Autoimmune Diseases and Inflammation

## **Chapter 10** Celiac Disease

*Nour Amin Elsahoryi*

#### **Abstract**

Celiac disease is chronic autoimmune-mediated small intestinal enteropathy. CD caused by ingestion of the dietary gluten that found in wheat, barley, and rye, in the individual who are predisposed genetically by having leucocyte antigen, (HLA)-DQ2 or -DQ8-positive. Rigorous adherence to a gluten-free diet is the only treatment for this condition to reduce the symptoms and the consequences at the short-term and the long term. The aim of this chapter is provide updates and comprehensive overview about the celiac disease epidemiology, pathogenetic information, clinical, and diagnostic methods, updated therapeutic strategy approaches that followed as a treatment and recommendations. Its challenge to understand all the domains that causes celiac disease. Finding alternative diet and trying different lifestyle still under debates. However, complete exclusion of the gluten-containing food from the patient's diet is the only effective treatment to avoid the disease complications.

**Keywords:** Celiac Disease, Epidemiology, Pathophysiology, Genetics, Diagnosis, Risk Factors, Complications, Treatment

#### **1. Introduction**

In most studies and publications, coeliac disease or Celiac disease (CD) is characterized as a serological and histological immune-mediated disease caused by dietary gluten in people who are genetically predisposed to it [1, 2]. Individuals who have positive HLA-DQ2-positive and/or HLA-DQ8 and consume glute-contains diet are susceptible to have CD because they have genetic and/or environmental factors [1, 2]. Gluten is the storage protein of wheat and founds in the endosperm of other cereals (secalins in rye, hordeins in barley and avenins in oats [3]. The gluten protein is a CD trigger that could found also in that cereals hybrids such as the spelt, and the kamut [4]. This protein is composed of two main fractions: prolamin and glutelin in various proportions among the causative cereals. For example, the prolamin is about 68% and glutelin is 82% in the wheat, 52% and 71% in the rye and 62% and 71% in the barley, respectively [4]. However, the other proteins component such as albumins and the globulins are related to the IgE-mediated allergic response in the genetically susceptible individuals that have positive leukocyte antigen HLA-DQ2-and/or HLA-DQ8 [4].

The small intestinal is the primary affected organ with CD, intraepithelial lymphocytes (IELs) count increase after gluten ingestion and the immune response leads to structural changes in the gut such as villi blunting or flattening (villous atrophy) and elongation of the crypts (crypt hyperplasia) [2]. CD could lead to various extraintestinal symptoms and manifestations (Gastrointestinal and Extraintestinal) [2] as shown in **Table 1**, but also it is very important to mention that some patients remain asymptomatic.

A few decades ago, CD was hidden and underdiagnosed disease, but recently this disease was well-diagnosed sequencE by positive celiac-specific serologic tests and small intestinal biopsy specimens to ensure the true incidence and to find the true prevalence globally [5]. In 2012, Gujral et al. [6] mentioned that approximately 1% of the global prevalence is suffering from CD. Recent systematic review and meta-analysis (2018) published that the pooled global prevalence of CD was 0.7% (95% CI, 0.5%–0.9%) [5]. The seroprevalence of CD from 96 studies was 1.4% (1.1%–1.7%, 95 CI) at significant heterogeneity (97.5%). Whereas the pooled global prevalence that was confirmed by biopsy was 0.7% (0.5%–0.9%, 95%CI). The highest CD prevalence was in Europe and Oceania (0.8%), followed by Asia (0.6%), Africa (0.5%) and 0.4% in South America as shown in **Figure 1**. Prevalence among children was higher than adults (0.9% and 0.5%, respectively) higher among the females than males (0.6% and 0.4%, respectively) [5].

To date, the only effective treatment for CD is a lifelong strict gluten-free diet, which lead to recovery of the mucosal damage recovery in the small intestine. Following restricted GFD improves the clinical symptoms and reduce the short and the long term complications that associated with CD [1, 2, 7–11].


