Interrupt HAV binding and entry into host cells

**References**

[23, 24, 28]

[3–5, 49]

[50]

[50]

[7, 15]

*Hepatitis A and Other Associated Hepatobiliary Diseases*

and recently as an antibacterial ingredient in some food products [68]. Taxifolin (dihydroquercetin) is a flavononol amply found in grapes, olive oil, citrus fruits and onions [69]. It has been shown to possess strong pharmacological activities, including antioxidative, hepatoprotective, cardioprotective, anti-diabetic, anti-inflammatory, antitumor, neuroprotective effects, and had played a remarkable role in the preclusion of Alzheimer's disease [69]. Atropine is naturally occurring compound (alkaloid) majorly found in belladonna (Solanaceae) plants. It is a muscuranic receptor antagonist and is used medically to modulate muscular contractions and

dilations which consequently regulate blood flow to cells and tissues [70].

HAV titer reduction by the compounds are not yet clearly elucidated.

**3. Adjunctive anti-HAV natural products**

ments for the control of acute hepatitis A infection.

**3.1 Japanese rice-koji miso extracts**

A previous study investigated the inhibitory potential of protamine, atropine and taxifolin against HAV replication in PLC/PRF/5 cells, and found out that the trio exhibited some significant but not drastic effects on HAV replication [2]. Atropine demonstrated a concentration–dependent reduction in the infectivity of HAV but the antigenicity of the virus was not affected. HAV titer was reduced at the maximum concentration of 50, 59 and 50 μg/ml of protamine, taxifolin and atropine, respectively. It was suggested that further studies be done to determine the effect of these compounds on several multiplicities of HAV infection and also investigate possible synergistic effects of these compounds with other substances that have potential for clinical use against HAV infection [2]. The mechanisms of

Koji, also known as *Aspergillus oryzae*, is a filamentous fungus employed by the Japanese to ferment certain kinds of food like soybeans, potatoes, rice and some other grains [71]. Miso is one of the by-products of the fermentation of Japanese rice by Koji. Miso is conventional Japanese seasoning used for preparing miso soup, a staple Japanese cuisine [71]. Previous studies showed that Japanese miso extract increases the expression of a heat-shock protein known as glucose-regulated protein 78 (GRP78) and suppresses ultraviolet C mutagenesis [72]. Some researchers observed that HAV replication was retarded upon expression of GRP78 [71]; hence GRP78 has become a potential host antiviral against HAV infection [73]. Recent post-infection assay examined miso extracts obtained from Japanese rice-koji for antiviral activity against HAV, and it was shown that the miso extracts inhibited HAV replication by enhancing the expression of GRP78 in human hepatocytes (Huh7 and PXB cells) [71]. These findings suggested that Japanese miso extracts may synergistically work as antivirals against HAV infection by partially modulating GRP78 expression [71]. Miso extracts may also serve as effective dietary supple-

Conventional Korean soy sauce is generally made with germinated soybean, salt and water [74]. The soy sauce is fermented after cooking and crushing soybean, then mold it into a block form (Meju) with concurrent addition of salt (NaCl) and water before exposing it to natural conditions [3, 4]. The percentage salt content of traditional Korean soy sauce is around 16.3–20.8% NaCl [75]. Studies have shown that soy sauce possesses diverse biological activities such as angiotensin inhibitory, anti-platelet, anticarcinogenic, and anti-oxidant activities [74]. Also, there is a report about the antibacterial activity of soy sauce against *Escherichia coli* O157:H7 [76]. The

**84**

**3.2 Korean soy sauce**

antimicrobial effects of soy sauce were attributed to the presence of a combination of ingredients and properties including NaCl, ethanol, pH, organic acids, and preservatives [74].

A study that evaluated the antiviral activity of the Korean soy sauce on HAV inoculated in raw fresh crabs (*Portunus trituberculantus*) to simulate storage conditions for homemade Ganjanggejang (a salted preserved raw seafood in Korean cuisine) revealed that there was an over 90% reduction of the HAV titer in the Ganjanggejang marinated in soy sauce containing 20% NaCl for at least 3 days [74]. Hence, the soy sauce was synergistically more effective at increasing salt concentrations. The antiviral activity of soy sauce is majorly due to the salt (NaCl) concentrations and partially attributable to its other constituents, such as ethanol, organic acids, and preservatives, and the pH of 5.11–6.98 [77]. Inhibition of HAV in crabs by NaCl in soy sauce might be due to changes in water activity which may affect virus survival [77]. In addition, antiviral mechanisms associated with NaCl may include altering the molecular structure of the viral RNA and inhibiting the viral enzymes' activity [74]. However, it's not likely that Korean soy sauce will be of relevance in clinical practice rather it may be instrumental for immediate food preservation and storage before consumption (**Table 1**).



#### **Table 1.**

*Summary of anti-HAV natural products.*

#### **4. Miscellaneous products**

Duck hepatitis A virus type-1(DHAV-1) is a variant of hepatitis A virus that attacks ducks. It has been proposed that duck hepatitis A is a small animal model for the human hepatitis A [78]. It may be correct to say that antiviral agents against DHAV-1 will also demonstrate appropriate antiviral activity against human hepatitis A virus. Several natural agents have been under study to explore their antiviral potentials against DHAV-1 and they include phosphorylated *Codonopsis pilosula* polysaccharide (pCPP), Raw Rehmannia Radix Polysaccharide (RRRP), Baicalin phospholipid complex (BAPC), flavonoid combinations—baicalin-linarin-icariinnotoginsenosideR1 (BLIN).

It was reported that **RRRP** could significantly reduce mortality rate, liver lesion scoring, alleviate visual liver lesion, and decrease the alterations of plasma biochemical evaluation indexes of hepatic injury induced by DHAV-1 infection [79]. **pCPP** was also reported to demonstrate a strong inhibitory effect on DHAV-1 replication, which led to a significant decrease on the number of viral particles [80]. Studies with DHAV-1-infected ducklings treated with **BAPC** showed that it significantly inhibited DHAV-1 adsorption, replication and release [81]. Furthermore, it was reported that BAPC played anti-oxidative and immuno-supportive roles during the treatment, and that the immuno-supportive role was critical to the treatment. Another study evaluated the anti-DHAV-1 activity of a flavonoid mix, **BLIN** [82]. At 20 μg/mL, DHAV-1 inhibitory rate of BLIN at 20 μg/mL was reported to be 69.3% in duck embryonic hepatocytes. It was demonstrated that the survival rate of ducklings treated by BLIN was about 35.5%, which was remarkably higher than that of virus control (0.0%) [82]. In addition, after the treatment with BLIN, both

**87**

**Author details**

Damian Chukwu Odimegwu1

Nigeria, Nsukka, Enugu State, Nigeria

provided the original work is properly cited.

\* and Uzochukwu Gospel Ukachukwu<sup>2</sup>

1 Faculty of Pharmaceutical Sciences, Department of Pharmaceutical Microbiology

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and Biotechnology, University of Nigeria, Nsukka, Enugu State, Nigeria

\*Address all correspondence to: damian.odimegwu@unn.edu.ng

against HAV will gain ample attention in the nearest future.

2 Faculty of Biological Sciences, Department of Biochemistry, University of

*Antiviral Natural Products against Hepatitis-A Virus DOI: http://dx.doi.org/10.5772/intechopen.91869*

injury indices and the oxidative stress indices.

**5. Future outlook**

the hepatic injury and the oxidative stress of the infected ducklings assuaged [82]. Concurrently, a significant positive correlation was said to exist between the hepatic

Currently, studies exploring potential anti-HAV natural products are still emerging and had attracted little attention, possibly because a vaccine has been developed to mitigate the spread of the viral infection to a considerable length of years. However, there is need for development of more efficient and effective anti-HAV therapeutic, prophylactic and adjunctive agents, and as at now, none has been licensed. Investigations into natural products with anti-HAV hold a promising outlook as several of them have demonstrated remarkable potential to control HAV infection and replication. In addition, studies should be aimed at mimicking more closely the features of the human hepatitis A virus *in vivo* than *in vitro* so as to clearly establish the basis for the application of these natural agents in a clinical setting. There is need to develop suitable animal models that could present very similar clinical manifestations as found in humans during hepatitis A virus infection, for more accurate interpretation and correlation of outcomes from pre-clinical studies involving natural products therapy. Hopefully, studies on antiviral natural products

the hepatic injury and the oxidative stress of the infected ducklings assuaged [82]. Concurrently, a significant positive correlation was said to exist between the hepatic injury indices and the oxidative stress indices.

### **5. Future outlook**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

100 mg/ml and 40 mg/ml at 37°C for 24 h

Protamine 50 μg/ml Reduced HAV

Taxifolin 59 μg/ml Reduced HAV

Atropine 50 μg/ml Reduced HAV

20% NaCl

10 μl at 37°C Inhibited the

— Inhibited HAV

**Evaluated natural products**

Aqueous extracts of *Hibiscus sabdariffa* Calyces

4-phenylcoumarin derivatives

Japanese rice-koji miso extracts

**Table 1.**

**4. Miscellaneous products**

*Summary of anti-HAV natural products.*

Korean Soy Sauce Containing

notoginsenosideR1 (BLIN).

Duck hepatitis A virus type-1(DHAV-1) is a variant of hepatitis A virus that attacks ducks. It has been proposed that duck hepatitis A is a small animal model for the human hepatitis A [78]. It may be correct to say that antiviral agents against DHAV-1 will also demonstrate appropriate antiviral activity against human hepatitis A virus. Several natural agents have been under study to explore their antiviral potentials against DHAV-1 and they include phosphorylated *Codonopsis pilosula* polysaccharide (pCPP), Raw Rehmannia Radix Polysaccharide (RRRP), Baicalin phospholipid complex (BAPC), flavonoid combinations—baicalin-linarin-icariin-

**Concentration Result Proposed** 

Reduced HAV titer to undetectable levels in suspension

activity of HAV 3C protease

infectivity

infectivity

infectivity

replication

over 90% reduction of the HAV titer

**mechanism of action**

Interrupt HAV adsorption on cell

Inhibited HAV replication by enhancing the expression of GRP78 in human hepatocytes

Inhibition of viral enzymes' activity

surface

— [22, 23]

— [2]

— [2]

— [2]

**References**

[63]

[71]

[74]

It was reported that **RRRP** could significantly reduce mortality rate, liver lesion scoring, alleviate visual liver lesion, and decrease the alterations of plasma biochemical evaluation indexes of hepatic injury induced by DHAV-1 infection [79]. **pCPP** was also reported to demonstrate a strong inhibitory effect on DHAV-1 replication, which led to a significant decrease on the number of viral particles [80]. Studies with DHAV-1-infected ducklings treated with **BAPC** showed that it significantly inhibited DHAV-1 adsorption, replication and release [81]. Furthermore, it was reported that BAPC played anti-oxidative and immuno-supportive roles during the treatment, and that the immuno-supportive role was critical to the treatment. Another study evaluated the anti-DHAV-1 activity of a flavonoid mix, **BLIN** [82]. At 20 μg/mL, DHAV-1 inhibitory rate of BLIN at 20 μg/mL was reported to be 69.3% in duck embryonic hepatocytes. It was demonstrated that the survival rate of ducklings treated by BLIN was about 35.5%, which was remarkably higher than that of virus control (0.0%) [82]. In addition, after the treatment with BLIN, both

**86**

Currently, studies exploring potential anti-HAV natural products are still emerging and had attracted little attention, possibly because a vaccine has been developed to mitigate the spread of the viral infection to a considerable length of years. However, there is need for development of more efficient and effective anti-HAV therapeutic, prophylactic and adjunctive agents, and as at now, none has been licensed. Investigations into natural products with anti-HAV hold a promising outlook as several of them have demonstrated remarkable potential to control HAV infection and replication. In addition, studies should be aimed at mimicking more closely the features of the human hepatitis A virus *in vivo* than *in vitro* so as to clearly establish the basis for the application of these natural agents in a clinical setting. There is need to develop suitable animal models that could present very similar clinical manifestations as found in humans during hepatitis A virus infection, for more accurate interpretation and correlation of outcomes from pre-clinical studies involving natural products therapy. Hopefully, studies on antiviral natural products against HAV will gain ample attention in the nearest future.

### **Author details**

Damian Chukwu Odimegwu1 \* and Uzochukwu Gospel Ukachukwu<sup>2</sup>

1 Faculty of Pharmaceutical Sciences, Department of Pharmaceutical Microbiology and Biotechnology, University of Nigeria, Nsukka, Enugu State, Nigeria

2 Faculty of Biological Sciences, Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria

\*Address all correspondence to: damian.odimegwu@unn.edu.ng

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[66] Bedoya LM, Beltran M, Sancho R, Olmedo DA, Sanchez-Palomino S, del Olmo E, et al. 4-Phenylcoumarins as HIV transcription inhibitors. Bioorganic

[67] Marquez N, Sancho R, Bedoya LM, Alcamí J, Lopez-Perez JL, Feliciano AS, et al. Mesuol, a natural occurring 4-phenylcoumarin, inhibits HIV-1 replication by targeting the NF-kappa B pathway. Antiviral Research.

[68] Gill TA, Singer DS, Thompson JW.

[69] Islam NMD, Oda H, Motohiro T. Changes in the cell morphology and the release of soluble constituents from washed cells of *Bacillus subtillis* by the action of protamine. Nippon Suisan

[70] Behçet A. The source-synthesishistory and use of atropine. The Journal of Academic Emergency Medicine.

[71] Win NN, Kanda T, Nakamoto S, Moriyama M, Jiang X, Suganami A, et al. Inhibitory effect of Japanese rice-koji miso extracts on hepatitis A virus replication in association with the elevation of glucose-regulated protein 78 expression. International Journal of Medical Sciences. 2018;**15**(11):1153-1159

[72] Jiang X, Ren Q, Chen SP, et al. UVC mutagenicity is suppressed in Japanese miso-treated human RSa cells, possibly via GRP78 expression. Bioscience,

Purification and analysis of protamine. Process Biochemistry.

Gakkaishi. 1987;**53**:297-303

& Medicinal Chemistry Letters.

2015;**12**:66-79

2005;**15**:4447-4450

2005;**66**:137-145

2006;**41**:1875-1882

2014;**13**:2-3

**92**

[80] Ming K, Chen Y, Yao F, Shi J, Yang J, Du H, et al. Phosphorylated *Codonopsis pilosula* polysaccharide could inhibit the virulence of duck hepatitis A virus compared with *Codonopsis pilosula* polysaccharide. International Journal of Biological Macromolecules. 2017;**94**:28-35

[81] Chen Y, Yang Y, Wang F, Yang X, Yao F, Ming K, et al. Antiviral effect of baicalin phospholipid complex against duck hepatitis Avirus type 1. Poultry Science. 2018;**97**(8):2722-2732

[82] Chen Y, Zeng L, Lu Y, Yang Y, Xu M, Wang Y, et al. Treatment effect of a flavonoid prescription on duck virus hepatitis by its hepatoprotective and antioxidative ability. Pharmaceutical Biology. 2017;**55**(1):198-205

**95**

Section 3

Other Hepatobiliary

Conditions

### Section 3

## Other Hepatobiliary Conditions

**97**

**Chapter 7**

**Abstract**

**1. Introduction**

Hepatic Involvement

in Hemophagocytic

Lymphohistiocytosis

*and Subash Chandra Samal*

liver transplantation in this clinical setting.

**Keywords:** cytokine storm, HLH, liver, transplant, outcome

*Somanath Padhi, RajLaxmi Sarangi, Susama Patra* 

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome which results in uncontrolled systemic proliferation of benign macrophages in all reticuloendothelial organs producing worsening peripheral blood cytopenia(s); hypercytokinemia leading to hepatic injury producing hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia; and if not diagnosed and treated early may lead to disseminated intravascular coagulation (DIC), multiorgan dysfunction, and death in nearly all individuals. It is postulated that hepatic injury/dysfunction starts early in the course of the disease which may mimic nonspecific hepatitis like prodrome to fulminant hepatic failure; possibly requiring liver transplant. While HLH as an entity is being increasingly recognized nowadays across wide specialties (both pediatric and adults); hepatic involvement in this setting has been poorly characterized. This chapter is aimed to highlight on the diagnosis and classification of HLH with a special emphasis on the pathophysiology of hepatic dysfunction, histomorphology of liver; and the current concept and controversies on the role of

Hemophagocytic lymphohistiocytosis (HLH) or hemophagocytic syndrome is a potentially catastrophic hyperinflammatory syndrome occurring in genetically susceptible individuals which results due to hyperactive, yet inappropriate, immune system going runamock [1]. This results due to impaired cytotoxic T lymphocyte (CTL)/natural killer (NK) cell activity producing uncontrolled proliferation of benign macrophages in all reticuloendothelial organs such as bone marrow, spleen, liver, and lymph nodes; causing histiocytic hemophagocytosis, worsening unexplained peripheral blood cytopenia (s), cytokine storm, cytokine mediated hepatic injury/ dysfunction producing spectrum of biochemical alteration such as hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, coagulopathy, disseminated intravascular coagulation (DIC), multi organ dysfunction (MOD); and if not diagnosed and treated early, may lead to death in virtually all case [2]. Since the first description of cases coined as "histiocytic medullary reticulocytosis" by Scott Robin and Smith in 1939, there has been a sequential change in nomenclature of this entity [3–6]. While HLH

#### **Chapter 7**

## Hepatic Involvement in Hemophagocytic Lymphohistiocytosis

*Somanath Padhi, RajLaxmi Sarangi, Susama Patra and Subash Chandra Samal*

#### **Abstract**

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome which results in uncontrolled systemic proliferation of benign macrophages in all reticuloendothelial organs producing worsening peripheral blood cytopenia(s); hypercytokinemia leading to hepatic injury producing hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia; and if not diagnosed and treated early may lead to disseminated intravascular coagulation (DIC), multiorgan dysfunction, and death in nearly all individuals. It is postulated that hepatic injury/dysfunction starts early in the course of the disease which may mimic nonspecific hepatitis like prodrome to fulminant hepatic failure; possibly requiring liver transplant. While HLH as an entity is being increasingly recognized nowadays across wide specialties (both pediatric and adults); hepatic involvement in this setting has been poorly characterized. This chapter is aimed to highlight on the diagnosis and classification of HLH with a special emphasis on the pathophysiology of hepatic dysfunction, histomorphology of liver; and the current concept and controversies on the role of liver transplantation in this clinical setting.

**Keywords:** cytokine storm, HLH, liver, transplant, outcome

#### **1. Introduction**

Hemophagocytic lymphohistiocytosis (HLH) or hemophagocytic syndrome is a potentially catastrophic hyperinflammatory syndrome occurring in genetically susceptible individuals which results due to hyperactive, yet inappropriate, immune system going runamock [1]. This results due to impaired cytotoxic T lymphocyte (CTL)/natural killer (NK) cell activity producing uncontrolled proliferation of benign macrophages in all reticuloendothelial organs such as bone marrow, spleen, liver, and lymph nodes; causing histiocytic hemophagocytosis, worsening unexplained peripheral blood cytopenia (s), cytokine storm, cytokine mediated hepatic injury/ dysfunction producing spectrum of biochemical alteration such as hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, coagulopathy, disseminated intravascular coagulation (DIC), multi organ dysfunction (MOD); and if not diagnosed and treated early, may lead to death in virtually all case [2]. Since the first description of cases coined as "histiocytic medullary reticulocytosis" by Scott Robin and Smith in 1939, there has been a sequential change in nomenclature of this entity [3–6]. While HLH

as an entity is being increasingly recognized nowadays across wide specialties (both pediatric and adults); hepatic involvement in this setting has been poorly characterized [7–9]. Hepatomegaly and hepatic dysfunction, including elevated serum transaminases and bilirubin, cholestasis, and coagulopathy typically occur early in the disease and are associated with marked hematologic and/or neurological abnormalities. In rare instances acute hepatic failure may dominate the clinical picture, which in combination with hyperferritinemia, may mimic neonatal hemochromatosis [10].

#### **2. Classification of HLH**

Traditionally, HLH has been broadly classified into two forms: (i) primary HLH which is known to harbor documented genetic abnormalities implicated in the cytotoxic functions of the NK cell/CTL; and (ii) a secondary form which occurs in adults/elderly population without any genetic abnormality. However, upon the realization that HLH defining genetic abnormality can occur in any age, that these defects may be uncommon even in pediatric age group, the designations *primary and secondary* have become less relevant; and stratification into *genetic and acquired* forms seems more appropriate [11]. The genetic variant is again subdivided into autosomal recessive familial HLH (FHL) involving the several mutations in the CTL/NK


**Table 1.**

*Classification of hemophagocytic lymphohistiocytosis (HLH) (adopted from Rosado et al. [1] and Gholam et al. [12]).*

**99**

*Hepatic Involvement in Hemophagocytic Lymphohistiocytosis*

cell cytotoxic pathways [PFR1/perforin 1 (20–50%), UNC13D/Munc 13-4 (20%), STX11/syntaxin 11 (1%), STXB2/syntaxin binding protein 2 or UNC18B (unknown)] and those associated with primary immunodeficiency syndromes such as Chédiak-Higashi syndrome (LYST), Griscelli syndrome type 2 (RAB27A/Rab27a), Hermansky-Pudlak syndrome type 2 (AP3B1), and X-linked proliferative syndrome (XLP) type 1

Acquired form of HLH is known to be triggered by diverse etiologies in susceptible individuals; and are segregated as (i) infection associated secondary to viruses (notably EVB, CMV, HHV-8, HSV, dengue, parvo B19, HAV, HBV, HCV, etc.; any bacteria, fungi, parasites such as Plasmodia, Leishmania, Strongyloides), (ii) malignancy associated (NK-T cell lymphoma/leukemia, anaplastic large cell lymphoma, plasma cell myeloma, Hodgkin lymphoma, B cell non Hodgkin lymphoma, acute lymphoid and myeloid leukemias; and solid malignancies such as lung cancer, hepatocellular carcinoma, etc.), and (iii) macrophage activation

(SHD2D1A/SAP protein) and type 2 (BIRC4/XIAP protein) [12] (**Table 1**).

syndrome or MAS (associated with autoimmune disorders) [1, 3, 13].

There has been a paradigm shift of focus in the diagnosis of HLH since 2004 (**Table 2**) [3]. The 2004 diagnostic criteria developed for the pediatric HLH have been widely adopted in adult medicine without systematic validation. Both 2004 and 2009 guidelines incorporated mutational/genetic analysis as a "major criterion" which has subsequently been taken out, especially for adult HLH case. Moreover, two important parameters that were incorporated in previous criteria such as "impaired NK cell activity" and "increased soluble interleukin 2 receptor" are likely to be removed sooner or later as these tests are available in only very few specialized centers all over the world and are very costly. Therefore, in practice, the necessary five out of eight criteria as per the HLH 2004 guidelines are actually five out of six parameters tested. In addition, the criteria of "bone marrow hemophagocytosis" is becoming increasingly less important nowadays as histiocytic hemophagocytosis has a poor specificity in the diagnosis of HLH and this may not even be evident during initial marrow evaluation [1]! In order to overcome these shortcomings, the French investigators proposed to adopt a new objective scoring system (HLH probability score or HScore) (**Table 3**). A total probability score of 169 was found to have a higher sensitivity and specificity for the diagnosis of HLH [6]. Furthermore, simpler routine laboratory parameters (extended variables) have been incorporated to diagnose the disease early. These include peripheral blood monocytosis, hyponatremia, elevated lactate dehydrogenase, elevated β2

microglobulin, impaired coagulation parameters, and CSF pleocytosis [14].

associated HLH (8.56 vs. 0.66, respectively, *P* = 0.0004) [19].