#### **Table 1.**

*The clinical gastrointestinal and extraintestinal manifestations.*

#### **Figure 1.**

*The pooled prevalence of biopsy-confirmed CD worldwide [5].*

### **2. Epidemiology**

In the last decades, prevalence of CD was investigated in many countries by determining the new incidents in a specific time (cross-sectional studies mainly) [3]. According to some reports, CD prevalence has increased in some countries, such as the United States, where CD prevalence is estimated to be between 0.5 and 1 percent of the general population [3]. In Finland and Italy, however, the incidence was lower [1]. Overall, the CD prevalence was found through screening the general population which includes the serological tests. The difference in the diagnosis method led to inaccurate estimation of the prevalence and it remains greatly unrecognized [2, 12]. Numerous studies calculated the prevalence based on the positive serology test with duodenal biopsies to confirm the incident, whereas other studies diagnose the incident based on the positive serology test only [1–3, 12]. Although the differences in the CD diagnosis and cases reporting confirmation, the prevalence of CD incidents increases worldwide [1–3, 12]. Moreover, recent evidence pointed to increase the morbidity and mortality with CD [12, 13] and diminished the quality of life [12].

Recent systematic review and meta-analysis revealed that the worldwide CD seroprevalence is 1.4% based on positive IgA anti-TG2 test and/or test of antiendomysial antibodies [5]. The pooled global prevalence based on the confirmation of the biopsy results CD was 0.7% with a different result among the countries. For example, the high prevalence was reported in Europe and Oceania (0.8%) compared with South America (0.4%), Africa (0.5%) and Asia (0.6%) [5]. The epidemiological studies of CD reported that the incidents among females are more than males and among children more than adults [2, 3, 5]. In addition, the prevalence among patients who have higher first-degree relatives is more [1]. Moreover, the prevalence of CD is higher among the patients who suffer from other chronic diseases such as diabetes Meletus type 1 (DMT1), Down syndrome, and IgA deficiency [1, 14]. Based on specific geographical areas, United States, Brazil, Italy and Russia have the highest prevalence (1.6–2.3%), but very rarely in some countries such as China, Indonesia, Pakistan and unknown in other countries such as Far East Asia and sub-Saharan Africa [2, 3]. In the Arab country, Ashraf El-Metwally et al. reported in a recent systematic review that the CD prevalence was varied and the highest estimation was in Saudi Arabia (3.2%) and the lowest prevalence was in Tunisia (0.1%) [8]. The results among the Arab population were in agreement with the other populations, the incidence was higher among females than males, among children more than adults, and it is associated with other chronic diseases mainly Down's syndrome and DMT1 [8]. Finally, some studies reported that CD prevalence is less than 0.5% such as china but Scherf et al. consider the prevalence in such countries is underdiagnosis and the remaining cases need more investigation [3].

### **3. Risk factors of celiac disease (CD)**

Pathogenesis of CD is identified predisposing the risk factors and it is includes genetic factors and environmental exposure to the gluten protein that stimulates the autoimmunity that cause the mucosal damage and villous atrophy [1–3, 6, 12]. CD is a unique autoimmune disease in that its key genetic elements (human leukocyte antigen (HLA)-DQ2 and HLA-DQ8), the autoantigen involved (tissue transglutaminase (tTG)). In the case of CD, there is an imbalance between T helper 1 and 2 cell responses. The genetic and environmental factors are both lead to impair the function of the intestine, inappropriate immune response, and an imbalanced gut microbiome [1, 2]. Overall, susceptibility to having CD is thought to be due to a combination of genetic and environmental factors as shown in **Figure 2**.