Another interesting change has been made in regard to the measurement of serum ferritin. A ≥ 500 μg/L cut off among pediatric population (up to 18 years of age) was found to have 84% sensitivity in HLH-1994 trial and therefore was included in the HLH 2004 guidelines [15]. Subsequently, pediatricians have revised their ferritin cut off value to ≥10,000 μg/L with a higher sensitivity and near 100% specificity for the diagnosis of HLH [16]. On the contrary, recent reports from adult intensive care units (ICUs) have suggested a lower ferritin cut off value of 3000 to 4000 μg/L with >80% sensitivity and specificity in HLH diagnosis [17]. While hyperferritinemia is not specific to HLH, the same in the clinical context of fever, worsening cytopenia (s), and splenomegaly is highly valuable in the ICUs where sepsis is the major overlapping clinical condition [18]. Recent studies have shown that a high serum soluble interleukin 2 receptor to ferritin ratio is an important biomarker in distinguishing lymphoma associated HLH compared to benign disease

*DOI: http://dx.doi.org/10.5772/intechopen.90238*

**3. Diagnostic criteria**

*Hepatic Involvement in Hemophagocytic Lymphohistiocytosis DOI: http://dx.doi.org/10.5772/intechopen.90238*

cell cytotoxic pathways [PFR1/perforin 1 (20–50%), UNC13D/Munc 13-4 (20%), STX11/syntaxin 11 (1%), STXB2/syntaxin binding protein 2 or UNC18B (unknown)] and those associated with primary immunodeficiency syndromes such as Chédiak-Higashi syndrome (LYST), Griscelli syndrome type 2 (RAB27A/Rab27a), Hermansky-Pudlak syndrome type 2 (AP3B1), and X-linked proliferative syndrome (XLP) type 1 (SHD2D1A/SAP protein) and type 2 (BIRC4/XIAP protein) [12] (**Table 1**).

Acquired form of HLH is known to be triggered by diverse etiologies in susceptible individuals; and are segregated as (i) infection associated secondary to viruses (notably EVB, CMV, HHV-8, HSV, dengue, parvo B19, HAV, HBV, HCV, etc.; any bacteria, fungi, parasites such as Plasmodia, Leishmania, Strongyloides), (ii) malignancy associated (NK-T cell lymphoma/leukemia, anaplastic large cell lymphoma, plasma cell myeloma, Hodgkin lymphoma, B cell non Hodgkin lymphoma, acute lymphoid and myeloid leukemias; and solid malignancies such as lung cancer, hepatocellular carcinoma, etc.), and (iii) macrophage activation syndrome or MAS (associated with autoimmune disorders) [1, 3, 13].

#### **3. Diagnostic criteria**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

**2. Classification of HLH**

**A.Genetic**

**B.Acquired**

ii. Associated with primary immunodeficiency syndromes

vi. Autoimmune disease associated (macrophage activation syndrome,

MAS)

*Gholam et al. [12]).*

as an entity is being increasingly recognized nowadays across wide specialties (both pediatric and adults); hepatic involvement in this setting has been poorly characterized [7–9]. Hepatomegaly and hepatic dysfunction, including elevated serum transaminases and bilirubin, cholestasis, and coagulopathy typically occur early in the disease and are associated with marked hematologic and/or neurological abnormalities. In rare instances acute hepatic failure may dominate the clinical picture, which in combination with hyperferritinemia, may mimic neonatal hemochromatosis [10].

Traditionally, HLH has been broadly classified into two forms: (i) primary HLH

which is known to harbor documented genetic abnormalities implicated in the cytotoxic functions of the NK cell/CTL; and (ii) a secondary form which occurs in adults/elderly population without any genetic abnormality. However, upon the realization that HLH defining genetic abnormality can occur in any age, that these defects may be uncommon even in pediatric age group, the designations *primary and secondary* have become less relevant; and stratification into *genetic and acquired* forms seems more appropriate [11]. The genetic variant is again subdivided into autosomal recessive familial HLH (FHL) involving the several mutations in the CTL/NK

**Type Examples with proportions in parentheses**

i. Autosomal recessive/familial HLH PFR1/perforin 1 (20-50%), UNC13D/Munc 13-4 (20%),

i. Virus associated Herpes viruses (EBV, CMV, HHV-8, HSV), HIV, HTLV,

iii. Fungal associated *Candida* spp., *Cryptococcus* spp., *Pneumocystis* spp.,

*Classification of hemophagocytic lymphohistiocytosis (HLH) (adopted from Rosado et al. [1] and* 

ii. Bacteria associated *Staphylococcus aureus*, *Campylobacter* spp., *Fusobacterium* spp.,

iv. Parasitic *Plasmodium falciparum*, *Plasmodium vivax*, *Toxoplasma* spp.,

v. Malignancy associated Peripheral T-cell/NK-cell lymphomas, ALCL, ALL, Hodgkin

spondyloarthropathies

or UNC18B (unknown)

(XLP) type 2 (BIRC4/XIAP protein)

*burgdorferi*, *Mycobacterium tuberculosis*

*Histoplasma* spp., *Aspergillus* spp., *Fusarium* spp.

*Babesia* spp., *Strongyloides* spp., *Leishmania* spp.

lymphoma, multiple myeloma, acute erythroid leukemia Prostate and lung cancer, hepatocellular carcinoma

Systemic-onset juvenile idiopathic arthritis, Kawasaki disease, systemic lupus erythematosus, seronegative

STX11/syntaxin 11 (1%), STXB2/syntaxin binding protein 2

Chediak-Higashi syndrome (LYST), Griscelli syndrome type 2 (RAB27A/Rab27a), Hermansky-Pudlak syndrome type 2 (AP3B1), X-linked proliferative syndrome (XLP) type 1 (SHD2D1A/SAP protein), X-linked proliferative syndrome

adenovirus, HAV, HBV, HCV, measles, mumps, rubella, dengue, hantavirus, parvovirus B19, Enterovirus, influenza

*Mycoplasma* spp., *Chlamydia* spp., *Legionella* spp., *Salmonella typhi*, *Rickettsia* spp., *Brucella* spp.*, Ehrlichia* spp.*, Borrelia* 

**98**

**Table 1.**

There has been a paradigm shift of focus in the diagnosis of HLH since 2004 (**Table 2**) [3]. The 2004 diagnostic criteria developed for the pediatric HLH have been widely adopted in adult medicine without systematic validation. Both 2004 and 2009 guidelines incorporated mutational/genetic analysis as a "major criterion" which has subsequently been taken out, especially for adult HLH case. Moreover, two important parameters that were incorporated in previous criteria such as "impaired NK cell activity" and "increased soluble interleukin 2 receptor" are likely to be removed sooner or later as these tests are available in only very few specialized centers all over the world and are very costly. Therefore, in practice, the necessary five out of eight criteria as per the HLH 2004 guidelines are actually five out of six parameters tested. In addition, the criteria of "bone marrow hemophagocytosis" is becoming increasingly less important nowadays as histiocytic hemophagocytosis has a poor specificity in the diagnosis of HLH and this may not even be evident during initial marrow evaluation [1]! In order to overcome these shortcomings, the French investigators proposed to adopt a new objective scoring system (HLH probability score or HScore) (**Table 3**). A total probability score of 169 was found to have a higher sensitivity and specificity for the diagnosis of HLH [6]. Furthermore, simpler routine laboratory parameters (extended variables) have been incorporated to diagnose the disease early. These include peripheral blood monocytosis, hyponatremia, elevated lactate dehydrogenase, elevated β2 microglobulin, impaired coagulation parameters, and CSF pleocytosis [14].

Another interesting change has been made in regard to the measurement of serum ferritin. A ≥ 500 μg/L cut off among pediatric population (up to 18 years of age) was found to have 84% sensitivity in HLH-1994 trial and therefore was included in the HLH 2004 guidelines [15]. Subsequently, pediatricians have revised their ferritin cut off value to ≥10,000 μg/L with a higher sensitivity and near 100% specificity for the diagnosis of HLH [16]. On the contrary, recent reports from adult intensive care units (ICUs) have suggested a lower ferritin cut off value of 3000 to 4000 μg/L with >80% sensitivity and specificity in HLH diagnosis [17]. While hyperferritinemia is not specific to HLH, the same in the clinical context of fever, worsening cytopenia (s), and splenomegaly is highly valuable in the ICUs where sepsis is the major overlapping clinical condition [18]. Recent studies have shown that a high serum soluble interleukin 2 receptor to ferritin ratio is an important biomarker in distinguishing lymphoma associated HLH compared to benign disease associated HLH (8.56 vs. 0.66, respectively, *P* = 0.0004) [19].


*¶ Hemoglobin; <90 g/L (in infants <4 weeks old, <100 g/L); Platelets <100 × 109 /L; Neutrophils <1.0 × 109 /L). ¶¶≥ 500 μg/L.*

*¶¶¶Fasting triglycerides ≥3.0 mmol/L (≥265 mg/dL).*

*≠ ≤1.5 g/L.*

*§In bone marrow aspirate.*

*§§Likely to be dropped as a criteria.*

*ǁ Coagulopathy, hyperbilirubinemia, hypoalbuminemia, hyponatremia, raised lactate dehydrogenase, elevated β2 microglobulin, peripheral blood monocytosis, CSF pleocytosis, etc. √ Included in the criteria. x Not included in the criteria.*

#### **Table 2.**

*Updated diagnostic criteria for HLH [3–6, 14].*

#### **4. Pathophysiology of HLH**

Genetic HLH results due to inability to clear the antigenic stimulus and thus turn off the inflammatory response is what ultimately leads to *cytokine storm* characteristic of HLH. In healthy individuals, viral and tumor antigenic stimuli leads to Th1 mediated cytokine response (IFN-γ, TNF-α, GM-CSF) which in turn, stimulates CTL and NK cells to clear off target cells (viral infected cells, tumor cells, etc.) through release of perforin and granzyme granules at the synaptic site. Perforin is a key cytolytic protein that acts by inserting itself in the membrane of the target cell and creating pores that lead to osmotic lysis of the target cell. The normal production of vesicle granule content requires orchestrated steps of maturation, polarization, docking, fusion, and finally degranulation in the immunological synapse. All the genetic defects described in FHL involve either inadequate levels of perforin itself (FHL2) or improper granule exocytosis (FHL3–5 and immunodeficiency syndromes) (see above in the classification) (**Figure 1**) [2, 20].

**101**

*Hepatic Involvement in Hemophagocytic Lymphohistiocytosis*

Known immunosuppression¶ 0 (no) or 18 (yes)

**Parameters Number of points (criteria for scoring)**

C) 0 (<38.4), 33 (38.4-39.4), or 49 (>39.4)

Number of cytopenia (s)± 0 (1 lineage), 24 (2 lineages), or 34 (3 lineages) Ferritin (μg/L) 0 (< 2000), 35 (2000–6000), or 50 (>6000)

*Hemophagocytic lymphohistiocytosis probability score (HScore) as proposed by Fardet et al. [6].*

Triglyceride (mmoles/L) 0 (<1.5), 44 (1.5–4), or 64 (>4)

Fibrinogen (g/L) 0 (>2.5), or 30 (≤ 2.5) Serum SGOT (IU/L) 0 (< 30), or 19 (≥ 30)

Organomegaly 0 (no), 23 (hepatomegaly or splenomegaly), or 38 (hepatomegaly and splenomegaly

0 (no) or 35 (yes)

*Human immunodeficiency virus or receiving long term immunosuppressive therapy (glucocorticoids, cyclosporine,* 

*, platelet count ≤ 110,000/mm3*

*.*

**5. Pathophysiology of hepatic dysfunction: the cytokine theory**

It is now postulated that hepatic injury/dysfunction HLH is mainly due to cytokine storm which results due to impaired NK/Cytotoxic T lymphocyte function in a *genetically susceptible* individual while triggering factors playing a crucial role. The up regulation of granulocytic monocytic colony stimulating factor receptor on the macrophages along with macrophage proliferation leads to splenohepatomegaly. The macrophage derived IL-2, IFN-γ, and TNF-α mediated inflammation is reported to be predominantly porto-sinusoidal rather than lobular without any significant alteration in lobular architecture; which in turn produces raised transaminases, hepatocyte hemosiderosis; sinusoidal dilatation and congestion, Kupffer cell hyperplasia and hypertrophy producing hemosiderosis and hemophagocytosis. Furthermore, lymphocyte or lymphohistiocyte mediated biliary ductular injury

*Immune response in healthy subjects and uncontrolled, ineffective immune response in patients with genetic* 

*DOI: http://dx.doi.org/10.5772/intechopen.90238*

Temperature (°

aspirate

*azathioprine).*

*¶*

*±*

**Table 3.**

**Figure 1.**

Hemophagocytosis in marrow

*Hb ≤ 92 g/L, total leukocyte count ≤ 5000/mm3*

*HLH. Adopted and modified from Janka GE [2].*

*Hepatic Involvement in Hemophagocytic Lymphohistiocytosis DOI: http://dx.doi.org/10.5772/intechopen.90238*


*Human immunodeficiency virus or receiving long term immunosuppressive therapy (glucocorticoids, cyclosporine, azathioprine).*

*± Hb ≤ 92 g/L, total leukocyte count ≤ 5000/mm3 , platelet count ≤ 110,000/mm3 .*

#### **Table 3.**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

**Diagnostic parameters HLH diagnostic criteria**

Immunosuppression (**Table 3**)

Decreased NK cell activity§§

Increased soluble IL2

Required number of

*§In bone marrow aspirate. §§Likely to be dropped as a criteria.*

 *Included in the criteria. x*

receptor§§

criteria

*¶¶≥ 500 μg/L.*

*¶*

*≠ ≤1.5 g/L.*

*ǁ*

*√*

**Table 2.**

**HLH-1994**

**HLH-1997**

**HLH-2004**

Molecular diagnosis x x √ √ x x

Fever √ √ √ √ √ √ Splenomegaly √ √ √ √ √ √ Cytopenia (s)¶ √ √ √ √ √ √ Hyperferritinemia¶¶ x x √ √ √ √ Hypertriglyceridemia¶¶¶ √ √ √ √ √ √ Hypofibrinogenemia<sup>≠</sup> √ √ √ √ √ √ Hemophagocytosis √ √ √ √ √ √§

Raised SGOT x x x x √ x

Supportive features<sup>ǁ</sup> x x x x √ √

*Coagulopathy, hyperbilirubinemia, hypoalbuminemia, hyponatremia, raised lactate dehydrogenase, elevated β2* 

molecular diagnosis

All All 5/8 or

*Hemoglobin; <90 g/L (in infants <4 weeks old, <100 g/L); Platelets <100 × 109*

 *Not included in the criteria.*

**HLH-2009**

x x x x √ x

x x √ √ x x

x x √ √ x x

2 major or 1 major and 4 minors

HScore (probability score) (**Table-3**)

*/L; Neutrophils <1.0 × 109*

*/L).*

**HLH-2014**

**HLH-2016**

**100**

**4. Pathophysiology of HLH**

*Updated diagnostic criteria for HLH [3–6, 14].*

*¶¶¶Fasting triglycerides ≥3.0 mmol/L (≥265 mg/dL).*

*microglobulin, peripheral blood monocytosis, CSF pleocytosis, etc.*

Genetic HLH results due to inability to clear the antigenic stimulus and thus turn off the inflammatory response is what ultimately leads to *cytokine storm* characteristic of HLH. In healthy individuals, viral and tumor antigenic stimuli leads to Th1 mediated cytokine response (IFN-γ, TNF-α, GM-CSF) which in turn, stimulates CTL and NK cells to clear off target cells (viral infected cells, tumor cells, etc.) through release of perforin and granzyme granules at the synaptic site. Perforin is a key cytolytic protein that acts by inserting itself in the membrane of the target cell and creating pores that lead to osmotic lysis of the target cell. The normal production of vesicle granule content requires orchestrated steps of maturation, polarization, docking, fusion, and finally degranulation in the immunological synapse. All the genetic defects described in FHL involve either inadequate levels of perforin itself (FHL2) or improper granule exocytosis (FHL3–5 and immunodeficiency

syndromes) (see above in the classification) (**Figure 1**) [2, 20].

*Hemophagocytic lymphohistiocytosis probability score (HScore) as proposed by Fardet et al. [6].*

#### **Figure 1.**

*Immune response in healthy subjects and uncontrolled, ineffective immune response in patients with genetic HLH. Adopted and modified from Janka GE [2].*

#### **5. Pathophysiology of hepatic dysfunction: the cytokine theory**

It is now postulated that hepatic injury/dysfunction HLH is mainly due to cytokine storm which results due to impaired NK/Cytotoxic T lymphocyte function in a *genetically susceptible* individual while triggering factors playing a crucial role. The up regulation of granulocytic monocytic colony stimulating factor receptor on the macrophages along with macrophage proliferation leads to splenohepatomegaly. The macrophage derived IL-2, IFN-γ, and TNF-α mediated inflammation is reported to be predominantly porto-sinusoidal rather than lobular without any significant alteration in lobular architecture; which in turn produces raised transaminases, hepatocyte hemosiderosis; sinusoidal dilatation and congestion, Kupffer cell hyperplasia and hypertrophy producing hemosiderosis and hemophagocytosis. Furthermore, lymphocyte or lymphohistiocyte mediated biliary ductular injury

#### **Figure 2.**

*Cytokine basis of HLH associated hepatic dysfunction. GM-CSF; granulocytic monocytic colony stimulating factor, IL; interleukin, IFN-γ; interferon gamma, TNF-α; tumor necrosis factor alpha, DIC; disseminated intravascular coagulation, MODS; multiorgan dysfunction syndrome. Note the parameters from no. 1 to 7 are incorporated in the HLH criteria. The pathophysiologic features assigned A to D are related to cytokine mediated liver parenchymal alteration (see below). Schematic representation summarized from de Kerguenec et al. [9] and Billiau et al. [21].*

and cytokine (IL 1, IL 6, and TNF-α) mediated impaired lipoprotein lipase activity causes cholestasis, hyperbilirubinemia and hypertriglyceridemia. Finally, hyperferritinemia so characteristic of HLH, is nothing but the result of acute phase reaction as well as increased erythrophagocytosis by Kupffer cells. All these cytokine basis of hepatic injury may culminate in severe hepatic functional compromise leading to hypofibrinogenemia, hypoalbuminemia, disseminated intravascular coagulation, and multiorgan dysfunction with a fatal outcome (**Figure 2**).

#### **6. Histology of liver in HLH**

The morphology of liver in HLH is not well characterized because of insufficient biopsy data, late diagnosis, sampling bias (needle biopsy vs. wedge biopsy); and associated triggering factors such as virus associated histological alterations; especially in acquired cases (**Table 4**).

Morphological changes as observed in several large series of liver biopsy specimens have shown relatively well-preserved hepatic parenchyma with a portal and sinusoidal lymphohistiocytic, CD 3+, CD8+, Granzyme B+, and variable perforin+ T cell-rich infiltrate [7–9, 21, 22]. Diverse histological patterns have been described in such cases (**Table 4**): (i) adult type *chronic hepatitis* like characterized by *mild* portal lymphocytic infiltrate with mild bile duct injury and endothelialitis, reported to be so *characteristic* of neonatal/childhood HLH; (ii) *leukemia like* pattern characterized by *extensive* portal, lymphohistiocytic infiltrate expanding the tracts and encroaching upon the lobular periphery blurring the portal limiting plate and infiltrating the sinusoids; (iii) *histiocytic storage disorder-like* pattern characterized by massive infiltration of histiocyte rich infiltrate plugging and distending the sinusoids and venules; (iv) *neonatal giant cell hepatitis-like* pattern characterized by extensive giant cell transformation of hepatocytes with prominent *architectural disarray*; (v) increased hepatic hemosiderosis along with marked hyperferritinemia and features of acute liver failure mimicking *neonatal hemochromatosis*; (vi) post stem cell transplantation *graft* 

**103**

**Sl.no, age, gender**

**C/F as per HLH-2004**

**Architecture disarray (present/absent);**

**Cholestasis/**

**Sinusoid(dilatation/**

**inflammation**

**Kupffer cell (number/ hemosiderosis**

**Hp**

**Venules (endothelialitis)**

**Outcome**

**steatosis**

**Lymphocyte mediated bile duct injury; nature of inflammation**

**Inflammation: location, nature,**

**Pattern, necrosis, hemosiderosis**

> 1. 12d, M

All

Present; Severe, lobular >> portal, *Giant cell hepatitis like*, spotty necrosis

++/x

++; *Giant cell hepatitis like*; CD8/

Yes/LH type

↑/x

+++

Yes

Death

Gr. B/Perforin+ T cell

++/x

+, fibrosis, Perforin+ T cells

Yes/lymphocytic

↑/+

Mild

No

Death

Absent; Moderate to severe, centrilobular hepatitis, *chronic active GVHD*

Present; lobular >> portal, *Giant* 

++/x

++, *Giant cell* 

Yes/LH type

↑/+++

+++

Yes

Death

*hepatitis like*;

CD8+/Perforin−

T cells

*cell hepatitis like*,

increased iron in

hepatocytes (+++)

*mimicking neonatal* 

*hemochromatosis*

Absent; Portal >>

+++

+++, *leukemia* 

Yes/dense lymphocytic

↑/x

+++

Yes, Congestion

*like,* CD8/Gr. B/

Perforin+

lobules; centrilobular

necrosis

4. 25d, - 5. 2m, M

6.2m, F

All

Absent; Portal >>

+/++

+++; *leukemia like;*

Yes/LH type

↑/+

++

Yes

Alive

Perforin+ T cells

lobules

ARF

Absent; Portal >>

+/++

+++; *chronic* 

Yes/lymphocytic

↑/+

+++

Yes

Death

*persistent hepatitis* 

*like*, CD8/Gr B/

Perforin+ T cells

lobules

ALF

3. 16d, M

All

2. 7m, M

All

*Hepatic Involvement in Hemophagocytic Lymphohistiocytosis*

*DOI: http://dx.doi.org/10.5772/intechopen.90238*

#### *Hepatic Involvement in Hemophagocytic Lymphohistiocytosis DOI: http://dx.doi.org/10.5772/intechopen.90238*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

and cytokine (IL 1, IL 6, and TNF-α) mediated impaired lipoprotein lipase activity causes cholestasis, hyperbilirubinemia and hypertriglyceridemia. Finally, hyperferritinemia so characteristic of HLH, is nothing but the result of acute phase reaction as well as increased erythrophagocytosis by Kupffer cells. All these cytokine basis of hepatic injury may culminate in severe hepatic functional compromise leading to hypofibrinogenemia, hypoalbuminemia, disseminated intravascular coagulation,

*Cytokine basis of HLH associated hepatic dysfunction. GM-CSF; granulocytic monocytic colony stimulating factor, IL; interleukin, IFN-γ; interferon gamma, TNF-α; tumor necrosis factor alpha, DIC; disseminated intravascular coagulation, MODS; multiorgan dysfunction syndrome. Note the parameters from no. 1 to 7 are incorporated in the HLH criteria. The pathophysiologic features assigned A to D are related to cytokine mediated liver parenchymal alteration (see below). Schematic representation summarized from de Kerguenec* 

The morphology of liver in HLH is not well characterized because of insufficient biopsy data, late diagnosis, sampling bias (needle biopsy vs. wedge biopsy); and associated triggering factors such as virus associated histological alterations;

Morphological changes as observed in several large series of liver biopsy specimens have shown relatively well-preserved hepatic parenchyma with a portal and sinusoidal lymphohistiocytic, CD 3+, CD8+, Granzyme B+, and variable perforin+ T cell-rich infiltrate [7–9, 21, 22]. Diverse histological patterns have been described in such cases (**Table 4**): (i) adult type *chronic hepatitis* like characterized by *mild* portal lymphocytic infiltrate with mild bile duct injury and endothelialitis, reported to be so *characteristic* of neonatal/childhood HLH; (ii) *leukemia like* pattern characterized by *extensive* portal, lymphohistiocytic infiltrate expanding the tracts and encroaching upon the lobular periphery blurring the portal limiting plate and infiltrating the sinusoids; (iii) *histiocytic storage disorder-like* pattern characterized by massive infiltration of histiocyte rich infiltrate plugging and distending the sinusoids and venules; (iv) *neonatal giant cell hepatitis-like* pattern characterized by extensive giant cell transformation of hepatocytes with prominent *architectural disarray*; (v) increased hepatic hemosiderosis along with marked hyperferritinemia and features of acute liver failure mimicking *neonatal hemochromatosis*; (vi) post stem cell transplantation *graft* 

and multiorgan dysfunction with a fatal outcome (**Figure 2**).

**6. Histology of liver in HLH**

*et al. [9] and Billiau et al. [21].*

**Figure 2.**

especially in acquired cases (**Table 4**).

**102**



**105**

**Sl.no, age, gender**

**C/F as per HLH-2004**

**Architecture disarray (present/absent);**

**Cholestasis/**

**Sinusoid(dilatation/**

**inflammation**

**Kupffer cell (number/ hemosiderosis**

**Hp**

**Venules (endothelialitis)**

**Outcome**

**steatosis**

**Lymphocyte mediated bile duct injury; nature of inflammation**

**Inflammation: location, nature,**

**Pattern, necrosis, hemosiderosis**

> 14. 3m, M

15. 4m, M

16. 8m, F 17. 8m, M

All, HCV

Absent,

++/+

+++; *leukemia* 

Yes/LH type

↑/++

+++

Yes

Lost

to follow-up

*like*, CD8/Gr B+/

Perforin+ T cells

portal>>lobular,

hepatic hemosiderosis

(+++)

18. 9m, M 19. 11m, F

All

Absent,

++/++

+++; *leukemia* 

Yes/LH type

↑/+

+++

Yes

Death

*like*, CD8/Gr B+/

Perforin+ T cells

portal>>lobular

All

Absent,

+/+

++; *chronic* 

Yes/LH type

↑/++

+++

Yes

Death

*persistent hepatitis* 

*like*, CD8/Gr B+/

Perforin+ T cells

portal>>lobular

positive

ALF

Sibling

Absent, Portal>>lobular; chronic hepatitis like

+/x

+++, *Leukemia like*, CD8/Gr B+/

Yes/LH type

↑/x

+++

Yes

Death

Perforin+ T cells

++/++

++, *Storage histiocytic like,*

Yes/histiocytic infiltrate

↑/++

+++

Yes

Death

like storage cells

CD8/Gr B+/

Perforin+ T cells,

perivenous fibrosis

Absent, centrilobular hemorrhage, atrophy of hepatic cords

All

Absent; Portal>>lobular; *chronic hepatitis like*

+/x

++, *chronic persistent hepatitis like*, CD8/Gr B+/

Yes/LH type

↑/x

+++

Yes

Death

Perforin+ T cells

*Hepatic Involvement in Hemophagocytic Lymphohistiocytosis*

*DOI: http://dx.doi.org/10.5772/intechopen.90238*

*Hepatitis A and Other Associated Hepatobiliary Diseases*


#### *Hepatic Involvement in Hemophagocytic Lymphohistiocytosis DOI: http://dx.doi.org/10.5772/intechopen.90238*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

**104**

**Sl.no,**

**C/F as per** 

**Architecture disarray** 

**Cholestasis/**

**Lymphocyte** 

**Sinusoid(dilatation/**

**Kupffer**

**Hp**

**Venules**

**Outcome**

**(endothelialitis)**

**cell**

**(number/**

**hemosiderosis**

**inflammation**

**mediated bile duct** 

**injury; nature of** 

**inflammation**

**steatosis**

**(present/absent);**

**Inflammation:** 

**location, nature,**

**Pattern, necrosis,** 

**hemosiderosis**

Absent; Portal >>

++/x

+++; *leukemia* 

Yes/LH type

↑/+

+

Yes

Death

*like;* CD8/Gr B/

Perforin+ T cells

lobules, hemosiderosis

(+)

8. 3m, M 9. 3m, M 10. 3m, M 11. 3m, M

12. 3m, F 13. 3m, M

All,

Absent;

+/x

+++, *Leukemia* 

Yes/LH type

↑/x

+++

Yes

Death

*like*, CD8/Gr B+/

Perforin+ T cells

Portal>>lobular

consanguinity

All

Present;

+/+

++, *chronic* 

Yes/LH type

↑/x

+++

Yes

*persistent hepatitis* 

*like* CD8/Gr B+/

Perforin+ T cells

Portal>>lobular; *Giant* 

*cell hepatitis like pattern*

All

Present,

+/+

++, CD8/Gr B+/

Yes/LH type

↑/x

+++

Yes

Death

Perforin+ T cells

Lobular>>portal; *Giant* 

*cell hepatitis*

Sibling

Absent;

++/x

+++, *leukemia* 

Yes/LH type

↑/x

+++

ND

Death

*like,* CD8/Gr B+/

Perforin+ T cells

Portal>>lobular

ALF

Absent;

++/+

+++, *leukemia* 

Yes/leukemia like

↑/+

+++

Yes

Death

*like*, CD8/Gr B+/

Perforin+ T cells

Portal>>lobular,

hepatic siderosis (+++)

All

Absent; lobular >>

+++/+

+; *chronic persistent* 

Yes/LH type

↑/++

+

Yes

Death

*hepatitis like*; CD8/

Gr B+/Perforin−

T cells

portal, *chronic hepatitis* 

*like*

7. 2m, M

All

**HLH-2004**

**age,** 

**gender**


### **Table4.**

**107**

*Hepatic Involvement in Hemophagocytic Lymphohistiocytosis*

*versus host disease related* changes; (vii) *lymphocyte depleted* morphology unrelated to prior immunosuppressive or immunomodulator therapy; especially later in the

Common to all specimens and helpful in diagnosing HLH are a constellation of *additional* features that included distinctive lymphocyte-mediated bile duct injury, significant endothelialitis of terminal portal and central veins, sinusoidal congestion and dilatation, increased Kupffer cell activity with or without hemosiderosis, erythrophagocytosis, and histiocytic hemophagocytosis which is reported as inconspicuous to florid. Steatosis and cholestasis were also usually present [8–10]. The *lymphocytemediated bile duct injury* is characterized by nests or circumferential sheaths of lymphomononuclear cells interposed between the epithelium and the basal lamina eliciting *little damage* to the epithelium. The portal inflammation with cholangitis observed in FHL is reminiscent of primary sclerosing cholangitis, primary biliary cirrhosis, and vanishing bile duct syndrome; though neutrophils, plasma cells, granulomatous inflammation, periductal sclerosis, or ductopenia common in latter conditions are reported to be rare in HLH cases [10]. *Endothelialitis* of terminal hepatic and portal veins may result in transmural phlebitis and hemorrhage and extensive apoptosis of perivenular hepatocytes. The degree of inflammation, bile duct damage, endotheliali-

course of the disease or as a part of aberrant cytokine modulation [10].

tis, cholestasis, and steatosis seem to reflect the clinical stage of the disease.

The mortality rate is very high in HLH associated acute liver failure cases. However, this association is extremely rare. Moreover, the presence of two clinical conditions (HLH and acute liver failure) together makes its further complicated and delays the diagnosis. The average time from earliest diagnosis of liver failure to a definitive diagnosis of HLH has been reported to be 17.27 days [23]. This suggests that HLH is a late occurring phenomenon in the process of ALF. On the contrary, there are reports which support the viewpoint of HLH causing liver injury and thus culminating in ALF [24, 25]. The exact mechanism is still not known, as far as HLH induced liver injury is concerned. It is most probably the infiltration of activated macrophage or over production of cytokine in HLH can explain the degree of liver injury. In a clinical scenario, where the patient present with prolonged fever, jaundice and pancytopenia; HLH should be considered as a differential diagnosis [23]. The role of liver transplantation in the treatment of HLH – ALF is controversial. It is so, because of the primarily systemic nature of the disease, the risk of hepatic recurrence of HLH during the post-transplant period, increased in rejection rate and poor general condition of the patient to tolerate the transplant procedure [26]. The post-transplant survival at the end of 6 months is only 33% for the primary HLH – ALF patient [27]. However, a small clinical series involving nine pediatric patients, reported a better survival rate among the secondary HLH – ALF group [26].

In the secondary form of HLH, the liver transplantation is also not very helpful in the situation such as absence of ALF (MELD score < 20–22); when the clinical severity is due to the combined effect of ALF and HLH, rather than ALF alone; and when the HLH is severe and highly likely to be irreversible. In these situations, high mortality from advanced and likely irreversible HLH may limit the benefits of liver transplantation [28]. Liver biopsy should be performed to decide the extent of the liver injury and the role played by the hepatic injury vs. systemic HLH in the patients with ALF. However, liver transplant is still an option in HLH – ALF cases with predominant liver involvement from HLH and this should be undertaken before the highly lethal complication of HLH, such as, septic shock, DIC, bone marrow failure,

explosive immune activation from HLH supervenes.

**7. Liver transplantation: current concept and controversies**

*DOI: http://dx.doi.org/10.5772/intechopen.90238*

 *Histopathology of liver in cases with hemophagocytic lymphohistiocytosis as described in several series (Chen et al. [10], n = 19; Ost et al. [8], n = 27; de Kerguenec et al. [9], n = 30).* *Hepatic Involvement in Hemophagocytic Lymphohistiocytosis DOI: http://dx.doi.org/10.5772/intechopen.90238*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

**106**

**Sl.no,**

**C/F as per** 

**Architecture disarray** 

**Cholestasis/**

**Lymphocyte** 

**Sinusoid(dilatation/**

**Kupffer**

**Hp**

**Venules**

**Outcome**

**(endothelialitis)**

**cell**

**(number/**

**hemosiderosis**

**inflammation**

**mediated bile duct** 

**injury; nature of** 

**inflammation**

**steatosis**

**(present/absent);**

**Inflammation:** 

**location, nature,**

**Pattern, necrosis,** 

**hemosiderosis**

Absent; portal >>>

Not described

++ to +++, *chronic* 

Yes/LH type

↑/x

+

Not studied

Autopsy

series [8]

*persistent hepatitis* 

*like*

*(characteristic)*

27.

1

0HLH

children

30. adults 2

0HLH

Absent; portal >>>

++/++

++ to +++; LH

Yes/LH type,

↑/++

++/+++

Not described

Ref. [9]

erythrophagocytosis

type to tumoral

infiltration,

no ductular

proliferation

or damage or

ductopenia, *no Hp* 

*in portal area*

*Footnotes: d; days, m; months, M; male, F; female, C/F; clinical feature, ALF; acute liver failure, ARF; acute renal failure, GVHD; graft versus host disease, +; mild/inconspicuous, ++; moderate, +++;* 

*Histopathology of liver in cases with hemophagocytic lymphohistiocytosis as described in several series (Chen et al. [10], n = 19; Ost et al. [8], n = 27; de Kerguenec et al. [9], n = 30).*

*marked, x; not present, Gr. B; granzyme B, LH; lymphohistiocytic,* ↑*; increase in number, ND; not described, Hp; hemophagocytosis.*

**Table 4.**

lobular; hepatocyte

necrosis (focal in 10;

diffuse in 4), siderosis

in 11

(19 M, 11 F)

ALF like in

19/29

(11 M, 16 F)

lobular

**HLH-2004**

**age,** 

**gender**

*versus host disease related* changes; (vii) *lymphocyte depleted* morphology unrelated to prior immunosuppressive or immunomodulator therapy; especially later in the course of the disease or as a part of aberrant cytokine modulation [10].

Common to all specimens and helpful in diagnosing HLH are a constellation of *additional* features that included distinctive lymphocyte-mediated bile duct injury, significant endothelialitis of terminal portal and central veins, sinusoidal congestion and dilatation, increased Kupffer cell activity with or without hemosiderosis, erythrophagocytosis, and histiocytic hemophagocytosis which is reported as inconspicuous to florid. Steatosis and cholestasis were also usually present [8–10]. The *lymphocytemediated bile duct injury* is characterized by nests or circumferential sheaths of lymphomononuclear cells interposed between the epithelium and the basal lamina eliciting *little damage* to the epithelium. The portal inflammation with cholangitis observed in FHL is reminiscent of primary sclerosing cholangitis, primary biliary cirrhosis, and vanishing bile duct syndrome; though neutrophils, plasma cells, granulomatous inflammation, periductal sclerosis, or ductopenia common in latter conditions are reported to be rare in HLH cases [10]. *Endothelialitis* of terminal hepatic and portal veins may result in transmural phlebitis and hemorrhage and extensive apoptosis of perivenular hepatocytes. The degree of inflammation, bile duct damage, endothelialitis, cholestasis, and steatosis seem to reflect the clinical stage of the disease.

#### **7. Liver transplantation: current concept and controversies**

The mortality rate is very high in HLH associated acute liver failure cases. However, this association is extremely rare. Moreover, the presence of two clinical conditions (HLH and acute liver failure) together makes its further complicated and delays the diagnosis. The average time from earliest diagnosis of liver failure to a definitive diagnosis of HLH has been reported to be 17.27 days [23]. This suggests that HLH is a late occurring phenomenon in the process of ALF. On the contrary, there are reports which support the viewpoint of HLH causing liver injury and thus culminating in ALF [24, 25]. The exact mechanism is still not known, as far as HLH induced liver injury is concerned. It is most probably the infiltration of activated macrophage or over production of cytokine in HLH can explain the degree of liver injury. In a clinical scenario, where the patient present with prolonged fever, jaundice and pancytopenia; HLH should be considered as a differential diagnosis [23]. The role of liver transplantation in the treatment of HLH – ALF is controversial. It is so, because of the primarily systemic nature of the disease, the risk of hepatic recurrence of HLH during the post-transplant period, increased in rejection rate and poor general condition of the patient to tolerate the transplant procedure [26]. The post-transplant survival at the end of 6 months is only 33% for the primary HLH – ALF patient [27]. However, a small clinical series involving nine pediatric patients, reported a better survival rate among the secondary HLH – ALF group [26].

In the secondary form of HLH, the liver transplantation is also not very helpful in the situation such as absence of ALF (MELD score < 20–22); when the clinical severity is due to the combined effect of ALF and HLH, rather than ALF alone; and when the HLH is severe and highly likely to be irreversible. In these situations, high mortality from advanced and likely irreversible HLH may limit the benefits of liver transplantation [28]. Liver biopsy should be performed to decide the extent of the liver injury and the role played by the hepatic injury vs. systemic HLH in the patients with ALF. However, liver transplant is still an option in HLH – ALF cases with predominant liver involvement from HLH and this should be undertaken before the highly lethal complication of HLH, such as, septic shock, DIC, bone marrow failure, explosive immune activation from HLH supervenes.

#### **Author details**

Somanath Padhi1 \*, RajLaxmi Sarangi2 , Susama Patra1 and Subash Chandra Samal3

1 Department of Pathology with Laboratory Medicine, All India Institute of Medical Sciences, Bhubaneswar, India

2 Department of Biochemistry, Kalinga Institute of Medical Sciences, Bhubaneswar, India

3 Department of Gastroenterology, All India Institute of Medical Sciences, Bhubaneswar, India

\*Address all correspondence to: pathol\_somanath@aiimsbhubaneswar.edu.in

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**109**

art.38690

*Hepatic Involvement in Hemophagocytic Lymphohistiocytosis*

[8] Ost A, Nilsson-Ardnor S, Henter JI. Autopsy findings in 27 children with haemophagocytic lymphohistiocytosis. Histopathology. 1998;**32**:310-316. DOI: 10.1046/j.1365-2559.1998.00377.x

[9] de Kerguenec C, Hillaire S, Molinié V,

[10] Chen JH, Fleming MD, Pinkus GS, Pinkus JL, Nichols KE, Mo JQ, et al. Pathology of the liver in familial hemophagocytic lymphohistiocytosis. The American Journal of Surgical Pathology. 2010;**34**:852-867. DOI: 10.1097/PAS.0b013e3181dbbb17

Hemophagocytic lymphohistiocytosis:

Hematology. 2013;**2013**:605-611. DOI: 10.1182/asheducation-2013.1.605

[11] Janka GE, Lehmberg K.

Pathogenesis and treatment.

[12] Gholam C, Grigoriadou S, Gilmour KC, Gaspar HB. Familial haemophagocytic lymphohistiocytosis:

Advances in the genetic basis, diagnosis and management. Clinical and Experimental

[13] Padhi S, Varghese RG,

Immunology. 2011;**163**:271-283. DOI: 10.1111/j.1365-2249.2010.04302.x

Ramdas A, Phansalkar MD, Sarangi R. Hemophagocytic lymphohistiocytosis: Critical reappraisal of a potentially under-recognized condition. Frontiers in Medicine. 2013;**7**:492-498. DOI: 10.1007/s11684-013-0292-0

[14] Tamamyan GN, Kantarjian HM, Ning J, Jain P, Sasaki K, McClain KL,

hemophagocytic lymphohistiocytosis in adults: Relation to hemophagocytosis, characteristics, and outcomes. Cancer.

et al. Malignancy-associated

Gardin C, Degott C, Erlinger S, et al. Hepatic manifestations of hemophagocytic syndrome: A study of 30 cases. The American Journal of Gastroenterology. 2001;**96**:852-857. DOI: 10.1111/j.1572-0241.2001.03632.x

*DOI: http://dx.doi.org/10.5772/intechopen.90238*

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[2] Janka GE. Familial and acquired hemophagocytic lymphohistiocytosis.

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**108**

India

**Author details**

Somanath Padhi1

Bhubaneswar, India

\*, RajLaxmi Sarangi2

Medical Sciences, Bhubaneswar, India

provided the original work is properly cited.

, Susama Patra1

1 Department of Pathology with Laboratory Medicine, All India Institute of

3 Department of Gastroenterology, All India Institute of Medical Sciences,

\*Address all correspondence to: pathol\_somanath@aiimsbhubaneswar.edu.in

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Department of Biochemistry, Kalinga Institute of Medical Sciences, Bhubaneswar,

and Subash Chandra Samal3

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[18] Sackett K, Cunderlik M, Sahni N, Killeen AA, Olson AP. Extreme hyperferritinemia: Causes and impact on diagnostic reasoning. American Journal of Clinical Pathology. 2016;**145**:646-650. DOI: 10.1093/ajcp/ aqw053

[19] Tsuji T, Hirano T, Yamasaki H, Tsuji M, Tsuda H. A high sIL-2R/ferritin ratio is a useful marker for the diagnosis of lymphoma-associated hemophagocytic syndrome. Annals of Hematology. 2014;**93**:821-826. DOI: 10.1007/s00277-013-1925-8

[20] Janka G. Hemophagocytic lymphohistiocytosis: When the immune system runs amok. Klinische Pädiatrie. 2009;**22**:278-285. DOI: 10.1055/s-0029-1237386

[21] Billiau AD, Roskams T, Van Damme-Lombaerts R, Matthys P, Wouters C. Macrophage activation syndrome: Characteristic findings on liver biopsy illustrating the key role

of activated, IFN-gamma-producing lymphocytes and IL-6- and TNFalpha-producing macrophages. Blood. 2005;**105**:1648-1651. DOI: 10.1182/ blood-2004-08-2997

[22] Kapelari K, Fruehwirth M, Heitger A, Königsrainer A, Margreiter R, Simma B, et al. Loss of intrahepatic bile ducts: An important feature of familial hemophagocytic lymphohistiocytosis. Virchows Archiv. 2005;**446**:619-625. DOI: 10.1007/ s00428-005-1238-y

[23] Dong J, Xie F, Jia L, Li J, Hu Z, Zhu Y, et al. Clinical characteristics of liver failure with hemophagocytic lymphohistiocytosis. Scientific Reports. 2019;**9**:8125. DOI: 10.1038/ s41598-019-43909-w

[24] Jagtap N, Sharma M, Rajesh G, Rao PN, Anuradha S, Tandan M, et al. Hemophagocytic lymphohistiocytosis masquerading as acute liver failure: A single center experience. Journal of Clinical and Experimental Hepatology. 2017;**7**:184-189. DOI: 10.1016/j. jceh.2017.01

[25] Lin S, Li Y, Long J, Liu Q, Yang F, He Y. Acute liver failure caused by hemophagocytic lymphohistiocytosis in adults: A case report and review of the literature. Medicine (Baltimore). 2016;**95**:e5431. DOI: 10.1097/ MD.0000000000005431

[26] Amir AZ, Ling SC, Naqvi A, Weitzman S, Fecteau A, Grant D, et al. Liver transplantation for children with acute liver failure associated with secondary hemophagocytic lymphohistiocytosis. Liver Transplantation. 2016;**22**:1245-1253. DOI: 10.1002/lt.24485

[27] Guthery SL, Heubi JE. Liver involvement in childhood histiocytic syndromes. Current Opinion in Gastroenterology. 2001;**17**:474-478

**111**

*Hepatic Involvement in Hemophagocytic Lymphohistiocytosis*

*DOI: http://dx.doi.org/10.5772/intechopen.90238*

[28] Capel MS, Hader I, Amin M. Acute liver failure secondary to severe systemic disease from fatal hemophagocytic lymphohistiocytosis: Case report and systematic literature review. World Journal of Hepatology. 2018;**10**:629-636.

DOI: 10.4254/wjh.v10.i9.629

*Hepatic Involvement in Hemophagocytic Lymphohistiocytosis DOI: http://dx.doi.org/10.5772/intechopen.90238*

[28] Capel MS, Hader I, Amin M. Acute liver failure secondary to severe systemic disease from fatal hemophagocytic lymphohistiocytosis: Case report and systematic literature review. World Journal of Hepatology. 2018;**10**:629-636. DOI: 10.4254/wjh.v10.i9.629

*Hepatitis A and Other Associated Hepatobiliary Diseases*

of activated, IFN-gamma-producing lymphocytes and IL-6- and TNFalpha-producing macrophages. Blood. 2005;**105**:1648-1651. DOI: 10.1182/

[22] Kapelari K, Fruehwirth M,

Heitger A, Königsrainer A, Margreiter R, Simma B, et al. Loss of intrahepatic bile ducts: An important feature of familial hemophagocytic

lymphohistiocytosis. Virchows Archiv. 2005;**446**:619-625. DOI: 10.1007/

[23] Dong J, Xie F, Jia L, Li J, Hu Z, Zhu Y, et al. Clinical characteristics of liver failure with hemophagocytic lymphohistiocytosis. Scientific Reports. 2019;**9**:8125. DOI: 10.1038/

[24] Jagtap N, Sharma M, Rajesh G, Rao PN, Anuradha S, Tandan M, et al. Hemophagocytic lymphohistiocytosis masquerading as acute liver failure: A single center experience. Journal of Clinical and Experimental Hepatology.

2017;**7**:184-189. DOI: 10.1016/j.

2016;**95**:e5431. DOI: 10.1097/ MD.0000000000005431

[26] Amir AZ, Ling SC, Naqvi A, Weitzman S, Fecteau A, Grant D, et al. Liver transplantation for children with acute liver failure associated with secondary hemophagocytic lymphohistiocytosis. Liver

Transplantation. 2016;**22**:1245-1253.

[27] Guthery SL, Heubi JE. Liver involvement in childhood histiocytic syndromes. Current Opinion in Gastroenterology. 2001;**17**:474-478

DOI: 10.1002/lt.24485

[25] Lin S, Li Y, Long J, Liu Q, Yang F, He Y. Acute liver failure caused by hemophagocytic lymphohistiocytosis in adults: A case report and review of the literature. Medicine (Baltimore).

blood-2004-08-2997

s00428-005-1238-y

s41598-019-43909-w

jceh.2017.01

2016;**122**:2857-2866. DOI: 10.1002/

[15] Allen CE, Yu X, Kozinetz CA, McClain KL. Highly elevated ferritin levels and the diagnosis of hemophagocytic lymphohistiocytosis. Pediatric Blood & Cancer. 2008;**50**:1227-

1235. DOI: 10.1002/pbc.21423

[16] Schram AM, Campigotto F, Mullally A, Fogerty A, Massarotti E,

hyperferritinemia does not predict for HLH in the adult population. Blood. 2015;**125**:1548-1552. DOI: 10.1182/

[17] Lachmann G, Spies C, Schenk T, Brunkhorst FM, Balzer F, La Rosée P, et al. Hemophagocytic lymphohistiocytosis: Potentially underdiagnosed in intensive care units. Shock. 2018;**50**:149-155. DOI: 10.1097/SHK.0000000000001048

Sahni N, Killeen AA, Olson AP. Extreme hyperferritinemia: Causes and impact on diagnostic reasoning. American Journal of Clinical Pathology.

2016;**145**:646-650. DOI: 10.1093/ajcp/

[19] Tsuji T, Hirano T, Yamasaki H, Tsuji M, Tsuda H. A high sIL-2R/ferritin

ratio is a useful marker for the diagnosis of lymphoma-associated hemophagocytic syndrome. Annals of Hematology. 2014;**93**:821-826. DOI:

10.1007/s00277-013-1925-8

10.1055/s-0029-1237386

[20] Janka G. Hemophagocytic lymphohistiocytosis: When the immune system runs amok. Klinische Pädiatrie. 2009;**22**:278-285. DOI:

[21] Billiau AD, Roskams T, Van Damme-Lombaerts R, Matthys P, Wouters C. Macrophage activation syndrome: Characteristic findings on liver biopsy illustrating the key role

Neuberg D, et al. Marked

blood-2014-10-602607

[18] Sackett K, Cunderlik M,

aqw053

cncr.30084

**110**

**113**

**Chapter 8**

**Abstract**

*Sabina Wiecek*

Primary Sclerosing Cholangitis

Primary sclerosing cholangitis (PSC) is a chronic liver disease of unknown aetiology affecting extrahepatic and/or intrahepatic bile ducts causing its inflammation and fibrosis with most frequent consequences including biliary cirrhosis and liver failure. The incidence of PSC in children and adolescents is 0.2 per 100,000 children per year, when in adults the reported incidence is higher and equals 0.5 to 1 in 100,000 individuals per year. PSC is more common among men and boys. The diagnosis is usually established in the second decade of life in the paediatric population with the mean age of diagnosis of 13.8 years. Many studies point out a strong correlation between IBD and PSC, especially ulcerative colitis. The prevalence of IBD among children with PSC diagnosis varies from 60 to 99%; however, the incidence of PSC is about 12% in patients with ulcerative colitis and fluctuates about 2–5% in Crohn's disease diagnosed patients. Clinical symptoms are present in approximately half of cases and they are unspecific in many of them. Elevated liver enzymes and biochemical markers of cholestasis are sometimes the only signs of PSC. Gold standard for PSC diagnosis is magnetic resonance cholangiopancreatography (MRCP) as a non-invasive procedure comparing to endoscopic retrograde cholangiopancreatography (ERCP) which is also used in some cases. The aim of the study was to review the risk factors, clinical symptoms, diagnostic methods and

treatment of paediatric patients with primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic liver disease of unknown aetiology affecting extrahepatic and/or intrahepatic bile ducts causing its inflammation and fibrosis with most frequent consequences including biliary cirrhosis and liver failure. The incidence of PSC in children and adolescents is 0.2 per 100,000 children per year when in adults the reported incidence is higher and equals 0.5 to 1 in 100,000 individuals per year. Several studies indicate the incidence of primary sclerosing cholangitis is increasing. A similar increase has been seen in most autoimmune diseases. PSC is more common among men and boys. The diagnosis is usually established in the second decade of life in the paediatric population with the

Many studies point out a strong correlation between IBD and PSC, especially ulcerative colitis. The prevalence of IBD among children with PSC diagnosis varies from 60 to 99%, however the incidence of PSC is about 12% in patients with ulcerative colitis and fluctuates about 2 to 5% in Crohn's disease diagnosed patients. In children, the diagnosis of IBD generally precedes the diagnosis of PSC [10–15].

**Keywords:** primary sclerosing cholangitis, children

mean age of diagnosis of 13.8 years [1–9].

(PSC) in Children

#### **Chapter 8**

## Primary Sclerosing Cholangitis (PSC) in Children

*Sabina Wiecek*

#### **Abstract**

Primary sclerosing cholangitis (PSC) is a chronic liver disease of unknown aetiology affecting extrahepatic and/or intrahepatic bile ducts causing its inflammation and fibrosis with most frequent consequences including biliary cirrhosis and liver failure. The incidence of PSC in children and adolescents is 0.2 per 100,000 children per year, when in adults the reported incidence is higher and equals 0.5 to 1 in 100,000 individuals per year. PSC is more common among men and boys. The diagnosis is usually established in the second decade of life in the paediatric population with the mean age of diagnosis of 13.8 years. Many studies point out a strong correlation between IBD and PSC, especially ulcerative colitis. The prevalence of IBD among children with PSC diagnosis varies from 60 to 99%; however, the incidence of PSC is about 12% in patients with ulcerative colitis and fluctuates about 2–5% in Crohn's disease diagnosed patients. Clinical symptoms are present in approximately half of cases and they are unspecific in many of them. Elevated liver enzymes and biochemical markers of cholestasis are sometimes the only signs of PSC. Gold standard for PSC diagnosis is magnetic resonance cholangiopancreatography (MRCP) as a non-invasive procedure comparing to endoscopic retrograde cholangiopancreatography (ERCP) which is also used in some cases. The aim of the study was to review the risk factors, clinical symptoms, diagnostic methods and treatment of paediatric patients with primary sclerosing cholangitis.

**Keywords:** primary sclerosing cholangitis, children

Primary sclerosing cholangitis (PSC) is a chronic liver disease of unknown aetiology affecting extrahepatic and/or intrahepatic bile ducts causing its inflammation and fibrosis with most frequent consequences including biliary cirrhosis and liver failure. The incidence of PSC in children and adolescents is 0.2 per 100,000 children per year when in adults the reported incidence is higher and equals 0.5 to 1 in 100,000 individuals per year. Several studies indicate the incidence of primary sclerosing cholangitis is increasing. A similar increase has been seen in most autoimmune diseases. PSC is more common among men and boys. The diagnosis is usually established in the second decade of life in the paediatric population with the mean age of diagnosis of 13.8 years [1–9].

Many studies point out a strong correlation between IBD and PSC, especially ulcerative colitis. The prevalence of IBD among children with PSC diagnosis varies from 60 to 99%, however the incidence of PSC is about 12% in patients with ulcerative colitis and fluctuates about 2 to 5% in Crohn's disease diagnosed patients. In children, the diagnosis of IBD generally precedes the diagnosis of PSC [10–15].

### **1. Aetiology**

The pathogenesis of PSC is unknown, but a number of mechanistic theories have been proposed. Despite the lack of scientifically proven aetiological factors, many components can be responsible for the PSC development.

#### **1.1 Genetic background**

Genetic background including an impact of HLA-A1, B8, DR3 haplotypes are one of the suspects as the diagnosis is made at a young age and family occurrence has been reported. Genome-wide comparisons of the frequency of genetic variants have provided a means of dissecting genetic risk in the many human diseases primary sclerosing cholangitis included. In the pathogenesis of PSC can play the role the presence of more non-HLA genes related to immunity and/or bile homeostasis. However most PSC genes appear to relate to adaptive immune reactions. There are limited genetic links between IBD and PSC. The HLA class 1 (expressed on all cells) and HLA class II (expressed on antigen- presenting cells) present potentially antigenic peptides derived from intra- and extracellular sources, to the T cell receptor (TCR) on CD\* and CD4 T cells. But the antigenic peptides are unknown. Data suggest the presence of PSC specific TCR in the livers of patients. The predominant cell type in the portal inflammatory infiltrate in liver patients with primary sclerosing cholangitis is the T cell. It is suggested that there is cross-reaction between cholangiocytes and T-cells. Some of scientists believe that genes as PRDX5, TGR5, PSMG1, NFKB1 may play a role in innate immune reactions [11, 16–18].

#### **1.2 Bile acids toxicity**

The concentric fibrosis around the bile ducts in PSC is found in a variety of conditions and likely represents a final pathway for bile ducts injury. Defects of mechanisms protecting against bile acid toxicity can be a factor playing an important role in PSC development. The biliary epithelium shows an activated phenotype in PSC, including an expansion of the peribiliary gland system [19–21].

#### **1.3 Autoimmunologic factors**

What is more, certain autoimmune reactions in genetically susceptible individuals seem to play an important role as well. The presence of non-specific autoantibodies such as ANA, ANCA (in >80% of patients) and anti-SMA (in >60% of patients) together with autoimmune diseases such as autoimmune hepatitis (overlap syndrome PSC/AIH in 25–35%), rheumatoid arthritis, autoimmune thyroiditis or type 1 diabetes mellitus suggests that PSC can be described as an autoimmune disease. However its prevalence among men (2:1) and the lack of response to immunosuppressive therapy contributed to the concept of PSC being rather an immunemediated disease. PSC with high immunoglobulin 4 (IgG4) levels and autoimmune hepatitis overlap syndrome have been described. But the lack of the efficacy of immunosuppressive treatment despite isolated autoimmune aetiology [11, 22–25].

#### **1.4 Role of microbiota**

The predominant coexistence of PSC and IBD led to a theory that dysregulation of gut microbiota in IBD patients causes liver T-cell activation provoking an inflammatory response in bile ducts. There is increasing appreciation of the co-metabolic functions of the gut microbiota in the bile homeostasis. The composition of the gut

**115**

*Primary Sclerosing Cholangitis (PSC) in Children DOI: http://dx.doi.org/10.5772/intechopen.90714*

often in patients [2, 8, 17, 22, 29, 30].

1.**Classical large-duct PSC**.

part of the biliary disease.

**3. Diagnostics**

5.**PSC with cholangiocarcinoma**.

and small -duct PSC in the presence of IBD.

**2. Clinical picture**

**2.1 Forms of PSC**

independent from clinical manifestations [26–29].

microbiota in PSC has been described using sequencing technologies. However, data from other diseases suggests that reduced bacterial diversity occurs prior to and

Clinical symptoms are present in approximately half of cases and they are unspecific in many of them. Most frequently patients complain of abdominal pain, fatigue and/or abdominal pain. Malaise, jaundice, splenomegaly or pruritus are reported less often. Elevated liver enzymes and biochemical markers of cholestasis are sometimes the only signs of PSC. The diagnosis of PSC may precede that of IBD, which may even present after liver transplantation for PSC. PSC may present in an IBD patients even after colectomy. Multiple gallbladder abnormalities in the course of primary sclerosing cholangitis including: dilatation (15%), gallstones (25%), cholecystitis, hydrops, polyps (4–6%), carcinoma (2.5–3.5%) are observed more

2.**Small-duct PSC**. A diagnosis of small-duct PSC is made upon histological findings characteristic of PSC and clinical and biochemical abnormalities suggestive of PSC. The HLA associations with IBD in small-duct PSC resemble those of large-duct PSC and suggest shared aetiologies between large-duct PSC

3.**PSC with high IgG4**. PSC patients with elevated IgG4 are less responsive and data suggest they may progress more rapidly than other PSC patients. IgG4 may be involved in the pathogenesis of autoimmune cholangitis and clinical

4.**PSC-AIH overlap syndrome**. Biochemical and histological features of autoimmune hepatitis are apparent in 7–14% of patients with PSC. Elevated transaminases and IgG may indicate autoimmune hepatitis, but may be elevated as a

The diagnosis is based on laboratory and imaging results as well as on elimination of other than PSC cholestatic diseases. When it comes to laboratory results among children, gamma-glutamyltransferase (GGT) is more specific cholestatic marker than alkaline phosphatase (ALP) as ALP levels tend to fluctuate during bone growth. Even though ultrasound (USG) is a cheap and simple way to visualise liver pathology, bile ducts abnormalities characteristic of PSC might not be visible. Gold standard for PSC diagnosis is magnetic resonance cholangiopancreatography (MRCP) with acceptable sensitivity and specificity as a non-invasive procedure comparing to endoscopic retrograde cholangiopancreatography (ERCP) which is also used in some cases. As typical cholangiographic changes define the diagnosis

response upon treatment with the anti-CD20 antibody (rituximab).

microbiota in PSC has been described using sequencing technologies. However, data from other diseases suggests that reduced bacterial diversity occurs prior to and independent from clinical manifestations [26–29].

### **2. Clinical picture**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

components can be responsible for the PSC development.

The pathogenesis of PSC is unknown, but a number of mechanistic theories have been proposed. Despite the lack of scientifically proven aetiological factors, many

Genetic background including an impact of HLA-A1, B8, DR3 haplotypes are one of the suspects as the diagnosis is made at a young age and family occurrence has been reported. Genome-wide comparisons of the frequency of genetic variants have provided a means of dissecting genetic risk in the many human diseases primary sclerosing cholangitis included. In the pathogenesis of PSC can play the role the presence of more non-HLA genes related to immunity and/or bile homeostasis. However most PSC genes appear to relate to adaptive immune reactions. There are limited genetic links between IBD and PSC. The HLA class 1 (expressed on all cells) and HLA class II (expressed on antigen- presenting cells) present potentially antigenic peptides derived from intra- and extracellular sources, to the T cell receptor (TCR) on CD\* and CD4 T cells. But the antigenic peptides are unknown. Data suggest the presence of PSC specific TCR in the livers of patients. The predominant cell type in the portal inflammatory infiltrate in liver patients with primary sclerosing cholangitis is the T cell. It is suggested that there is cross-reaction between cholangiocytes and T-cells. Some of scientists believe that genes as PRDX5, TGR5,

PSMG1, NFKB1 may play a role in innate immune reactions [11, 16–18].

in PSC, including an expansion of the peribiliary gland system [19–21].

The concentric fibrosis around the bile ducts in PSC is found in a variety of conditions and likely represents a final pathway for bile ducts injury. Defects of mechanisms protecting against bile acid toxicity can be a factor playing an important role in PSC development. The biliary epithelium shows an activated phenotype

What is more, certain autoimmune reactions in genetically susceptible individu-

The predominant coexistence of PSC and IBD led to a theory that dysregulation of gut microbiota in IBD patients causes liver T-cell activation provoking an inflammatory response in bile ducts. There is increasing appreciation of the co-metabolic functions of the gut microbiota in the bile homeostasis. The composition of the gut

als seem to play an important role as well. The presence of non-specific autoantibodies such as ANA, ANCA (in >80% of patients) and anti-SMA (in >60% of patients) together with autoimmune diseases such as autoimmune hepatitis (overlap syndrome PSC/AIH in 25–35%), rheumatoid arthritis, autoimmune thyroiditis or type 1 diabetes mellitus suggests that PSC can be described as an autoimmune disease. However its prevalence among men (2:1) and the lack of response to immunosuppressive therapy contributed to the concept of PSC being rather an immunemediated disease. PSC with high immunoglobulin 4 (IgG4) levels and autoimmune hepatitis overlap syndrome have been described. But the lack of the efficacy of immunosuppressive treatment despite isolated autoimmune aetiology [11, 22–25].

**1. Aetiology**

**1.1 Genetic background**

**1.2 Bile acids toxicity**

**1.3 Autoimmunologic factors**

**1.4 Role of microbiota**

**114**

Clinical symptoms are present in approximately half of cases and they are unspecific in many of them. Most frequently patients complain of abdominal pain, fatigue and/or abdominal pain. Malaise, jaundice, splenomegaly or pruritus are reported less often. Elevated liver enzymes and biochemical markers of cholestasis are sometimes the only signs of PSC. The diagnosis of PSC may precede that of IBD, which may even present after liver transplantation for PSC. PSC may present in an IBD patients even after colectomy. Multiple gallbladder abnormalities in the course of primary sclerosing cholangitis including: dilatation (15%), gallstones (25%), cholecystitis, hydrops, polyps (4–6%), carcinoma (2.5–3.5%) are observed more often in patients [2, 8, 17, 22, 29, 30].

### **2.1 Forms of PSC**

### 1.**Classical large-duct PSC**.


### **3. Diagnostics**

The diagnosis is based on laboratory and imaging results as well as on elimination of other than PSC cholestatic diseases. When it comes to laboratory results among children, gamma-glutamyltransferase (GGT) is more specific cholestatic marker than alkaline phosphatase (ALP) as ALP levels tend to fluctuate during bone growth. Even though ultrasound (USG) is a cheap and simple way to visualise liver pathology, bile ducts abnormalities characteristic of PSC might not be visible. Gold standard for PSC diagnosis is magnetic resonance cholangiopancreatography (MRCP) with acceptable sensitivity and specificity as a non-invasive procedure comparing to endoscopic retrograde cholangiopancreatography (ERCP) which is also used in some cases. As typical cholangiographic changes define the diagnosis

#### *Hepatitis A and Other Associated Hepatobiliary Diseases*

of PSC, prognostic scoring system. To diagnose small duct PSC which is a type of PSC affecting intrahepatic ducts only, as well as to confirm the presence of PSC/ AIH overlap syndrome it is necessary to perform liver histology. In recent years, there has been interest in the development of noninvasive tests of liver fibrosis for stratification and prognosis in PSC. Serum tests of liver fibrosis reflect fibrogenesis (APRI, Fib4 score). Liver stiffness measurement by transient elastography has been validated for the assessment of liver fibrosis in the liver diseases. Elastography in patients with PSC well correlate with the degree of fibrosis, performing best at the extremes of histological stage [5, 11, 31–34].

### **3.1 Differential diagnosis of PSC**


#### **3.2 Patients with PSC need control every 6 months**


### **4. Prognosis and complications**

PSC is a progressive disease where bile ducts fibrosis lead to cirrhosis and liver failure. PSC has a highly variable natural history. Asymptomatic patients have been shown to have a better prognosis than patients with symptoms at diagnosis. Comparing to adults, PSC in children seems milder, yet 15–45% of paediatric patients will require liver transplantation within 6–12 years after the diagnosis. The increased risk of biliary cancer and colorectal cancer in PSC is firmly established and of major clinical importance. The risk of cholangiocarcinoma (CCA) is about 160 times higher than in the general population. In spite of that, only 1% of patients experience this serious complication. In a multi-centre study of 7000 PSC patients hepatobiliary malignancy was diagnosed in 10.9%. Up to 50% of

**117**

*Primary Sclerosing Cholangitis (PSC) in Children DOI: http://dx.doi.org/10.5772/intechopen.90714*

ing and photodynamic therapy.

of the patients [2, 35–46].

**5.1 Ursodeoxycholic acid**

**5. Treatment**

cholangiocarcinoma are detected within a year of PSC diagnosis. Unfortunately, PSC diagnosed children may also develop other types of cancers such as gallbladder, colon or hepatocellular cancer. In the majority of cases, the early stages of cholangiocarcinoma are asymptomatic. Sometimes are observed abdominal pain, weight loss, increasing jaundice. Diagnosis of cholangiocarcinoma is based on tumour marker Ca19-9, imaging modalities, biliary brush cytology. The indication for liver transplantation in patients with dysplasia and no signs of cholangiocarcinoma remain controversial. Presence of dysplasia of any grade has been reported in 83% of explant livers with PSC-cholangiocarcinoma and 36% without cholangiocarcinoma. MRI and CT may visualise early features of cholangiocarcinoma in PSC but difficulties in distinguishing inflammatory, bening and malignant lesions lead to suboptimal diagnostic accuracy. Combined MRI/cholangioMRI has the highest sensitivity and specificity and is preferred for detection of small focus cholangiocarcinoma. Liver transplantation or surgery with complete resection is the only treatments with curative intent for cholangiocarcinoma. Liver transplantation with neoadjuvant therapy (external beam radiotherapy, endoluminal brachytherapy, chemotherapy) can be considered in patients with unresectable, perihilar early stage. Systemic chemotherapy remains the palliative treatment for patients not eligible for surgery. Other palliative treatment strategies include endoscopic stent-

PSC-IBD whether considered UC or Crohn's disease is almost universally colonic (usually a pancolitis) with a right-sided predominance, backwash ileitis and rectal sparing. The risk of colorectal cancer is fivefold higher than in IBD without PSC and may occur at any time from diagnosis. Colonoscopy should be performed in patients with PSC regularly from the moment of diagnosis. Chromoendoscopy is being increasingly recommended to facilitate detection of flat lesions with dysplasia. Four quadrant biopsies from all colonic segments and the terminal ileum should be performed. Hepatocellular and pancreatic cancer also occur in patients with PSC, but frequencies are lower than in cirrhosis liver from other causes. Currently there are no established prognostic tools that reliably estimate prognosis

Effective ways of PSC treatment are still lacking. Immunosuppressive medications did not show any benefits, while oral vancomycin therapy might be an option although more data is required. Symptomatic treatment of PSC also consists of supplementing the deficiencies of fat-soluble vitamins, preventing the development of osteoporosis and combating chronic itching: by using additional cholestyramine at a dose of 6–8 g/24 h and/or rifampicin. For patients refractory to the abovementioned treatment, oral naloxone therapy (50 mg/24 h) may be effective.

Ursodeoxycholic acid (UDCA) is commonly used and has been proved to reduce

GTP and AP levels which are both good prognostic factors improving patients' survival. However, UDCA treatment did not result in improved outcomes compared to no intervention. New therapeutic applications have been derived from this research in the form of norUDCA, to enhance general resistance to bile acid induced biliary injury. NorUDCA is slightly amidated in the liver, it is secreted into the bile in both unbound and glucuronic acid form. The biliary-hepatic flow of unbound norUDCA induces excessive secretion of bile rich in bicarbonate. Studies in mouse

#### *Primary Sclerosing Cholangitis (PSC) in Children DOI: http://dx.doi.org/10.5772/intechopen.90714*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

extremes of histological stage [5, 11, 31–34].

2.Congenital abnormalities of bile duct.

4.Traumatic/ischemic changes in bile ducts.

**3.2 Patients with PSC need control every 6 months**

PSC is a progressive disease where bile ducts fibrosis lead to cirrhosis and liver failure. PSC has a highly variable natural history. Asymptomatic patients have been shown to have a better prognosis than patients with symptoms at diagnosis. Comparing to adults, PSC in children seems milder, yet 15–45% of paediatric patients will require liver transplantation within 6–12 years after the diagnosis. The increased risk of biliary cancer and colorectal cancer in PSC is firmly established and of major clinical importance. The risk of cholangiocarcinoma (CCA) is about 160 times higher than in the general population. In spite of that, only 1% of patients experience this serious complication. In a multi-centre study of 7000 PSC patients hepatobiliary malignancy was diagnosed in 10.9%. Up to 50% of

3.Cholangiocarcinoma without PSC.

6.Infestation (ascaris, lambliosis)

• Tumour marker: Ca 19-9, AFP

• Ultrasonography examination

**4. Prognosis and complications**

• MRI/MRC if cirrhosis

**3.1 Differential diagnosis of PSC**

1.Choledocholithiasis.

5.HIV infection.

7.Sarcoidosis.

• Clinical review

• Serum liver tests

8.Pyogenic cholangitis

of PSC, prognostic scoring system. To diagnose small duct PSC which is a type of PSC affecting intrahepatic ducts only, as well as to confirm the presence of PSC/ AIH overlap syndrome it is necessary to perform liver histology. In recent years, there has been interest in the development of noninvasive tests of liver fibrosis for stratification and prognosis in PSC. Serum tests of liver fibrosis reflect fibrogenesis (APRI, Fib4 score). Liver stiffness measurement by transient elastography has been validated for the assessment of liver fibrosis in the liver diseases. Elastography in patients with PSC well correlate with the degree of fibrosis, performing best at the

**116**

cholangiocarcinoma are detected within a year of PSC diagnosis. Unfortunately, PSC diagnosed children may also develop other types of cancers such as gallbladder, colon or hepatocellular cancer. In the majority of cases, the early stages of cholangiocarcinoma are asymptomatic. Sometimes are observed abdominal pain, weight loss, increasing jaundice. Diagnosis of cholangiocarcinoma is based on tumour marker Ca19-9, imaging modalities, biliary brush cytology. The indication for liver transplantation in patients with dysplasia and no signs of cholangiocarcinoma remain controversial. Presence of dysplasia of any grade has been reported in 83% of explant livers with PSC-cholangiocarcinoma and 36% without cholangiocarcinoma. MRI and CT may visualise early features of cholangiocarcinoma in PSC but difficulties in distinguishing inflammatory, bening and malignant lesions lead to suboptimal diagnostic accuracy. Combined MRI/cholangioMRI has the highest sensitivity and specificity and is preferred for detection of small focus cholangiocarcinoma. Liver transplantation or surgery with complete resection is the only treatments with curative intent for cholangiocarcinoma. Liver transplantation with neoadjuvant therapy (external beam radiotherapy, endoluminal brachytherapy, chemotherapy) can be considered in patients with unresectable, perihilar early stage. Systemic chemotherapy remains the palliative treatment for patients not eligible for surgery. Other palliative treatment strategies include endoscopic stenting and photodynamic therapy.

PSC-IBD whether considered UC or Crohn's disease is almost universally colonic (usually a pancolitis) with a right-sided predominance, backwash ileitis and rectal sparing. The risk of colorectal cancer is fivefold higher than in IBD without PSC and may occur at any time from diagnosis. Colonoscopy should be performed in patients with PSC regularly from the moment of diagnosis. Chromoendoscopy is being increasingly recommended to facilitate detection of flat lesions with dysplasia. Four quadrant biopsies from all colonic segments and the terminal ileum should be performed. Hepatocellular and pancreatic cancer also occur in patients with PSC, but frequencies are lower than in cirrhosis liver from other causes. Currently there are no established prognostic tools that reliably estimate prognosis of the patients [2, 35–46].

#### **5. Treatment**

Effective ways of PSC treatment are still lacking. Immunosuppressive medications did not show any benefits, while oral vancomycin therapy might be an option although more data is required. Symptomatic treatment of PSC also consists of supplementing the deficiencies of fat-soluble vitamins, preventing the development of osteoporosis and combating chronic itching: by using additional cholestyramine at a dose of 6–8 g/24 h and/or rifampicin. For patients refractory to the abovementioned treatment, oral naloxone therapy (50 mg/24 h) may be effective.

#### **5.1 Ursodeoxycholic acid**

Ursodeoxycholic acid (UDCA) is commonly used and has been proved to reduce GTP and AP levels which are both good prognostic factors improving patients' survival. However, UDCA treatment did not result in improved outcomes compared to no intervention. New therapeutic applications have been derived from this research in the form of norUDCA, to enhance general resistance to bile acid induced biliary injury. NorUDCA is slightly amidated in the liver, it is secreted into the bile in both unbound and glucuronic acid form. The biliary-hepatic flow of unbound norUDCA induces excessive secretion of bile rich in bicarbonate. Studies in mouse

models have shown that the drug is less toxic, more effectively prevents peripheral fibrosis, proliferation of hepatocytes and cholangiocytes, reduces the content of hydroxyproline and infiltrating immune cells. In addition, it improves cholestasis parameters [6, 31, 47–48].

#### **5.2 Vankomycin**

Antibiotics, particularly vancomycin, may have a positive effect on PSC either via direct effects on the microbiome or via host-mediated mechanisms. In addition vancomycin has possible immunomodulatory and anti-inflammatory mechanisms, But there is not currently sufficient evidence to support treatment recommendations. Further research is needed to establish if vancomycin is a PSC treatment [48–50].

#### **5.3 ERCP**

Bile duct strictures are possible complications in the course of the disease that can be treated with prothesis during ERCP. The generally accepted arbitrary definition is stenosis of <1.5 mm in the common bile duct or <1 mmin the hepatic duct within 2 cm of the hilum. The incidence of complications associated with ERCP in patients with PSC is 4–18% [44, 46, 48].

#### **5.4 Liver transplantation**

Liver transplantation is a life-saving procedure with generally good outcomes, however, up to 16% of paediatric patients are affected by recurrent primary sclerosing cholangitis (rPSC) after transplantation. The indications for liver transplantation in PSC are similar to other liver diseases and transplanted with a qualifying MELD/PELD score in a patient with cirrhosis [11, 29, 51].

#### **5.5 Treatment of bacterial cholangitis**

Cholangitis occurs frequently but symptoms may be atypical. Prophylactic antibiotics should be ordered prior to and following biliary interventions. Positive bacterial or fungal cultures of bile can be associated with worse prognosis. Sometimes patients with recurrent cholangitis require long-term, rotating antibiotics.

### **Author details**

Sabina Wiecek Department of Paediatrics, Medical University of Silesia, Katowice, Poland

\*Address all correspondence to: sabinawk@wp.pl

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**119**

*Primary Sclerosing Cholangitis (PSC) in Children DOI: http://dx.doi.org/10.5772/intechopen.90714*

Primary sclerosing cholangitis in children versus adults: Lessons for the clinic. Expert Review of Gastroenterology &

[9] Valentino PL, Wiggins S, Harney S, et al. The natural history of primary sclerosing cholangitis in children: A large single-center longitudinal cohort study. Journal of Pediatric Gastroenterology and Nutrition.

[10] de Vries AB, Janse M, Blokzijl H, et al. Distinctive inflammatory bowel

sclerosing cholangitis. World Journal of Gastroenterology. 2015;**21**:1956-1971

[11] Karlsen T, Melum E, Franke A, et al. The utility of genome-wide association studies in hepatology. Hepatology.

[13] Mertz A, Nguyen NA, Katsanos KH, et al. Primary sclerosing cholangitis and inflammatory bowel disease comorbidity: An update of the

evidence. Annals of Gastroenterology.

[14] Naess S, Bjornsson E, Anmarkrud J, et al. Small-duct primary sclerosing cholangitis without inflammatory bowel disease is genetically different from large duct disese. Liver International.

[15] Palmela C, Peerani F, Castaneda D, et al. Inflammatory bowel disease and primary sclerosing cholangitis: A review of the phenotype and associated

Thorburn D, et al. Primary sclerosing

specific features. Gut and Liver.

[16] Karlsen TH, Folseraas T,

cholangitis a comprehensive review. Journal of Hepatology.

2017;**67**(6):1298-1323

disease phenotype in primary

[12] Loftus EV Jr, Harewood GC, Loftus CG, et al. PSC-IBD: A unique form of inflammatory bowel disease associated with primary sclerosing cholangitis. Gut. 2005;**54**(1):91-96

2016;**63**(6):603-609

2010;**51**:1833-1842

2019;**32**(2):124-133

2014;**34**:1488-1495

2018;**12**(1):17-29

[1] Adike A, Carey EJ, Lindor KD.

Hepatology. 2018;**12**:1025-1032

[2] Deneau M, Jensen MK, Holmen J, et al. Primary sclerosing cholangitis, autoimmune hepatitis, and overlap in Utah children: Epidemiology and natural history. Hepatology. 2013;**58**:

[3] Fagundes E, Ferreira A, Hosken C, et al. Primary sclerosing cholangitis in children and adolescent. Arquivos de Gastroenterologia. 2017;**54**:286-290

[4] Jankowska I. Pierwotne stwardniające

zapalenie dróg żółciowych. In: Pediatria I, editor. Redakcja naukowa Wanda Kawalec, Ryszard Grenda, Marek Kulus. Warszawa: PZWL Wydawnictwo Lekarskie; 2018. p. 575

[5] Lindor KD, Kowdley KV, Harrison ME. American college of gastroenterology. ACG clinical guideline: Primary sclerosing

cholangitis. The American Journal of Gastroenterology. 2015;**110**:646-659

[7] Miloh T, Arnon R, Shneider B, et al. A retrospective single-center review of primary sclerosing cholangitis in children. Clinical Gastroenterology and

[8] Weismuller T, Trivedy P, Bergquist A, et al. Patient age, sex and inflammatory bowel disease phenotypeassociate with course of primary sclerosing cholangitis. Gastroenterology. 2017;**152**:1975-1984

Hepatology. 2009;**7**:239-245

[6] Mieli-Vergani G, Vergani D, Baumann U, et al. Diagnosis and management of paediatric autoimmune liver disease: ESPGHAN Hepatology committee position statement. Journal of Pediatric Gastroenterology and Nutrition. 2018;**66**:345-360

**References**

1392-1400

*Primary Sclerosing Cholangitis (PSC) in Children DOI: http://dx.doi.org/10.5772/intechopen.90714*

#### **References**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

patients with PSC is 4–18% [44, 46, 48].

**5.5 Treatment of bacterial cholangitis**

\*Address all correspondence to: sabinawk@wp.pl

provided the original work is properly cited.

MELD/PELD score in a patient with cirrhosis [11, 29, 51].

**5.4 Liver transplantation**

parameters [6, 31, 47–48].

**5.2 Vankomycin**

**5.3 ERCP**

models have shown that the drug is less toxic, more effectively prevents peripheral fibrosis, proliferation of hepatocytes and cholangiocytes, reduces the content of hydroxyproline and infiltrating immune cells. In addition, it improves cholestasis

Antibiotics, particularly vancomycin, may have a positive effect on PSC either via direct effects on the microbiome or via host-mediated mechanisms. In addition vancomycin has possible immunomodulatory and anti-inflammatory mechanisms, But there is not currently sufficient evidence to support treatment recommendations. Further research is needed to establish if vancomycin is a PSC treatment [48–50].

Bile duct strictures are possible complications in the course of the disease that can be treated with prothesis during ERCP. The generally accepted arbitrary definition is stenosis of <1.5 mm in the common bile duct or <1 mmin the hepatic duct within 2 cm of the hilum. The incidence of complications associated with ERCP in

Liver transplantation is a life-saving procedure with generally good outcomes, however, up to 16% of paediatric patients are affected by recurrent primary sclerosing cholangitis (rPSC) after transplantation. The indications for liver transplantation in PSC are similar to other liver diseases and transplanted with a qualifying

Cholangitis occurs frequently but symptoms may be atypical. Prophylactic antibiotics should be ordered prior to and following biliary interventions. Positive bacterial or fungal cultures of bile can be associated with worse prognosis. Sometimes

patients with recurrent cholangitis require long-term, rotating antibiotics.

Department of Paediatrics, Medical University of Silesia, Katowice, Poland

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

**118**

**Author details**

Sabina Wiecek

[1] Adike A, Carey EJ, Lindor KD. Primary sclerosing cholangitis in children versus adults: Lessons for the clinic. Expert Review of Gastroenterology & Hepatology. 2018;**12**:1025-1032

[2] Deneau M, Jensen MK, Holmen J, et al. Primary sclerosing cholangitis, autoimmune hepatitis, and overlap in Utah children: Epidemiology and natural history. Hepatology. 2013;**58**: 1392-1400

[3] Fagundes E, Ferreira A, Hosken C, et al. Primary sclerosing cholangitis in children and adolescent. Arquivos de Gastroenterologia. 2017;**54**:286-290

[4] Jankowska I. Pierwotne stwardniające zapalenie dróg żółciowych. In: Pediatria I, editor. Redakcja naukowa Wanda Kawalec, Ryszard Grenda, Marek Kulus. Warszawa: PZWL Wydawnictwo Lekarskie; 2018. p. 575

[5] Lindor KD, Kowdley KV, Harrison ME. American college of gastroenterology. ACG clinical guideline: Primary sclerosing cholangitis. The American Journal of Gastroenterology. 2015;**110**:646-659

[6] Mieli-Vergani G, Vergani D, Baumann U, et al. Diagnosis and management of paediatric autoimmune liver disease: ESPGHAN Hepatology committee position statement. Journal of Pediatric Gastroenterology and Nutrition. 2018;**66**:345-360

[7] Miloh T, Arnon R, Shneider B, et al. A retrospective single-center review of primary sclerosing cholangitis in children. Clinical Gastroenterology and Hepatology. 2009;**7**:239-245

[8] Weismuller T, Trivedy P, Bergquist A, et al. Patient age, sex and inflammatory bowel disease phenotypeassociate with course of primary sclerosing cholangitis. Gastroenterology. 2017;**152**:1975-1984

[9] Valentino PL, Wiggins S, Harney S, et al. The natural history of primary sclerosing cholangitis in children: A large single-center longitudinal cohort study. Journal of Pediatric Gastroenterology and Nutrition. 2016;**63**(6):603-609

[10] de Vries AB, Janse M, Blokzijl H, et al. Distinctive inflammatory bowel disease phenotype in primary sclerosing cholangitis. World Journal of Gastroenterology. 2015;**21**:1956-1971

[11] Karlsen T, Melum E, Franke A, et al. The utility of genome-wide association studies in hepatology. Hepatology. 2010;**51**:1833-1842

[12] Loftus EV Jr, Harewood GC, Loftus CG, et al. PSC-IBD: A unique form of inflammatory bowel disease associated with primary sclerosing cholangitis. Gut. 2005;**54**(1):91-96

[13] Mertz A, Nguyen NA, Katsanos KH, et al. Primary sclerosing cholangitis and inflammatory bowel disease comorbidity: An update of the evidence. Annals of Gastroenterology. 2019;**32**(2):124-133

[14] Naess S, Bjornsson E, Anmarkrud J, et al. Small-duct primary sclerosing cholangitis without inflammatory bowel disease is genetically different from large duct disese. Liver International. 2014;**34**:1488-1495

[15] Palmela C, Peerani F, Castaneda D, et al. Inflammatory bowel disease and primary sclerosing cholangitis: A review of the phenotype and associated specific features. Gut and Liver. 2018;**12**(1):17-29

[16] Karlsen TH, Folseraas T, Thorburn D, et al. Primary sclerosing cholangitis a comprehensive review. Journal of Hepatology. 2017;**67**(6):1298-1323

[17] Boberg K, Chapman R, Hirschfield G, et al. Overlap syndrome : The International Autoimmune Hepatitis Group (IAIHG) position statement on a controversial issue. Journal of Hepatology. 2011;**54**:374-385

[18] Pollheimer MJ, Halilbasic E, Fickert P, et al. Pathogenesis of primary sclerosing cholangitis. Best Practice & Research. Clinical Gastroenterology. 2011;**25**(6):727-739

[19] Alvaro D, Gigliozzi A, Artili A. Regulation and deregulation of cholangiocyte proliferation. Journal of Hepatology. 2000;**33**:333-340

[20] Bilhartz L. Gallstones Disease and its Complications. 6th ed. Saunders; 1998. p. 1-27

[21] Carpino C, Cardinale V, Renzi A, et al. Activation of biliary tree stem cells within peribiliary glands in primary sclerosing cholangitis. Journal of Hepatology. 2015;**63**:1220-1228

[22] Deneau MR, Mack C, Abdou R, et al. Gamma glutamyltransferase reduction is associated with favorable outcomes in pediatric primary sclerosing cholangitis. Hepatology Communications. 2018;**2**:1369-1378

[23] Maillette de Buy Wenniger L, Rauws E, Beuers U. What an endoscopist should know about immunoglobulin-G4 associated disease of the pancreas and biliary tree. Endoscopy. 2012;**44**:66-73

[24] Ponsioen C, Kuiper H, Ten K, et al. Immunohistochemical analysis of inflammation in primary sclerosing cholangitis. European Journal of Gastroenterology & Hepatology. 1999;**11**:769-774

[25] Whiteside T, Lasky S, Si L, et al. Immunologic analysis of mononuclear cells in liver tissues and blood of patients with primary sclerosing cholangitis. Hepatology. 1985;**5**:468-474 [26] Kevans D, Tyler A, Holm K, et al. Characterization of intestinal microbiota in ulcerative colitis patients with and without primary sclerosing cholangitis. Journal of Crohn's & Colitis. 2016;**10**:330-337

[27] Kummen M, Holm K, Anmarkrud J, et al. The gut microbial profile in patients with primary sclerosing cholangitis is distinct from patients with ulcerative colitis without biliary disease and healthy controls. Gut. 2017;**66**:611-619

[28] Quraishi M, Sergeant M, Kay G, et al. The gut-adherent microbiota of PSC–IBD is distinct to that of IBD. Gut. 2017;**66**:386-388

[29] Laborda TJ, Kyle Jense M, et al. Treatment of primary sclerosing cholangitis in children. World Journal of Hepatology. 2019;**11**(1):19-36

[30] Bjimsson E. Small-duct primary sclerosing cholangitis. Current Gastroenterology Reports. 2009;**11**:37-41

[31] Chapman R, Fevery J, Kalloo A, et al. Diagnosis and management of primary sclerosing cholangitis. Hepatology. 2010;**51**(2):660-678

[32] Corpechot C, Gaouar F, El Naggar A, et al. Baseline values and changes in the liver stiffness measured by transient elastogtaphy are associated with severity of fibrosis and outcome of patients with primary sclerosing cholangitis. Gastroenterology. 2014;**146**:970-979

[33] Deneau MR, El-Matary W, Valentino PL, et al. The natural history of primary sclerosing cholangitis in 781 children: A multicenter, international collaboration. Hepatology. 2017;**66**(2):518-527

**121**

*Primary Sclerosing Cholangitis (PSC) in Children DOI: http://dx.doi.org/10.5772/intechopen.90714*

Digestive Diseases and Sciences.

Surgery. 2007;**11**:1488-1496

[44] Rudolf G, Gotthardt D,

[43] Nathan H, Pawlik T, Wolfgang C, et al. Trends in survivalafter surgery for cholangiocarcinoma: A 30 year population-based SEER database analysis. Journal of Gastrointestinal

Kloters-Plachky P, et al. Influence of dominant bile duct stenosis and biliary infections on outcome in primary sclerosing cholangitis. Journal of Hepatology. 2009;**51**:149-155

[45] Subramanian V, Mannath J, Ragunath K, et al. Meta-analysis: The diagnostic of chronoendoscopy for detecting dysplasia in patients with colonic inflammatory bowel disease. Alimentary Pharmacology & Therapeutics. 2011;**33**:304-312

[46] Stiehl A, Rudolf G, Kloters-Plachky P, et al. Development of dominant bile duct stenosis in patients with primary sclerosing cholangitis treated with ursodeoxycholic acid: Outcome after endoscopic treatment. Journal of Hepatology. 2002;**36**:151-156

[47] Lindor K, Kowdley R, Luketic V, et al. High-dose ursodeoxycholic acid

[48] Damman JL, Rodriguez EA, Ali AH, et al. Review article: The evidence that vancomycin is a therapeutic option for primary sclerosing cholangitis. Alimentary Pharmacology & Therapeutics. 2018;**47**:886-895

[49] Hey P, Lokan J, Johnson P, et al. Efficacy of oral vancomycin in

following liver transplantation. BML Case Reports. 2017;**25**:2017. pii:bcr-2017-221165. DOI: 10.1136/

recurrent primary sclerosing cholangitis

for the treatment of primary sclerosing cholangitis. Hepatology.

2009;**50**:808-814

bcr-2017-221165

2005;**50**:1734-1740

transient elastography and comparison with spleen length measurement for staging of fibrosis and clinical prognosis in primary sclerosing cholangitis. PLoS

One. 2016;**11**:e0164224

[35] Blechacz B, Gores G.

1996;**38**:610-615

2004;**99**:523-526

Cholangiocarcinoma: Advances in pathogenesis, diagnosis and treatment.

Hepatology. 2008;**48**:308-321

[36] Boberg K, Ling G. Primary

sclerosing cholangitis and malignancy. Best Practice & Research. Clinical Gastroenterology. 2011;**25**:753-764

[37] Broome U, Olsson R, Loof L, et al. Natural history and prognostic factors in 305 Swedish patients with with primary sclerosing cholangitis. Gut.

[38] Burak K, Angulo P, Pasha TM, et al. Incidence and risk factors for cholangiocarcinoma in primary sclerosing cholangitis. The American

[39] Charatcharoenwitthaya P, Enders F, Halling K, et al. Utility of serum tumor markers, imaging and biliary cytology for detecting cholangiocarcinoma in primary sclerosing cholangitis. Hepatology. 2008;**48**:1106-1117

El-Youssif M, et al. Primary sclerosing

[41] Kaminski M, Hassan C, Bisschops R, et al. Advanced imaging for detection and differentiation of colorectal neoplasia: European Society of Gastrointestinal Endoscopy (ESGE) Guideline. Endoscopy. 2014;**46**:435-449

[42] Levy C, Lymp J, Angulo P, et al. The value of serum Ca 19-9 in predicting of cholangiocarcinomas in patients with primary sclerosing cholangitis.

Journal of Gastroenterology.

[40] Feldstein AE, Perrault J,

cholangitis in children: A longterm follow-up study. Hepatology.

2003;**38**(1):210-217

[34] Ehlken H, Wroblewski R, Corpechot C, et al. Validation of *Primary Sclerosing Cholangitis (PSC) in Children DOI: http://dx.doi.org/10.5772/intechopen.90714*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

[26] Kevans D, Tyler A, Holm K, et al. Characterization of intestinal microbiota in ulcerative colitis patients with and without primary sclerosing cholangitis. Journal of Crohn's & Colitis.

[27] Kummen M, Holm K, Anmarkrud J, et al. The gut microbial profile in patients with primary sclerosing cholangitis is distinct from patients with ulcerative colitis without biliary disease and healthy controls. Gut.

[28] Quraishi M, Sergeant M, Kay G, et al. The gut-adherent microbiota of PSC–IBD is distinct to that of IBD. Gut.

[29] Laborda TJ, Kyle Jense M, et al. Treatment of primary sclerosing

[31] Chapman R, Fevery J, Kalloo A, et al. Diagnosis and management of primary sclerosing cholangitis. Hepatology. 2010;**51**(2):660-678

[32] Corpechot C, Gaouar F, El Naggar A, et al. Baseline values and changes in the liver stiffness measured by transient elastogtaphy are associated with severity of fibrosis and outcome of patients with primary sclerosing cholangitis. Gastroenterology.

[33] Deneau MR, El-Matary W,

[34] Ehlken H, Wroblewski R, Corpechot C, et al. Validation of

Valentino PL, et al. The natural history of primary sclerosing cholangitis in 781 children: A multicenter,

international collaboration. Hepatology.

Hepatology. 2019;**11**(1):19-36

[30] Bjimsson E. Small-duct primary sclerosing cholangitis. Current Gastroenterology Reports.

cholangitis in children. World Journal of

2016;**10**:330-337

2017;**66**:611-619

2017;**66**:386-388

2009;**11**:37-41

2014;**146**:970-979

2017;**66**(2):518-527

Hirschfield G, et al. Overlap syndrome :

Fickert P, et al. Pathogenesis of primary sclerosing cholangitis. Best Practice & Research. Clinical Gastroenterology.

[17] Boberg K, Chapman R,

The International Autoimmune Hepatitis Group (IAIHG) position statement on a controversial issue. Journal of Hepatology. 2011;**54**:374-385

[18] Pollheimer MJ, Halilbasic E,

[19] Alvaro D, Gigliozzi A, Artili A. Regulation and deregulation of cholangiocyte proliferation. Journal of

[20] Bilhartz L. Gallstones Disease and its Complications. 6th ed. Saunders;

[21] Carpino C, Cardinale V, Renzi A, et al. Activation of biliary tree stem cells within peribiliary glands in primary sclerosing cholangitis. Journal of Hepatology. 2015;**63**:1220-1228

[22] Deneau MR, Mack C, Abdou R, et al. Gamma glutamyltransferase reduction is associated with favorable outcomes in pediatric primary sclerosing cholangitis. Hepatology Communications. 2018;**2**:1369-1378

[23] Maillette de Buy Wenniger L, Rauws E, Beuers U. What an endoscopist should know about immunoglobulin-G4 associated disease of the pancreas and biliary tree. Endoscopy. 2012;**44**:66-73

[24] Ponsioen C, Kuiper H, Ten K, et al. Immunohistochemical analysis of inflammation in primary sclerosing cholangitis. European Journal of Gastroenterology & Hepatology.

[25] Whiteside T, Lasky S, Si L, et al. Immunologic analysis of mononuclear cells in liver tissues and blood of patients with primary sclerosing cholangitis. Hepatology. 1985;**5**:468-474

Hepatology. 2000;**33**:333-340

2011;**25**(6):727-739

1998. p. 1-27

**120**

1999;**11**:769-774

transient elastography and comparison with spleen length measurement for staging of fibrosis and clinical prognosis in primary sclerosing cholangitis. PLoS One. 2016;**11**:e0164224

[35] Blechacz B, Gores G. Cholangiocarcinoma: Advances in pathogenesis, diagnosis and treatment. Hepatology. 2008;**48**:308-321

[36] Boberg K, Ling G. Primary sclerosing cholangitis and malignancy. Best Practice & Research. Clinical Gastroenterology. 2011;**25**:753-764

[37] Broome U, Olsson R, Loof L, et al. Natural history and prognostic factors in 305 Swedish patients with with primary sclerosing cholangitis. Gut. 1996;**38**:610-615

[38] Burak K, Angulo P, Pasha TM, et al. Incidence and risk factors for cholangiocarcinoma in primary sclerosing cholangitis. The American Journal of Gastroenterology. 2004;**99**:523-526

[39] Charatcharoenwitthaya P, Enders F, Halling K, et al. Utility of serum tumor markers, imaging and biliary cytology for detecting cholangiocarcinoma in primary sclerosing cholangitis. Hepatology. 2008;**48**:1106-1117

[40] Feldstein AE, Perrault J, El-Youssif M, et al. Primary sclerosing cholangitis in children: A longterm follow-up study. Hepatology. 2003;**38**(1):210-217

[41] Kaminski M, Hassan C, Bisschops R, et al. Advanced imaging for detection and differentiation of colorectal neoplasia: European Society of Gastrointestinal Endoscopy (ESGE) Guideline. Endoscopy. 2014;**46**:435-449

[42] Levy C, Lymp J, Angulo P, et al. The value of serum Ca 19-9 in predicting of cholangiocarcinomas in patients with primary sclerosing cholangitis.

Digestive Diseases and Sciences. 2005;**50**:1734-1740

[43] Nathan H, Pawlik T, Wolfgang C, et al. Trends in survivalafter surgery for cholangiocarcinoma: A 30 year population-based SEER database analysis. Journal of Gastrointestinal Surgery. 2007;**11**:1488-1496

[44] Rudolf G, Gotthardt D, Kloters-Plachky P, et al. Influence of dominant bile duct stenosis and biliary infections on outcome in primary sclerosing cholangitis. Journal of Hepatology. 2009;**51**:149-155

[45] Subramanian V, Mannath J, Ragunath K, et al. Meta-analysis: The diagnostic of chronoendoscopy for detecting dysplasia in patients with colonic inflammatory bowel disease. Alimentary Pharmacology & Therapeutics. 2011;**33**:304-312

[46] Stiehl A, Rudolf G, Kloters-Plachky P, et al. Development of dominant bile duct stenosis in patients with primary sclerosing cholangitis treated with ursodeoxycholic acid: Outcome after endoscopic treatment. Journal of Hepatology. 2002;**36**:151-156

[47] Lindor K, Kowdley R, Luketic V, et al. High-dose ursodeoxycholic acid for the treatment of primary sclerosing cholangitis. Hepatology. 2009;**50**:808-814

[48] Damman JL, Rodriguez EA, Ali AH, et al. Review article: The evidence that vancomycin is a therapeutic option for primary sclerosing cholangitis. Alimentary Pharmacology & Therapeutics. 2018;**47**:886-895

[49] Hey P, Lokan J, Johnson P, et al. Efficacy of oral vancomycin in recurrent primary sclerosing cholangitis following liver transplantation. BML Case Reports. 2017;**25**:2017. pii:bcr-2017-221165. DOI: 10.1136/ bcr-2017-221165

*Hepatitis A and Other Associated Hepatobiliary Diseases*

[50] Shah A, Crawford D, Burger D, Martin N, et al. Effects of antibiotic therapy in primary sclerosing cholangitis with and without inflammatory bowel disease: A systematic review and metaanalysis. Seminars in Liver Disease. 2019;**39**(4):432-441

[51] Soufi N, Bazerbachi F, Deneau M. Post-transplant disease recurrence in pediatric PSC. Current Gastroenterology Reports. 2018;**20**:44. DOI: 10.1007/s11894-018-0649-2

**123**

common bile duct.

**Chapter 9**

**Abstract**

Cholestasis: The Close

*Manuela R. Martinefski, Silvia E. Lucangioli,* 

*Liliana G. Bianciotti and Valeria P. Tripodi*

CoQ10 supplementation to current traditional therapies.

**Keywords:** cholestasis, coenzyme Q10, bile acids

**therapeutic approaches**

and Coenzyme Q10

Relationship between Bile Acids

Cholestasis is defined as the impairment in formation or excretion of bile from the liver to the intestine. It may result from defects in intrahepatic production of bile, impairment of hepatic transmembrane transporters, or mechanical obstruction to bile flow. In cholestasis, hepatocytes are exposed to high levels of bile acids, particularly those bearing hydrophobic properties. The increase in bile acids

induces oxidative stress, leading to an imbalance in the prooxidant:antioxidant ratio which determines the final cellular redox status. This chapter will focus on the close relationship between bile acids and the most powerful endogenous antioxidant, coenzyme Q10 in cholestasis, and the eventual alternative therapeutic option of

**1. Cholestasis: types, clinical presentation, diagnosis and current** 

Bile is a nonenzymatic secretion produced by hepatocytes. The main components of bile include bile salts necessary for enzymatic fat digestion and absorption, bilirubin, and cholesterol. Drugs and other xenobiotics are also excreted into bile following hepatic metabolization. Bile flow is dependent on the active canalicular transport of bile acids and other substrates mediated by the bile salt export pump (Bsep), which transports osmotically active monoanionic bile salts into the bile canaliculus and multidrug resistance-associated protein 2 (Mrp2), which exports oxidized and reduced glutathione. Bile secreted by the hepatocytes is stored and concentrated in the gallbladder, which contracts in the presence of the hormone cholecystokinin resulting in bile release into the duodenum through the cystic and

Cholestasis is defined as the decrease or suppression of bile flow due to impaired

secretion by hepatocytes or to obstruction of bile at any level of the excretory pathway, from the hepatocyte canalicular membrane to the ampulla of Vater in the duodenum. Cholestasis leads to the retention of the major constituents of bile, bilirubin, and bile acids, in blood. By convention, cholestasis is chronic when it lasts more than 6 months. Prevalence of cholestasis is not significantly different between males and females. Nevertheless, women are at lighter risk of developing

#### **Chapter 9**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

[50] Shah A, Crawford D, Burger D, Martin N, et al. Effects of antibiotic therapy in primary sclerosing cholangitis with and without inflammatory bowel disease: A systematic review and metaanalysis. Seminars in Liver Disease.

[51] Soufi N, Bazerbachi F, Deneau M. Post-transplant disease recurrence

Gastroenterology Reports. 2018;**20**:44. DOI: 10.1007/s11894-018-0649-2

2019;**39**(4):432-441

in pediatric PSC. Current

**122**

## Cholestasis: The Close Relationship between Bile Acids and Coenzyme Q10

*Manuela R. Martinefski, Silvia E. Lucangioli, Liliana G. Bianciotti and Valeria P. Tripodi*

#### **Abstract**

Cholestasis is defined as the impairment in formation or excretion of bile from the liver to the intestine. It may result from defects in intrahepatic production of bile, impairment of hepatic transmembrane transporters, or mechanical obstruction to bile flow. In cholestasis, hepatocytes are exposed to high levels of bile acids, particularly those bearing hydrophobic properties. The increase in bile acids induces oxidative stress, leading to an imbalance in the prooxidant:antioxidant ratio which determines the final cellular redox status. This chapter will focus on the close relationship between bile acids and the most powerful endogenous antioxidant, coenzyme Q10 in cholestasis, and the eventual alternative therapeutic option of CoQ10 supplementation to current traditional therapies.

**Keywords:** cholestasis, coenzyme Q10, bile acids

#### **1. Cholestasis: types, clinical presentation, diagnosis and current therapeutic approaches**

Bile is a nonenzymatic secretion produced by hepatocytes. The main components of bile include bile salts necessary for enzymatic fat digestion and absorption, bilirubin, and cholesterol. Drugs and other xenobiotics are also excreted into bile following hepatic metabolization. Bile flow is dependent on the active canalicular transport of bile acids and other substrates mediated by the bile salt export pump (Bsep), which transports osmotically active monoanionic bile salts into the bile canaliculus and multidrug resistance-associated protein 2 (Mrp2), which exports oxidized and reduced glutathione. Bile secreted by the hepatocytes is stored and concentrated in the gallbladder, which contracts in the presence of the hormone cholecystokinin resulting in bile release into the duodenum through the cystic and common bile duct.

Cholestasis is defined as the decrease or suppression of bile flow due to impaired secretion by hepatocytes or to obstruction of bile at any level of the excretory pathway, from the hepatocyte canalicular membrane to the ampulla of Vater in the duodenum. Cholestasis leads to the retention of the major constituents of bile, bilirubin, and bile acids, in blood. By convention, cholestasis is chronic when it lasts more than 6 months. Prevalence of cholestasis is not significantly different between males and females. Nevertheless, women are at lighter risk of developing

drug-induced cholestasis and intrahepatic cholestasis of pregnancy. Despite that, cholestasis may affect people of every age group, newborns and infants are more prone due to the immaturity of the liver.

The morphologic features of cholestasis are dependent on the severity, duration, and the underlying cause. Cholestasis is classified as intrahepatic or extrahepatic cholestasis depending on the cause that leads to impaired bile flow. Intrahepatic cholestasis is due to a disease affecting the hepatocytes and/or the intrahepatic bile ducts, whereas extrahepatic cholestasis or obstructive cholestasis results from the obstruction of the extrahepatic biliary ducts.

Obstruction of bile ducts can be caused by gallstones, cysts, stenosis, or tumors. The most frequent causes of extrahepatic cholestasis in adults include cholelithiasis and malignancies of the biliary tree or the head of the pancreas. However, in children, biliary atresia and cystic fibrosis are the main causes. Intermittent or partial obstruction may lead to ascending cholangitis, a secondary bacterial infection of the biliary tree. The typical morphological changes are reversible if the obstruction is corrected, but if it persists it can lead to biliary cirrhosis.

Causes of intrahepatic or hepatocellular cholestasis include viral and autoimmune hepatitis, inborn errors of bile acid synthesis, primary biliary cirrhosis, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, total parenteral nutrition, and drug toxicity. The drug class mostly implicated in cholestasis is antibiotics. However, anti-inflammatory drugs, highly active antiretroviral therapy, psychotropes, some chemotherapy agents, oral contraceptives, and anabolic steroids have also been reported to cause cholestasis [1]. Although primary sclerosing cholangitis affects intrahepatic bile ducts, it can also affect extrahepatic bile ducts.

Clinical presentation of cholestasis includes jaundice, pruritus, skin xanthomas, or symptoms associated with intestinal malabsorption. Jaundice and pruritus are present in all types of cholestasis whether acute or chronic, whereas the other clinical features are more associated with chronic cholestasis.

Jaundice is the clinical expression of bilirubin retention. Excretion of conjugated bilirubin is the rate-limiting step of bilirubin clearance. During cholestasis, conjugation of bilirubin continues but the excretion is significantly reduced. Jaundice is observed by scleral icterus at a concentration as low as 2 mg/dL accompanied by dark urine. The concentration of conjugated bilirubin in blood depends on its production rate and excretion pathways, as well as cholestasis degree. Non conjugated bilirubin is also increased in patients with cholestasis. The magnitude of the increase in serum bilirubin concentration does not correlated with the type or severity of cholestasis. Pruritus is a frequent clinical manifestation of cholestasis, which has been long associated with increased serum bile acids. However, its origin is multifactorial and diverse studies show that not only bile acids but also lysophosphatidic acid, and bilirubin are potential mediators of cholestatic itch [2]. Retention of bile acids and their conjugated salts results in biological membrane injury, particularly in the liver due to their detergent properties. Increased hydrophobic bile salts favor their incorporation into membranes, altering membrane fluidity and function. Enhanced secondary bile acids like lithocholic acid result in further membrane injury. The transport of bile salts from plasma to bile is the principal driving force for bile formation and it is mediated by several hepatic transporters, mostly belonging to the ABC family of transporters. Numerous studies support that the failure to excrete bile salts into the canaliculus is the main mechanism underlying cholestasis. In this sense retrieval of the canalicular transporters Bsep and Mrp2 from hepatocyte plasma membrane to endosomal compartments in different types of cholestasis has been well documented [3, 4]. However, other works consider that the endocytic retrieval of canalicular transporter is the result of cholestasis on the

**125**

unclear.

opiate antagonists [9].

**2.1 Bile acids physicochemical properties**

*Cholestasis: The Close Relationship between Bile Acids and Coenzyme Q10*

hepatocyte function. In either case, the retention of bile salts in the liver induces down-regulation of bile acid synthesis, overall reduction in the total pool size and

Skin xanthomas and signs of malabsorption are associated with chronic cholestasis. Skin xanthomas result from focal accumulation of cholesterol in the dermis and usually appear around the eyes, but may be present in other parts of the body. Malabsorption occurs due to the failure of enough bile salts to reach the duodenum, so the digestion and absorption of dietary fat is impaired. Fat soluble vitamins like A, E, D, and K are poorly absorbed in cholestasis leading to clinical symptoms and

In all types of cholestasis, characteristic laboratory findings are elevated serum alkaline phosphatase and γ-glutamyltranspeptidase, enzymes present on the canalicular membranes of hepatocytes, and bile duct epithelial cells. Alkaline phosphatase is also elevated in bone growth or disease, pregnancy, or intestinal diseases. λ-Glutamyltranspeptidase is a sensitive marker of cholestasis [5], although no specific since it can be elevated in other liver diseases [6]. Furthermore, its elevation may reflect enzyme induction by drugs or alcohol. Serum 5′-nucleotidase, an enzyme located in canalicular membranes and lining the sinusoids is also elevated in cholestasis, although it appears to be less sensitive than alkaline phosphatase. Serum elevation of hepatic enzymes is accompanied by increased serum bilirubin and bile acids. An increase in serum bile acids is an early marker of cholestasis. In the diagnosis of cholestasis, the first key step is to identify whether it is intrahepatic, extrahepatic, or both. The patient history and physical examination usually provide useful information. Elevation of both hepatic enzymes (alkaline phosphatase and λ-glutamyl transpeptidase) is a hallmark of cholestasis although the identification of the type of cholestasis requires imaging studies and additional biochemical studies. Imaging studies include first an abdominal ultrasonography to exclude dilated intra and extrahepatic ducts. When bile duct alterations are observed, further imaging studies like magnetic resonance cholangiopancreatography or endoscopic retrograde cholangiopancreatography should be performed. A diagnostic of intrahepatic cholestasis can be made when imaging studies exclude mechanical obstruction. Then, further biochemical studies are necessary to identify the intrahepatic cause of cholestasis, including liver biopsies when the diagnosis is

The therapeutic intervention for cholestasis may differ depending on the etiology [7]. Based on controlled clinical trials, ursodeoxycholic acid (UDCA) is the treatment of choice for diverse cholestatic disorders like primary biliary cirrhosis and intrahepatic cholestasis of pregnancy due to its anticholestatic properties. However, UDCA treatment is not so effective in other cholestatic disorders like in primary sclerosing cholangitis. No therapy of proven benefit for the long-term prognosis of genetic cholestatic liver disease exists. In drug-induced cholestasis, withdrawal of the drug is the only effective treatment [8]. Pruritus is a common manifestation of cholestasis, which can be of serious severity. Management of pruritus includes cholestyramine as first line-treatment and then rifampicin, and

**2. Bile acids: physicochemical properties, synthesis, and therapeutics**

Bile acids (BA) are steroid compounds, hydroxyl derivatives of 5β-cholan-24 oic acid. Primary BA are cholic acid (CA) and chenodeoxycholic acid (CDCA);

*DOI: http://dx.doi.org/10.5772/intechopen.90831*

damage to hepatocytes.

signs of their deficiency.

*Hepatitis A and Other Associated Hepatobiliary Diseases*

prone due to the immaturity of the liver.

obstruction of the extrahepatic biliary ducts.

is corrected, but if it persists it can lead to biliary cirrhosis.

cal features are more associated with chronic cholestasis.

drug-induced cholestasis and intrahepatic cholestasis of pregnancy. Despite that, cholestasis may affect people of every age group, newborns and infants are more

The morphologic features of cholestasis are dependent on the severity, duration, and the underlying cause. Cholestasis is classified as intrahepatic or extrahepatic cholestasis depending on the cause that leads to impaired bile flow. Intrahepatic cholestasis is due to a disease affecting the hepatocytes and/or the intrahepatic bile ducts, whereas extrahepatic cholestasis or obstructive cholestasis results from the

Obstruction of bile ducts can be caused by gallstones, cysts, stenosis, or tumors. The most frequent causes of extrahepatic cholestasis in adults include cholelithiasis and malignancies of the biliary tree or the head of the pancreas. However, in children, biliary atresia and cystic fibrosis are the main causes. Intermittent or partial obstruction may lead to ascending cholangitis, a secondary bacterial infection of the biliary tree. The typical morphological changes are reversible if the obstruction

Causes of intrahepatic or hepatocellular cholestasis include viral and autoimmune hepatitis, inborn errors of bile acid synthesis, primary biliary cirrhosis, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, total parenteral nutrition, and drug toxicity. The drug class mostly implicated in cholestasis is antibiotics. However, anti-inflammatory drugs, highly active antiretroviral therapy, psychotropes, some chemotherapy agents, oral contraceptives, and anabolic steroids have also been reported to cause cholestasis [1]. Although primary sclerosing cholangitis affects intrahepatic bile ducts, it can also affect extrahepatic

Clinical presentation of cholestasis includes jaundice, pruritus, skin xanthomas, or symptoms associated with intestinal malabsorption. Jaundice and pruritus are present in all types of cholestasis whether acute or chronic, whereas the other clini-

Jaundice is the clinical expression of bilirubin retention. Excretion of conjugated

bilirubin is the rate-limiting step of bilirubin clearance. During cholestasis, conjugation of bilirubin continues but the excretion is significantly reduced. Jaundice is observed by scleral icterus at a concentration as low as 2 mg/dL accompanied by dark urine. The concentration of conjugated bilirubin in blood depends on its production rate and excretion pathways, as well as cholestasis degree. Non conjugated bilirubin is also increased in patients with cholestasis. The magnitude of the increase in serum bilirubin concentration does not correlated with the type or severity of cholestasis. Pruritus is a frequent clinical manifestation of cholestasis, which has been long associated with increased serum bile acids. However, its origin is multifactorial and diverse studies show that not only bile acids but also lysophosphatidic acid, and bilirubin are potential mediators of cholestatic itch [2]. Retention of bile acids and their conjugated salts results in biological membrane injury, particularly in the liver due to their detergent properties. Increased hydrophobic bile salts favor their incorporation into membranes, altering membrane fluidity and function. Enhanced secondary bile acids like lithocholic acid result in further membrane injury. The transport of bile salts from plasma to bile is the principal driving force for bile formation and it is mediated by several hepatic transporters, mostly belonging to the ABC family of transporters. Numerous studies support that the failure to excrete bile salts into the canaliculus is the main mechanism underlying cholestasis. In this sense retrieval of the canalicular transporters Bsep and Mrp2 from hepatocyte plasma membrane to endosomal compartments in different types of cholestasis has been well documented [3, 4]. However, other works consider that the endocytic retrieval of canalicular transporter is the result of cholestasis on the

**124**

bile ducts.

hepatocyte function. In either case, the retention of bile salts in the liver induces down-regulation of bile acid synthesis, overall reduction in the total pool size and damage to hepatocytes.

Skin xanthomas and signs of malabsorption are associated with chronic cholestasis. Skin xanthomas result from focal accumulation of cholesterol in the dermis and usually appear around the eyes, but may be present in other parts of the body. Malabsorption occurs due to the failure of enough bile salts to reach the duodenum, so the digestion and absorption of dietary fat is impaired. Fat soluble vitamins like A, E, D, and K are poorly absorbed in cholestasis leading to clinical symptoms and signs of their deficiency.

In all types of cholestasis, characteristic laboratory findings are elevated serum alkaline phosphatase and γ-glutamyltranspeptidase, enzymes present on the canalicular membranes of hepatocytes, and bile duct epithelial cells. Alkaline phosphatase is also elevated in bone growth or disease, pregnancy, or intestinal diseases. λ-Glutamyltranspeptidase is a sensitive marker of cholestasis [5], although no specific since it can be elevated in other liver diseases [6]. Furthermore, its elevation may reflect enzyme induction by drugs or alcohol. Serum 5′-nucleotidase, an enzyme located in canalicular membranes and lining the sinusoids is also elevated in cholestasis, although it appears to be less sensitive than alkaline phosphatase. Serum elevation of hepatic enzymes is accompanied by increased serum bilirubin and bile acids. An increase in serum bile acids is an early marker of cholestasis.

In the diagnosis of cholestasis, the first key step is to identify whether it is intrahepatic, extrahepatic, or both. The patient history and physical examination usually provide useful information. Elevation of both hepatic enzymes (alkaline phosphatase and λ-glutamyl transpeptidase) is a hallmark of cholestasis although the identification of the type of cholestasis requires imaging studies and additional biochemical studies. Imaging studies include first an abdominal ultrasonography to exclude dilated intra and extrahepatic ducts. When bile duct alterations are observed, further imaging studies like magnetic resonance cholangiopancreatography or endoscopic retrograde cholangiopancreatography should be performed. A diagnostic of intrahepatic cholestasis can be made when imaging studies exclude mechanical obstruction. Then, further biochemical studies are necessary to identify the intrahepatic cause of cholestasis, including liver biopsies when the diagnosis is unclear.

The therapeutic intervention for cholestasis may differ depending on the etiology [7]. Based on controlled clinical trials, ursodeoxycholic acid (UDCA) is the treatment of choice for diverse cholestatic disorders like primary biliary cirrhosis and intrahepatic cholestasis of pregnancy due to its anticholestatic properties. However, UDCA treatment is not so effective in other cholestatic disorders like in primary sclerosing cholangitis. No therapy of proven benefit for the long-term prognosis of genetic cholestatic liver disease exists. In drug-induced cholestasis, withdrawal of the drug is the only effective treatment [8]. Pruritus is a common manifestation of cholestasis, which can be of serious severity. Management of pruritus includes cholestyramine as first line-treatment and then rifampicin, and opiate antagonists [9].

#### **2. Bile acids: physicochemical properties, synthesis, and therapeutics**

#### **2.1 Bile acids physicochemical properties**

Bile acids (BA) are steroid compounds, hydroxyl derivatives of 5β-cholan-24 oic acid. Primary BA are cholic acid (CA) and chenodeoxycholic acid (CDCA);

secondary BA such as deoxycholic acid (DCA) and lithocholic acid (LCA), all of them in 3α-position, and ursodeoxycholic acid (UDCA) is a hydroxyl derivative in 3β-position (**Figure 1**) [10].

BA have different physicochemical properties according to the number, position, and orientation of their hydroxy groups and the conjugation with glycine and taurine (**Figure 1**). In this sense, this characteristic influence their solubility, detergency, and hydrophobicity [11].

BA have an important role in biological systems under physiological and pathological conditions [12]. Their functions are associated with lipid digestion and absorption, solubilization of cholesterol and bile formation. In this case, BA influence in volume and composition of the bile.

The number, position, and orientation of the hydroxy groups of the BA impact directly on the hydrophobicity and detergency property and the relationship to the toxicity. In the case of BA with hydroxy groups in 3-α position, the higher the number of hydroxy groups, less hydrophobicity and lower detergency and, as a result, lower toxicity.

It must be pointed out that the orientation of the hydroxy group rules over the properties in the molecule. This can be seen on the CDCA (7α) and its epimer, the UDCA (7β), where the UDCA showed a strong reduction of detergency and hydrophobicity. Also, the BA toxicity is directly related to its hydrophobicity and detergency, because those interact with the cellular membranes in different ways, including the union, the insertion in the lipidic bilayer and its solubilization increasing its fluidity [10].

Therefore, UDCA is administered as therapeutic agent for the treatment of hepatobiliary disorders such as cholestasis, biliary dyspepsia, primary biliary cirrhosis, and different cholestatic conditions.

#### **2.2 Bile acids synthesis**

The synthesis of BA is produced exclusively in the liver, based on a series of enzymatic reactions in the hepatocyte, in which 17 enzymes are involved. The cholesterol (hydrophobic compound) turns into the primary BA, also known as colic acid (CA) and chenodeoxycholic (CDCA), through the first step and limiting of the called "classic" or "neutral" way of the BA biosynthesis, where the hydroxylation of the cholesterol is produced, catalyzed by the enzyme cytochrome P450

**127**

*Cholestasis: The Close Relationship between Bile Acids and Coenzyme Q10*

cholesterol 7a-hydroxylasa (CYP7A1). The BA synthesis can also occur through an "alternative" or "acidic" way, where the CYP27A1 intervenes and changes the BA oxysterols. Unlike the CYP7A1, the CYP27A1 is not regulated by the BA and is estimated only the 6% of the synthesis of BA is produced through this way. Before its secretion in the canalicular biliary light for the storage in the biliary gold bladder as mixed micelles with phospholipids and cholesterol, the primary BA are mainly conjugated with taurine and glycine, forming the conjugated BA, that with the Na<sup>+</sup>

form the biliary salts. When ingesting a food, the contraction of biliary gold

BA as therapeutic agent are appropriated in the chronic cholestasis deceases. BA can be orally administered following two strategies, the "displacement therapy" and/or "replacement therapy." UDCA may be used to displace endogenous BA to decrease the intrahepatic concentration of potentially cytotoxic BA accumulated in cholestasis. On the other hand, primary BA such as cholic acid (CA) might be used to replace a depleted BA pool resulted from defective biosynthesis on consequence

UDCA (3α-7β-hydroxy-5β-cholan-24-oic acid) is naturally occurring BA, that normally constitutes 1–2% of the BA in human bile. UDCA is obtained by 7α-epimerization of the primary BA chenodeoxycholic acid (CDCA), by intestinal bacteria. [18] UDCA and CDCA differ in the hydroxyl group orientation at seventh

UDCA is a weak acid (pKa = 5), and poorly water soluble, however, its solubility increases directly to the increase of the solution pH. After orally administrations, UDCA must be solubilized in mixed micelles present in small intestinal content in order to allow absorption [19, 20]. During the cholestasis disease, the UDCA bioavailability is limited due to the reduction of endogenous BA micelles in the duodenal lumen. Unconjugated UDCA is absorbed by passive diffusion in the proximal jejunum and in the ileum, thus extracted from portal venous blood by the liver and conjugated with glycine or taurine. Conjugated UDCA is secreted into the bile.

It is worth mentioning that in the UDCA oral administration, the half-life of the UDCA in the portal circulation is short, thus the maximum concentrations in liver/

UDCA is the BA of choice in view of the proven efficacy and lack of side effects in the treatment of cholestasis diseases. In the case of CDCA, its inherent toxicity is related to the fact that CDCA undergoes bacterial conversion dihydroxylation to a toxic, monohydroxy BA, like lithocholic acid (LCA), unlike UDCA, which is more

position, allowing higher hydrophilicity of UDCA in comparison to CDCA.

bile achieved by dividing the dose equally over 24 h are adequate.

resistant to bacterial dihydroxylation [21, 22].

bladder expels the micellar BA to the intestinal light to help digestion. In the gut, the intestinal bacteria deconjugate and dehydroxylate the primary BA, resulting in other species, denominated secondary BA: deoxycholic acid (DCA), a CA derivative, and ursodeoxycholic acid (UDCA), a CDCA derivative. The enterohepatic circulation allows the 95% of the BA to be reabsorbed from the distal ileum and transported back to the liver through portal circulation. Only 5% of the BA are not reabsorbed and are eliminated through feces. This small amount of loss is recovered through the novo synthesis of the BA in the liver. The size of the BA reserve is strictly regulated by the liver and gut to avoid a cytotoxic accumulation. When the reserve of BA increases, a feedback mechanism is activated, ruled by the interaction of several nuclear receptors, mainly the farnesoid X nuclear receptor (FXR) to inhibit the novo synthesis of BA. Therefore, the FXR is a "BA sensor," when the BA are joined to this receptor, they mediate their own synthesis control to provide a

*DOI: http://dx.doi.org/10.5772/intechopen.90831*

strict regulation of its reserve [13–15].

**2.3 Bile acid therapy in hepatobiliary disease: role of UDCA**

to restore the physiological function of BA [16, 17].

and K<sup>+</sup>

**Figure 1.** *Bile acids: chemical structure.*

*Cholestasis: The Close Relationship between Bile Acids and Coenzyme Q10 DOI: http://dx.doi.org/10.5772/intechopen.90831*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

influence in volume and composition of the bile.

3β-position (**Figure 1**) [10].

result, lower toxicity.

increasing its fluidity [10].

**2.2 Bile acids synthesis**

rhosis, and different cholestatic conditions.

detergency, and hydrophobicity [11].

secondary BA such as deoxycholic acid (DCA) and lithocholic acid (LCA), all of them in 3α-position, and ursodeoxycholic acid (UDCA) is a hydroxyl derivative in

BA have different physicochemical properties according to the number, position, and orientation of their hydroxy groups and the conjugation with glycine and taurine (**Figure 1**). In this sense, this characteristic influence their solubility,

BA have an important role in biological systems under physiological and pathological conditions [12]. Their functions are associated with lipid digestion and absorption, solubilization of cholesterol and bile formation. In this case, BA

The number, position, and orientation of the hydroxy groups of the BA impact directly on the hydrophobicity and detergency property and the relationship to the toxicity. In the case of BA with hydroxy groups in 3-α position, the higher the number of hydroxy groups, less hydrophobicity and lower detergency and, as a

It must be pointed out that the orientation of the hydroxy group rules over the properties in the molecule. This can be seen on the CDCA (7α) and its epimer, the UDCA (7β), where the UDCA showed a strong reduction of detergency and hydrophobicity. Also, the BA toxicity is directly related to its hydrophobicity and detergency, because those interact with the cellular membranes in different ways, including the union, the insertion in the lipidic bilayer and its solubilization

Therefore, UDCA is administered as therapeutic agent for the treatment of hepatobiliary disorders such as cholestasis, biliary dyspepsia, primary biliary cir-

The synthesis of BA is produced exclusively in the liver, based on a series of enzymatic reactions in the hepatocyte, in which 17 enzymes are involved. The cholesterol (hydrophobic compound) turns into the primary BA, also known as colic acid (CA) and chenodeoxycholic (CDCA), through the first step and limiting of the called "classic" or "neutral" way of the BA biosynthesis, where the hydroxylation of the cholesterol is produced, catalyzed by the enzyme cytochrome P450

**126**

**Figure 1.**

*Bile acids: chemical structure.*

cholesterol 7a-hydroxylasa (CYP7A1). The BA synthesis can also occur through an "alternative" or "acidic" way, where the CYP27A1 intervenes and changes the BA oxysterols. Unlike the CYP7A1, the CYP27A1 is not regulated by the BA and is estimated only the 6% of the synthesis of BA is produced through this way. Before its secretion in the canalicular biliary light for the storage in the biliary gold bladder as mixed micelles with phospholipids and cholesterol, the primary BA are mainly conjugated with taurine and glycine, forming the conjugated BA, that with the Na<sup>+</sup> and K<sup>+</sup> form the biliary salts. When ingesting a food, the contraction of biliary gold bladder expels the micellar BA to the intestinal light to help digestion. In the gut, the intestinal bacteria deconjugate and dehydroxylate the primary BA, resulting in other species, denominated secondary BA: deoxycholic acid (DCA), a CA derivative, and ursodeoxycholic acid (UDCA), a CDCA derivative. The enterohepatic circulation allows the 95% of the BA to be reabsorbed from the distal ileum and transported back to the liver through portal circulation. Only 5% of the BA are not reabsorbed and are eliminated through feces. This small amount of loss is recovered through the novo synthesis of the BA in the liver. The size of the BA reserve is strictly regulated by the liver and gut to avoid a cytotoxic accumulation. When the reserve of BA increases, a feedback mechanism is activated, ruled by the interaction of several nuclear receptors, mainly the farnesoid X nuclear receptor (FXR) to inhibit the novo synthesis of BA. Therefore, the FXR is a "BA sensor," when the BA are joined to this receptor, they mediate their own synthesis control to provide a strict regulation of its reserve [13–15].

#### **2.3 Bile acid therapy in hepatobiliary disease: role of UDCA**

BA as therapeutic agent are appropriated in the chronic cholestasis deceases. BA can be orally administered following two strategies, the "displacement therapy" and/or "replacement therapy." UDCA may be used to displace endogenous BA to decrease the intrahepatic concentration of potentially cytotoxic BA accumulated in cholestasis. On the other hand, primary BA such as cholic acid (CA) might be used to replace a depleted BA pool resulted from defective biosynthesis on consequence to restore the physiological function of BA [16, 17].

UDCA (3α-7β-hydroxy-5β-cholan-24-oic acid) is naturally occurring BA, that normally constitutes 1–2% of the BA in human bile. UDCA is obtained by 7α-epimerization of the primary BA chenodeoxycholic acid (CDCA), by intestinal bacteria. [18] UDCA and CDCA differ in the hydroxyl group orientation at seventh position, allowing higher hydrophilicity of UDCA in comparison to CDCA.

UDCA is a weak acid (pKa = 5), and poorly water soluble, however, its solubility increases directly to the increase of the solution pH. After orally administrations, UDCA must be solubilized in mixed micelles present in small intestinal content in order to allow absorption [19, 20]. During the cholestasis disease, the UDCA bioavailability is limited due to the reduction of endogenous BA micelles in the duodenal lumen. Unconjugated UDCA is absorbed by passive diffusion in the proximal jejunum and in the ileum, thus extracted from portal venous blood by the liver and conjugated with glycine or taurine. Conjugated UDCA is secreted into the bile.

It is worth mentioning that in the UDCA oral administration, the half-life of the UDCA in the portal circulation is short, thus the maximum concentrations in liver/ bile achieved by dividing the dose equally over 24 h are adequate.

UDCA is the BA of choice in view of the proven efficacy and lack of side effects in the treatment of cholestasis diseases. In the case of CDCA, its inherent toxicity is related to the fact that CDCA undergoes bacterial conversion dihydroxylation to a toxic, monohydroxy BA, like lithocholic acid (LCA), unlike UDCA, which is more resistant to bacterial dihydroxylation [21, 22].

The versatility presented by UDCA in the treatment of cholestatic diseases is due to its multiple action mechanisms:


#### *2.3.1 Biliary stones dissolution*

UDCA reduces the content of cholesterol in the bile by reducing the hepatic synthesis of cholesterol and its absorption by the gut itself. In addition to solubilizing the cholesterol into micelles, it causes the cholesterol to scatter into liquid crystals in an aqueous medium causing a favorable environment for the dissolution of biliary stones. In addition to this, reduces the viscosity and improves the bile flow.

#### *2.3.2 Changes in the BA reserve levels of hydrophobicity*

In the cholestasis, the increase of hydrophobic BA produces the cytolysis of plasmatic membrane. In normal individuals, the UDCA represents not more than 4% of the complete endogenous BA reserve. Under a treatment with UDCA, this percentage increases to 40–60% under a conventional dosage of 13–15 mg/kg/day, becoming the UDCA the predominant BA, which shifts the more hydrophobic endogenous BA. Therefore, the substitution of the potentially toxic hydrophobic endogenous BA in the total BA group to a hydrophilic turns the bile more hydrophilic and less cytotoxic, reducing the hepatic lesion.

#### **2.4 UDCA and oxidative stress**

It has been proposed that UDCA antioxidative action is due to the induction of glutathione (GSH) synthesis and in this way, mitochondrial injury apoptosis is prevented [23]. UDCA activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and induces the translocation of nuclear factor-E2-related factor 2 (Nrf2) into the nucleus. Hence, it could be hypothesized that UDCA increases the gene expression of enzymes associated with GSH synthesis and induces the down-regulation of intracellular ROS levels [24]. In a similar fashion, insulin reduces oxidative stress by the activation of PI3K and extracellular signal-regulated protein kinase in HepG2 cells [25]. Therefore, both UDCA and insulin may exert a cytoprotective effects against oxidative stress and. Noteworthy, UDCA may reduce fatty acids-induced insulin resistance.

#### **3. Coenzyme Q10: generalities, clinical approaches and its relation to intrahepatic cholestasis of pregnancy**

Coenzyme Q (CoQ ) is an endogenous lipophilic compound synthetized in all tissues and cells. The biosynthetic pathway of CoQ in eukaryotes has been

**129**

**Figure 2.**

*Cholestasis: The Close Relationship between Bile Acids and Coenzyme Q10*

characterized by studies of mutants deficient in CoQ in *Saccharomyces cerevisiae*. The biosynthesis of CoQ initiates with the hydroxybenzoic acid to which a polyisoprenoid lipid tail is attached. Thus, CoQ is the product of two different converging biosynthetic pathways: the synthesis of 4-hydroxybenzoate, derived from the metabolism of tyrosine and the synthesis of the isoprene side chain that begins with the conversion of acetyl-coenzyme A (CoA) through the mevalonate route and regulated by the HMG CoA reductase. Formerly, the trans-prenyl transferase catalyzes the condensation of farnesyl pyrophosphate with numerous trans isopentenyl pyrophosphates, to form the long isoprenoid chain. Finally, these two pathways converge in a terminal step, where 4-hydroxybenzoate and polyprenyl pyrophosphate are linked by a condensation reaction catalyzed by the enzyme polyprenyl

Due to its ubiquitous distribution, CoQ is also called ubiquinone. In mammals,

CoQ10, mainly placed in the inner mitochondrial membrane, plays its principal

Although its biosynthesis is not completely dilucidated, it is well known that different mutations in some genes which codify for proteins within its biosynthetic pathway have been identified. These mutations define the primary CoQ10 deficiencies [34–40]. At this time, from the 13 known CoQ genes direct or indirect related to CoQ biosynthesis, it is recognize that eight of them can cause CoQ10 deficiency and disease [41]. Primary CoQ10 deficiencies are a group of rare diseases of clinically heterogeneous appearance suggesting an autosomal recessive inheritance, because relatives are often affected, whereas parents are characteristically unaffected. The four most frequent clinical phenotypes associated with primary CoQ10 deficiencies are encephalomiophaty, cerebellar ataxia, multisystemic infantile form, and glomerulophaty and myophaty, all of them having a muscular and neurologic compromise [27]. Patients affected with primary CoQ10 deficiency, although its clinical severity, highly respond to CoQ10 supplementation being most effective the

On the contrary, secondary CoQ10 deficiency is more frequent and of less severe clinical presentation. However, its treatment only ameliorates the symptoms although improve life quality. Secondary CoQ10 deficiency is associated to different pathologies such as neuro-muscular degenerative pathologies, cardiovascular, thyroid

ubiquinone contains a 2,3-dimethoxy-5-methylbenzoquinone core with, predominantly, a hydrophobic 10 isoprenyl units, so it is designated as coenzyme Q10

role in promoting the electron transfer from complexes I and II to complex III within the mitochondrial respiratory chain to finally obtain cellular energy [27]. Taking into account its redox properties, CoQ10 also acts as a potent lipophilic antioxidant, scavenging oxygen reactive species, protecting lipids, protein, and cellular DNA and being involved in multiple steps of vital cellular metabolism such as the electron transfer in plasmatic membranes [28] and lysosomes [29], modulation of apoptosis [30, 31] and proton transport between uncoupled proteins [32]. CoQ10 also has an important intracellular signaling role in modulating the mitochondrial

*DOI: http://dx.doi.org/10.5772/intechopen.90831*

4-hydroxybenzoate transferase [26].

permeability transition pore [33].

sooner the treatment begins [35, 42].

*Coenzyme Q10: (A) oxidized form and (B) reduced form.*

(CoQ10, **Figure 2**).

#### *Cholestasis: The Close Relationship between Bile Acids and Coenzyme Q10 DOI: http://dx.doi.org/10.5772/intechopen.90831*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

• Changes in the BA reserve hydrophobicity level

*2.3.2 Changes in the BA reserve levels of hydrophobicity*

cytotoxic, reducing the hepatic lesion.

**intrahepatic cholestasis of pregnancy**

**2.4 UDCA and oxidative stress**

• Protection against the cellular death induced by cytotoxic BA

to its multiple action mechanisms:

• Immunoregulatory effects

*2.3.1 Biliary stones dissolution*

• Biliary stones dilution

systems

The versatility presented by UDCA in the treatment of cholestatic diseases is due

• Modulation of the expression of the transporters and the liver's enzymatic

• Normalization of the altered cellular location of hepatocellular transporters

UDCA reduces the content of cholesterol in the bile by reducing the hepatic synthesis of cholesterol and its absorption by the gut itself. In addition to solubilizing the cholesterol into micelles, it causes the cholesterol to scatter into liquid crystals in an aqueous medium causing a favorable environment for the dissolution of biliary

In the cholestasis, the increase of hydrophobic BA produces the cytolysis of plasmatic membrane. In normal individuals, the UDCA represents not more than 4% of the complete endogenous BA reserve. Under a treatment with UDCA, this percentage increases to 40–60% under a conventional dosage of 13–15 mg/kg/day, becoming the UDCA the predominant BA, which shifts the more hydrophobic endogenous BA. Therefore, the substitution of the potentially toxic hydrophobic endogenous BA in the total BA group to a hydrophilic turns the bile more hydrophilic and less

It has been proposed that UDCA antioxidative action is due to the induction of glutathione (GSH) synthesis and in this way, mitochondrial injury apoptosis is prevented [23]. UDCA activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and induces the translocation of nuclear factor-E2-related factor 2 (Nrf2) into the nucleus. Hence, it could be hypothesized that UDCA increases the gene expression of enzymes associated with GSH synthesis and induces the down-regulation of intracellular ROS levels [24]. In a similar fashion, insulin reduces oxidative stress by the activation of PI3K and extracellular signal-regulated protein kinase in HepG2 cells [25]. Therefore, both UDCA and insulin may exert a cytoprotective effects against oxidative stress and. Noteworthy, UDCA may reduce fatty acids-induced insulin resistance.

**3. Coenzyme Q10: generalities, clinical approaches and its relation to** 

Coenzyme Q (CoQ ) is an endogenous lipophilic compound synthetized in all tissues and cells. The biosynthetic pathway of CoQ in eukaryotes has been

stones. In addition to this, reduces the viscosity and improves the bile flow.

**128**

characterized by studies of mutants deficient in CoQ in *Saccharomyces cerevisiae*. The biosynthesis of CoQ initiates with the hydroxybenzoic acid to which a polyisoprenoid lipid tail is attached. Thus, CoQ is the product of two different converging biosynthetic pathways: the synthesis of 4-hydroxybenzoate, derived from the metabolism of tyrosine and the synthesis of the isoprene side chain that begins with the conversion of acetyl-coenzyme A (CoA) through the mevalonate route and regulated by the HMG CoA reductase. Formerly, the trans-prenyl transferase catalyzes the condensation of farnesyl pyrophosphate with numerous trans isopentenyl pyrophosphates, to form the long isoprenoid chain. Finally, these two pathways converge in a terminal step, where 4-hydroxybenzoate and polyprenyl pyrophosphate are linked by a condensation reaction catalyzed by the enzyme polyprenyl 4-hydroxybenzoate transferase [26].

Due to its ubiquitous distribution, CoQ is also called ubiquinone. In mammals, ubiquinone contains a 2,3-dimethoxy-5-methylbenzoquinone core with, predominantly, a hydrophobic 10 isoprenyl units, so it is designated as coenzyme Q10 (CoQ10, **Figure 2**).

CoQ10, mainly placed in the inner mitochondrial membrane, plays its principal role in promoting the electron transfer from complexes I and II to complex III within the mitochondrial respiratory chain to finally obtain cellular energy [27]. Taking into account its redox properties, CoQ10 also acts as a potent lipophilic antioxidant, scavenging oxygen reactive species, protecting lipids, protein, and cellular DNA and being involved in multiple steps of vital cellular metabolism such as the electron transfer in plasmatic membranes [28] and lysosomes [29], modulation of apoptosis [30, 31] and proton transport between uncoupled proteins [32]. CoQ10 also has an important intracellular signaling role in modulating the mitochondrial permeability transition pore [33].

Although its biosynthesis is not completely dilucidated, it is well known that different mutations in some genes which codify for proteins within its biosynthetic pathway have been identified. These mutations define the primary CoQ10 deficiencies [34–40]. At this time, from the 13 known CoQ genes direct or indirect related to CoQ biosynthesis, it is recognize that eight of them can cause CoQ10 deficiency and disease [41]. Primary CoQ10 deficiencies are a group of rare diseases of clinically heterogeneous appearance suggesting an autosomal recessive inheritance, because relatives are often affected, whereas parents are characteristically unaffected. The four most frequent clinical phenotypes associated with primary CoQ10 deficiencies are encephalomiophaty, cerebellar ataxia, multisystemic infantile form, and glomerulophaty and myophaty, all of them having a muscular and neurologic compromise [27]. Patients affected with primary CoQ10 deficiency, although its clinical severity, highly respond to CoQ10 supplementation being most effective the sooner the treatment begins [35, 42].

On the contrary, secondary CoQ10 deficiency is more frequent and of less severe clinical presentation. However, its treatment only ameliorates the symptoms although improve life quality. Secondary CoQ10 deficiency is associated to different pathologies such as neuro-muscular degenerative pathologies, cardiovascular, thyroid

**Figure 2.** *Coenzyme Q10: (A) oxidized form and (B) reduced form.*

and reproductive diseases as well as cancer among others [43–46]. Coenzyme Q10 deficiency is commonly found associated to mitochondrial oxidative phosphorylation impairment, probably as an adaptive mechanism to maintain a balance in mitochondrial redox status. However, in spite of the high incidence of secondary CoQ deficiencies, the precise mechanisms underlying these secondary deficiencies remain unidentified specially in non-mitochondrial oxidative phosphorylation disorders [47].

What is certain is that final cellular CoQ10 concentration is related to the balance existing between biosynthetic and dietary supply on one side and energetic consumption on the other [48].

In a previous work, we have demonstrated a reduced plasmatic level of CoQ10 in mothers with intrahepatic cholestasis of pregnancy (ICP) as well as in an animal model, being the first report connecting CoQ10 deficiency to this disorder [49]. Later, it was confirmed in another study, which analyzed fetal CoQ10 levels in cord blood from ICP mothers [50]. It is well known that ICP is a high-risk pregnancy disease characterized by the accumulation of total serum bile acids, with an enhanced proportion of the hydrophobic bile acids which are highly cytotoxic. During the last decade, it was found many evidences suggesting that hydrophobic bile acids increase is responsible for the higher oxidative stress observed in ICP [51–53]. Thus, it was reasonable to suspect that CoQ10 levels could be diminished, secondary to the oxidative stress and/or mediated by a metabolic feedback [50]. Furthermore, a depleted CoQ9 levels (the predominant form of ubiquinone in rodents) was also observed in plasma, brain and muscle in a cholestatic rat model together with a positive correlation between CoQ9 and ursodeoxycholic/lithocholic acid ratio (UDCA/LCA). The latter suggests that increased plasma LCA may be closely related to CoQ9 decrease in blood and tissues [49].

CoQ10 decrease in ICP possibly reveals a disturbance on the delicate balance between oxidative stress and antioxidant defenses, thus accumulating large amounts of free radicals, imparing energy production, and increasing risk for the fetus. Although the relationship between CoQ10 and serum bile acids is not well established, it is possible that reduced CoQ10 levels result from enhanced ubiquinone extraction from blood because of higher cellular demand. As it was previously mentioned, it is also probable that CoQ10 depletion may be caused by increased proportion of circulating and intracellular hydrophobic bile acids and enhanced consumption of the CoQ10 by free radicals and/or a metabolic down regulation. The relationship between CoQ and bile acids will be discussed in the next section.

Since CoQ10 is a potent antioxidant and is even proposed as the first line of defense against oxidative insult [54], its tissue distribution and plasma levels will be dependent on its susceptibility to the oxidative stress induced by cholestasis.

#### **4. Bile acids and coenzyme Q10: possible relationship**

Several studies have provided evidence that oxidative stress may play an important role in the pathogenesis of hepatic injury in animal models of cholestasis [52, 55–58] and in humans [59–61].

Hepatic mitochondria have been proposed as the most important cellular source of reactive oxygen species (ROS) induced by bile acids (BA). Hydrophobic BA impair respiration and electron transport in hepatic mitochondria. Krähenbühl et al. reported that hydrophobic BA decrease the activities of several enzyme complexes involved in the electron transport chain, such as complexes I, III, and IV but not affected complex II in isolated rat liver mitochondria. Furthermore, hydrophobic BA decrease the mitochondrial membrane potential developed upon succinate energization and decrease state three and enhance state four in mitochondria [62].

**131**

CoQ synthesis [72].

secondary CoQ deficiency.

*Cholestasis: The Close Relationship between Bile Acids and Coenzyme Q10*

Yerushalmi et al. [63] proposed that ROS are generated at the ubiquinone-complex III interaction of the respiratory chain in hepatic mitochondria upon exposure to BA. Additionally, Botla et al. [64] reported that hydrophobic BA initiates the membrane permeability transition in hepatic mitochondria. In this context, oxidative stress results from an imbalance between increased free radical and impairment

Therefore, the link between BA and CoQ has recently achieved clinical relevance and open to potential therapeutics challenges. As it was aforementioned, a study with a validated animal model of ICP, which shows similar biochemical hepatic alterations as observed in ICP patients, showed a significant decrease in CoQ9 and α-tocopherol in plasma that correlated negatively with the increase in LCA levels in the animal model of ICP [49]. Stocker and Bowry reported that CoQ acts earlier than α-tocopherol in the antioxidant system, thus the reduction of plasmic CoQ could be considered as an early marker of oxidative insult [54]. The decrease in these antioxidants may contribute to increase oxidative stress in ICP [49]. CoQ plasmatic levels more likely reflect the degree of metabolic request; in this case decreased levels may be related to consumption by free radicals or by increasing cellular demand. On the other hand, tissue CoQ levels are related to the balance between biosynthesis, dietary supply and energetic consumption [48]. The increase in BA has different effect over CoQ tissue levels. It was observed that skeletal muscle and brain were more susceptible to oxidative stress and showed a decrease in CoQ levels in ICP animals, whereas liver and heart content of CoQ remained unchanged. An hepatic paradox described in animal model of cholestasis including EE cholestasis, where the activity of HMG-CoA reductase and 7 alpha hydroxylase is increased despite the increase of BA, could possible explain this finding [65–68]. Thus, taking into account, that CoQ is synthesized via HMG-CoA reductase, it is

possible that levels were maintained by an increase in its synthesis [49].

by Martinefski et al. demonstrated a highly prooxidant environment.

Nowadays, since the relationship between CoQ and BA is not well established, two explanations have been hypothesized. On one hand, during ICP, cholesterol levels could possibly be maintained due to a mevalonate pathway deviation flow that absorbs another branch of the metabolic flow including those required to support

On the other hand, hydrophobic BA stimulate the generation of ROS leading to a consumption of different antioxidants, including CoQ. Both scenarios led to a

In the field of cholestasis therapeutics, CoQ10 synthetic analog (idebenone) has shown to prevent BA stimulation of ROS from hepatic mitochondria and isolated

In accordance with those results, a significant decrease in CoQ10 and vitamin E levels was also observed in ICP patients respect to control pregnancies, coupled to an increase in total serum BA with a more hydrophobic profile [49]. It is worth mentioning that neonates are highly susceptible to oxidative damage caused by ROS, since the extrauterine environment is richer in oxygen than the intrauterine environment [69]. This problem is further aggravated by the low efficiency of natural antioxidant systems in the neonate that could be worsened if the antioxidant capacity of mother is deficient [48]. In addition, the direction of placental BA gradient, in normal pregnancy occurs from the fetus to the mother in order to promote toxic compounds elimination from the fetal compartment, while in ICP, this gradient is inverted allowing to accumulate BA in the fetal compartment [70, 71]. Thus, decreased CoQ10 levels in mothers with ICP may pose a risk for the newborn. Recently, another study which evaluates umbilical cord blood of newborn from ICP mothers showed a decrease in cholesterol normalized CoQ10 content and an increase in total serum BA respect to normal pregnancy [50]. The results obtained

*DOI: http://dx.doi.org/10.5772/intechopen.90831*

of antioxidant systems.

#### *Cholestasis: The Close Relationship between Bile Acids and Coenzyme Q10 DOI: http://dx.doi.org/10.5772/intechopen.90831*

*Hepatitis A and Other Associated Hepatobiliary Diseases*

consumption on the other [48].

to CoQ9 decrease in blood and tissues [49].

[52, 55–58] and in humans [59–61].

and reproductive diseases as well as cancer among others [43–46]. Coenzyme Q10 deficiency is commonly found associated to mitochondrial oxidative phosphorylation impairment, probably as an adaptive mechanism to maintain a balance in

mitochondrial redox status. However, in spite of the high incidence of secondary CoQ deficiencies, the precise mechanisms underlying these secondary deficiencies remain unidentified specially in non-mitochondrial oxidative phosphorylation disorders [47]. What is certain is that final cellular CoQ10 concentration is related to the balance existing between biosynthetic and dietary supply on one side and energetic

In a previous work, we have demonstrated a reduced plasmatic level of CoQ10 in mothers with intrahepatic cholestasis of pregnancy (ICP) as well as in an animal model, being the first report connecting CoQ10 deficiency to this disorder [49]. Later, it was confirmed in another study, which analyzed fetal CoQ10 levels in cord blood from ICP mothers [50]. It is well known that ICP is a high-risk pregnancy disease characterized by the accumulation of total serum bile acids, with an enhanced proportion of the hydrophobic bile acids which are highly cytotoxic. During the last decade, it was found many evidences suggesting that hydrophobic bile acids increase is responsible for the higher oxidative stress observed in ICP [51–53]. Thus, it was reasonable to suspect that CoQ10 levels could be diminished, secondary to the oxidative stress and/or mediated by a metabolic feedback [50]. Furthermore, a depleted CoQ9 levels (the predominant form of ubiquinone in rodents) was also observed in plasma, brain and muscle in a cholestatic rat model together with a positive correlation between CoQ9 and ursodeoxycholic/lithocholic acid ratio (UDCA/LCA). The latter suggests that increased plasma LCA may be closely related

CoQ10 decrease in ICP possibly reveals a disturbance on the delicate balance between oxidative stress and antioxidant defenses, thus accumulating large amounts of free radicals, imparing energy production, and increasing risk for the fetus. Although the relationship between CoQ10 and serum bile acids is not well established, it is possible that reduced CoQ10 levels result from enhanced ubiquinone extraction from blood because of higher cellular demand. As it was previously mentioned, it is also probable that CoQ10 depletion may be caused by increased proportion of circulating and intracellular hydrophobic bile acids and enhanced consumption of the CoQ10 by free radicals and/or a metabolic down regulation. The relationship between CoQ and bile acids will be discussed in the next section. Since CoQ10 is a potent antioxidant and is even proposed as the first line of defense against oxidative insult [54], its tissue distribution and plasma levels will be

dependent on its susceptibility to the oxidative stress induced by cholestasis.

tant role in the pathogenesis of hepatic injury in animal models of cholestasis

of reactive oxygen species (ROS) induced by bile acids (BA). Hydrophobic BA impair respiration and electron transport in hepatic mitochondria. Krähenbühl et al. reported that hydrophobic BA decrease the activities of several enzyme complexes involved in the electron transport chain, such as complexes I, III, and IV but not affected complex II in isolated rat liver mitochondria. Furthermore, hydrophobic BA decrease the mitochondrial membrane potential developed upon succinate energization and decrease state three and enhance state four in mitochondria [62].

Several studies have provided evidence that oxidative stress may play an impor-

Hepatic mitochondria have been proposed as the most important cellular source

**4. Bile acids and coenzyme Q10: possible relationship**

**130**

Yerushalmi et al. [63] proposed that ROS are generated at the ubiquinone-complex III interaction of the respiratory chain in hepatic mitochondria upon exposure to BA. Additionally, Botla et al. [64] reported that hydrophobic BA initiates the membrane permeability transition in hepatic mitochondria. In this context, oxidative stress results from an imbalance between increased free radical and impairment of antioxidant systems.

Therefore, the link between BA and CoQ has recently achieved clinical relevance and open to potential therapeutics challenges. As it was aforementioned, a study with a validated animal model of ICP, which shows similar biochemical hepatic alterations as observed in ICP patients, showed a significant decrease in CoQ9 and α-tocopherol in plasma that correlated negatively with the increase in LCA levels in the animal model of ICP [49]. Stocker and Bowry reported that CoQ acts earlier than α-tocopherol in the antioxidant system, thus the reduction of plasmic CoQ could be considered as an early marker of oxidative insult [54]. The decrease in these antioxidants may contribute to increase oxidative stress in ICP [49]. CoQ plasmatic levels more likely reflect the degree of metabolic request; in this case decreased levels may be related to consumption by free radicals or by increasing cellular demand. On the other hand, tissue CoQ levels are related to the balance between biosynthesis, dietary supply and energetic consumption [48]. The increase in BA has different effect over CoQ tissue levels. It was observed that skeletal muscle and brain were more susceptible to oxidative stress and showed a decrease in CoQ levels in ICP animals, whereas liver and heart content of CoQ remained unchanged. An hepatic paradox described in animal model of cholestasis including EE cholestasis, where the activity of HMG-CoA reductase and 7 alpha hydroxylase is increased despite the increase of BA, could possible explain this finding [65–68]. Thus, taking into account, that CoQ is synthesized via HMG-CoA reductase, it is possible that levels were maintained by an increase in its synthesis [49].

In accordance with those results, a significant decrease in CoQ10 and vitamin E levels was also observed in ICP patients respect to control pregnancies, coupled to an increase in total serum BA with a more hydrophobic profile [49]. It is worth mentioning that neonates are highly susceptible to oxidative damage caused by ROS, since the extrauterine environment is richer in oxygen than the intrauterine environment [69]. This problem is further aggravated by the low efficiency of natural antioxidant systems in the neonate that could be worsened if the antioxidant capacity of mother is deficient [48]. In addition, the direction of placental BA gradient, in normal pregnancy occurs from the fetus to the mother in order to promote toxic compounds elimination from the fetal compartment, while in ICP, this gradient is inverted allowing to accumulate BA in the fetal compartment [70, 71]. Thus, decreased CoQ10 levels in mothers with ICP may pose a risk for the newborn.

Recently, another study which evaluates umbilical cord blood of newborn from ICP mothers showed a decrease in cholesterol normalized CoQ10 content and an increase in total serum BA respect to normal pregnancy [50]. The results obtained by Martinefski et al. demonstrated a highly prooxidant environment.

Nowadays, since the relationship between CoQ and BA is not well established, two explanations have been hypothesized. On one hand, during ICP, cholesterol levels could possibly be maintained due to a mevalonate pathway deviation flow that absorbs another branch of the metabolic flow including those required to support CoQ synthesis [72].

On the other hand, hydrophobic BA stimulate the generation of ROS leading to a consumption of different antioxidants, including CoQ. Both scenarios led to a secondary CoQ deficiency.

In the field of cholestasis therapeutics, CoQ10 synthetic analog (idebenone) has shown to prevent BA stimulation of ROS from hepatic mitochondria and isolated

hepatocytes [63]. Therefore, taking into account the deficiency of CoQ found in ICP, supplementation with CoQ10 could represent a new complementary therapeutic proposal for ICP in order to protect both the mother and the newborn. However, further studies are required to obtain a deeper conclusion.

### **Author details**

Manuela R. Martinefski, Silvia E. Lucangioli, Liliana G. Bianciotti and Valeria P. Tripodi\* Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Argentina

\*Address all correspondence to: vtripodi@ffyb.uba.ar

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**133**

2009:237-267

2019;**21**(9):48

*Cholestasis: The Close Relationship between Bile Acids and Coenzyme Q10*

[10] Roda A, Gioacchini AM,

1995;**27**:327-331

[12] Hoffman A, Roda A.

1984;**25**:1477-1488

2009;**32**(2):S237-S245

Manetta AC, Cerrè C, Montagnani M, Fini A. Bile acids: Physico-chemical properties, function and activity. Journal of Gastroenterology.

[11] Lucangioli S, Carducci C, Tripodi V, Kenndler E. Retention of bile salts in micellar electrokinetic chromatography: Relation of capacity factor to octanol– water partition coefficient and critical micellar concentration. Journal of Chromatography B. 2001;**765**:113-120

Physicochemical properties of bile acids and their relationshiio to biological properties: An overview of the problem. Journal of Lipid Research.

[13] Stales B, Fonseca V. Bile acids and metabolic regulation: Mechanisms and clinical responses to bile acid sequestration. Diabetes Care.

[14] Tonin F, Arends IWCE. Latest development in the synthesis of ursodeoxycholic acid (UDCA): A critical review. Beilstein Journal of Organic Chemistry. 2018;**14**:470-483

[15] Tripodi V, Lucangioli S,

Scioscia S, Carducci C. Simultaneous determination of free and conjugated bile acids in serum by cyclodextrinmodified micellar electrokinetic chromatography. Journal of

Chromatography B. 2003;**785**:147-155

[16] Balistreri W. Fetal and neonatal bile aids synthesis and metabolism-clinical implications. Journal of Inherited Metabolic Disease. 1991;**14**:459-477

[17] Hofmann A. Targeting drugs to the enterohepatic circulation: Lessons from bile acids and other endobiotics. Journal of Controlled Release. 1985;**2**:3-11

*DOI: http://dx.doi.org/10.5772/intechopen.90831*

[1] Fernández-Murga LM, Petrov PD, Conde I, Castell JV, Goméz-Lechón MJ, Jover R. Advances in drug-induced cholestasis: Clinical perspectives, potential mechanisms and in vitro systems. Food and Chemical Toxicology.

[2] Patel SP, Vasavda C, Ho B, Meixiong J, Dong X, Kwatra SG. Cholestatic pruritus:

therapeutics. Journal of the American Academy of Dermatology. 2019

Larocca MC, Marrone J, Marinelli RA, Boaglio AC, et al. Mechanisms of canalicular transporter endocytosis in the cholestatic rat liver. Biochimica et Biophysica Acta - Molecular Basis of Disease. 2018;**1864**:1072-1085

[4] Roma MG, Barosso IR, Miszczuk G, Crocenzi FA, Pozzi EJS. Dynamic localization of hepatocellular

transporters: Role in biliary excretion and impairment in cholestasis. Current Medicinal Chemistry. 2019;**26**:1113-1154

[5] Ozer J, Ratner M, Shaw M, Bailey W, Schomaker S. The current state of serum biomarkers of hepatotoxicity. Toxicology. 2008;**245**:194-205

[6] Giannini EG, Testa R, Savarino V. Liver enzyme alteration: A guide for clinicians. CMAJ. 2005;**72**:367-379

[7] EASL. European Association for the Study of the liver EASL clinical practice guidelines: Management of cholestatic liver diseases. Journal of Hepatology.

[8] Navarro VJ, Senior JR. Drug-related hepatotoxicity. The New England Journal of Medicine. 2006;**354**:731-739

[9] Düll MM, Kremer AE. Treatment of pruritus secondary to liver disease. Current Gastroenterology Reports.

**References**

2018;**120**:196-212

Emerging mechanisms and

[3] Miszczuk GS, Barosso IR,

*Cholestasis: The Close Relationship between Bile Acids and Coenzyme Q10 DOI: http://dx.doi.org/10.5772/intechopen.90831*

#### **References**

*Hepatitis A and Other Associated Hepatobiliary Diseases*

further studies are required to obtain a deeper conclusion.

hepatocytes [63]. Therefore, taking into account the deficiency of CoQ found in ICP, supplementation with CoQ10 could represent a new complementary therapeutic proposal for ICP in order to protect both the mother and the newborn. However,

**132**

**Author details**

and Valeria P. Tripodi\*

Manuela R. Martinefski, Silvia E. Lucangioli, Liliana G. Bianciotti

\*Address all correspondence to: vtripodi@ffyb.uba.ar

provided the original work is properly cited.

Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Argentina

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

[1] Fernández-Murga LM, Petrov PD, Conde I, Castell JV, Goméz-Lechón MJ, Jover R. Advances in drug-induced cholestasis: Clinical perspectives, potential mechanisms and in vitro systems. Food and Chemical Toxicology. 2018;**120**:196-212

[2] Patel SP, Vasavda C, Ho B, Meixiong J, Dong X, Kwatra SG. Cholestatic pruritus: Emerging mechanisms and therapeutics. Journal of the American Academy of Dermatology. 2019

[3] Miszczuk GS, Barosso IR, Larocca MC, Marrone J, Marinelli RA, Boaglio AC, et al. Mechanisms of canalicular transporter endocytosis in the cholestatic rat liver. Biochimica et Biophysica Acta - Molecular Basis of Disease. 2018;**1864**:1072-1085

[4] Roma MG, Barosso IR, Miszczuk G, Crocenzi FA, Pozzi EJS. Dynamic localization of hepatocellular transporters: Role in biliary excretion and impairment in cholestasis. Current Medicinal Chemistry. 2019;**26**:1113-1154

[5] Ozer J, Ratner M, Shaw M, Bailey W, Schomaker S. The current state of serum biomarkers of hepatotoxicity. Toxicology. 2008;**245**:194-205

[6] Giannini EG, Testa R, Savarino V. Liver enzyme alteration: A guide for clinicians. CMAJ. 2005;**72**:367-379

[7] EASL. European Association for the Study of the liver EASL clinical practice guidelines: Management of cholestatic liver diseases. Journal of Hepatology. 2009:237-267

[8] Navarro VJ, Senior JR. Drug-related hepatotoxicity. The New England Journal of Medicine. 2006;**354**:731-739

[9] Düll MM, Kremer AE. Treatment of pruritus secondary to liver disease. Current Gastroenterology Reports. 2019;**21**(9):48

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[12] Hoffman A, Roda A. Physicochemical properties of bile acids and their relationshiio to biological properties: An overview of the problem. Journal of Lipid Research. 1984;**25**:1477-1488

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[59] Aboutwerat A, Pemberton PW, Smith A, Burrows PC, McMahon RF, Jain SK, et al. Oxidant stress is a significant feature of primary biliary cirrhosis. Biochimica et Biophysica Acta. 2003;**1637**(2):142-150

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[67] Heuman DM, Hernandez CR, Hylemon PB, Kubaska WM, Hartman C, Vlahcevic ZR. Regulation of bile acid synthesis. I. Effects of conjugated ursodeoxycholate and cholate on bile acid synthesis in chronic bile fistula rat.

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[63] Yerushalmi B, Dahl R, Devereaux MW, Gumpricht E, Sokol RJ. Bile acid-induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition. Hepatology. 2001;**33**(3):616-626

*Hepatitis A and Other Associated Hepatobiliary Diseases*

long-standing cholestasis. Free Radical Biology & Medicine. 1996;**20**(3):351-359

[56] Singh S, Shackleton G, Ah-Sing E, Chakraborty J, Bailey ME. Antioxidant defenses in the bile duct-ligated rat. Gastroenterology. 1992;**103**(5):1625-1629

Khandwala RA. Effect of dietary lipid and vitamin E on mitochondrial lipid peroxidation and hepatic injury in the bile duct-ligated rat. Journal of Lipid Research. 1991;**32**(8):1349-1357

[58] Sokol RJ, Winklhofer-Roob BM, Devereaux MW, McKim JM Jr. Generation of hydroperoxides in isolated rat hepatocytes and hepatic mitochondria exposed to hydrophobic

[59] Aboutwerat A, Pemberton PW, Smith A, Burrows PC, McMahon RF, Jain SK, et al. Oxidant stress is a significant feature of primary biliary cirrhosis. Biochimica et Biophysica Acta.

[60] Sokol RJ, Dahl R, Devereaux MW, Yerushalmi B, Kobak GE, Gumpricht E. Human hepatic mitochondria generate reactive oxygen species and undergo the permeability transition in response to hydrophobic bile acids. Journal of Pediatric Gastroenterology and Nutrition. 2005;**41**(2):235-243

[61] Vendemiale G, Grattagliano I, Lupo L, Memeo V, Altomare E. Hepatic oxidative alterations in patients with extra-hepatic cholestasis. Effect of surgical drainage. Journal of Hepatology. 2002;**37**(5):601-605

[62] Krähenbühl S, Talos C, Fischer S, Reichen J. Toxicity of bile acids on the electron transport chain of isolated rat liver mitochondria. Hepatology.

1994;**19**(2):471-479

bile acids. Gastroenterology. 1995;**109**(4):1249-1256

2003;**1637**(2):142-150

[57] Sokol RJ, Devereaux M,

delivery. Biology of the Neonate.

[49] Martinefski MR, Contin MD, Rodriguez MR, Geréz EM, Galleano ML, Lucangioli SE, et al. Coenzyme Q in pregnant women and rats with intrahepatic cholestasis. Liver International. 2014;**34**(7):1040-1048

[50] Martinefski MR, Cocucci SE, Di Carlo MB, Vega HR, Lucangioli SE, Perazzi BE, et al. Fetal coenzyme Q10 deficiency in intrahepatic cholestasis of pregnancy. Clinics and Research in Hepatology and Gastroenterology. 2019;**S2210-7401**(19):30171-30178

[51] Perez MJ, Velasco E, Monte MJ, Gonzalez-Buitrago JM, Marin JJ. Maternal ethanol consumption during pregnancy enhances bile acidinduced oxidative stress and apoptosis in fetal rat liver. Toxicology. 2006a;**225**:183-194

[52] Perez MJ, Macias RI, Duran C, Monte MJ, Gonzalez-Buitrago JM, Marin JJ. Oxidative stress and apoptosis in fetal rat liver induced by maternal cholestasis. Protective effect of ursodeoxycholic acid. Journal of Hepatology. 2005;**43**:324-332

[53] Perez MJ, Macias RI, Marin J. Maternal cholestasis induces placental

[54] Stocker R, Bowry VW. Frei B Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does alpha-tocopherol. Proceedings of the National Academy of Sciences of the United States of America.

[55] Parola M, Leonarduzzi G, Robino G, Albano E, Poli G, Dianzani MU. On the role of lipid peroxidation in the pathogenesis of liver damage induced by

1991;**88**(5):1646-1650

oxidative stress and apoptosis. Protective effect of ursodeoxycholic acid. Placenta. 2006b;**27**:34-41

2004;**86**:104-107

**136**

[64] Botla R, Spivey JR, Aguilar H, Bronk SF, Gores GJ. Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: A mechanism of UDCA cytoprotection. The Journal of Pharmacology and Experimental Therapeutics. 1995;**272**(2):930-938

[65] Dueland S, Reichen J, Everson GT, Davis R. Regulation of cholesterol and bile acid homoeostasis in bileobstructed rats. The Biochemical Journal. 1991;**280**(Pt 2):373-377

[66] Erickson SK, Jaeckle S, Lear SR, Brady SM, Havel RJ. Regulation of hepatic cholesterol and lipoprotein metabolism in ethinyl estradioltreated rats. Journal of Lipid Research. 1989;**30**(11):1763-1771

[67] Heuman DM, Hernandez CR, Hylemon PB, Kubaska WM, Hartman C, Vlahcevic ZR. Regulation of bile acid synthesis. I. Effects of conjugated ursodeoxycholate and cholate on bile acid synthesis in chronic bile fistula rat. Hepatology. 1988;**8**(2):358-365

[68] Shefer S, Nguyen L, Salen G, Batta AK, Brooker D, Zaki FG, et al. Feedback regulation of bile-acid synthesis in the rat. Differing effects of taurocholate and tauroursocholate. The Journal of Clinical Investigation. 1990;**85**(4):1191-1198

[69] Davis JM, Auten RL. Maturation of the antioxidant system and the effects on preterm birth. Seminars in Fetal & Neonatal Medicine. 2010;**15**(4): 191-195

[70] Colombo C, Roda A, Roda E, Buscaglia M, dell'gnola CA, Filippetti P, et al. Correlation between fetal and maternal serum bile acid concentrations. Pediatric Research. 1985;**19**(2):227-231

[71] Geenes V, Lövgren-Sandblom A, Benthin L, Lawrance D, Chambers J, Gurung V, et al. The reversed fetomaternal bile acid gradient in intrahepatic cholestasis of pregnancy is corrected by ursodeoxycholic acid. PLoS One. 2014;**9**(1):e83828

[72] Yubero D, Montero R, Armstrong J, Espinós C, Palau F, Santos-Ocaña C, et al. Molecular diagnosis of coenzyme Q10 deficiency. Expert Review of Molecular Diagnostics. 2015;**15**(8):1049-1059

*Edited by Costin Teodor Streba, Cristin Constantin Vere, Ion Rogoveanu, Valeria Tripodi and Silvia Lucangioli*

Hepatitis A is a major health concern throughout the world. Its impact has largely been limited in recent times by the large-scale use of vaccines. It is, however, still rampant in various parts of the world, partly due to lack of medication, poor water access, and contaminated food products. This book provides comprehensive information on various aspects of hepatitis A with a focus on three of the most important biliary diseases: cholestasis, primary sclerosing cholangitis, and hemophagocytic lymphohistiocytosis. Chapters cover such topics as pathology, epidemiology, at-risk populations, animal research models, and future trends.

Published in London, UK © 2020 IntechOpen © Dr\_Microbe / iStock

Hepatitis A and Other Associated Hepatobiliary Diseases

Hepatitis A and Other

Associated Hepatobiliary

Diseases

*Edited by Costin Teodor Streba, Cristin Constantin Vere, Ion Rogoveanu,* 

*Valeria Tripodi and Silvia Lucangioli*