**Figure 2.**

*Risk factors required for celiac disease development.*

#### **3.1 Genetic factor**

In the heritability of CD, there is a relevant role between the incidence and HLA haplotypes class II heterodimers, specifically DQ2 and DQ8 by ~25–40% of the genetic risk. Lindfors et al. reported that class II is histocompatibility complex molecules stated on the antigen-presenting cells (APCs) surface; they consist of an α-chain and a β-chain encoded by specific variants of the HLA-DQA1 and HLA-DQB1 genes, respectively [2]. HLA-DQ2 is encoded by the HLADQA1\*05:01 and HLADQB1\*02:01 (also called HLA-DQ2.5) alleles, whereas HLA-DQ8 is encoded by the HLADQA1\*03 and HLADQB1\*03:02 alleles [2]. Most CD patients (around 90%) have positive-HLA-DQ2 and the rest of them carry HLA-DQ8 [2]. HLA-DQ2 homozygosis form a higher risk of the early appearance of the disease in the children within the first relatives [1] and the average prevalence of CD among the first degree is more than the general population. It's important to mention that HLADQ2 and HLA-DQ8 are common among the general peoples (25–35%), and only 3% of this HLA compatible person have CD [1]. Most studies reported that the other HLA-DQ variants form a fairly modest risk effect (~15%) on CD because they are infrequently associated with this condition [1, 2, 12].

#### **3.2 Environmental factor**

Pro-autoimmune genetic background, viral infections not only the factors that could lead to the incident of CD [1–3, 12]. Ingestion gluten protein and early termination feeding practices are considered viral in CD development, autoimmunity, and then damage the mucosal tissue [1–3, 12]. Gluten is the storage protein in wheat that gives the dough viscoelastic properties [2]. Gluten is composed of alcohol-soluble constituents (gliadins) that consist of α-gliadins, γ-gliadinsand ω-gliadins, whereas the alcohol-insoluble glutenin consists of high-molecular-mass and low-molecular-mass glutenins [2]. Both segments (Gliadins and glutenins) are high in proline and glutamine amino acids which are resistant to proteolytic processing by gastric and pancreatic enzymes as well as mammalian small intestinal brush-border membrane enzymes [2]. Gluten is called CD trigger; it is a harmful dietary factor for CD patients, but to date, it is not clear why not all peoples who are genetically predisposed have CD and why some cases are diagnosed later in life [12]. The prevalence of CD is higher among the population that is characterized by higher consumption of wheat [12]. The recent epidemiological studies show the difference in the prevalence based on the region [12]. The effect of the environmental factor still varies among the studies. For example, three systematic reviews

and meta-analysis [15–17] reported that is no association between timing of gluten introduction and CD and the age of the patient, whereas other studies reported conflicting data [18, 19]. Incomplete digestion of the gluten in the human gut led to producing the gluten peptide that accesses the lamina propria through the epithelial barrier via the transcellular or paracellular route. Among CD patients, gluten peptides activate both adaptive and innate immune responses [1, 2]. Small intestinal mucosal gluten specific CD4+ T cell is an immune response in CD patients in addition to producing antibodies towards wheat gliadin and the enzyme TG2 (encoded by *TGM2*). The amino acids that available in the gluten peptide at key positions are selectively deamidated by TG2 [2].

#### **3.3 Feeding practice**

Numerous studies indicated that CD is associated with feeding practice, time of gluten introduction to the infant (age of gluten intake) and the infant diet type [1–3, 6, 12]. Consuming gluten-containing food in the first three months of the infant age is significantly associated with CD autoantibodies development compared with the latest months [2, 12, 20]. On the other hand, recent meta-analyses reported that there is no association between CD and breastfeeding [15]. Regards the time of the gluten intake, large prospective studies show that no association between CD and gluten introduction time among the high-risk populations [17, 18]. One study reported that high gluten doses in the infancy stage associated with CD [19]. The results still contradictory in this regard and more research is required.

#### **3.4 Infection**

An increased risk of CD has been linked to repeated rotavirus infection in a previous longitudinal prospective study [21]. Therefore, a recently study reported that rotavirus vaccination could have a protective effect on developing CD [22]. Furthermore, early childhood infections with enterovirus A and B, especially those with a high titer and a long duration, were linked to later CD, while adenovirus infections were not. Surprisingly [3, 23]. The prevalence of acute respiratory infections seems to be a factor as well [3, 23].

#### **3.5 Microorganisms**

CD is like all autoimmune diseases, reducing the risk of microorganisms, decrease exposure to various microorganisms and increase the hygiene aspects which could be related to reducing the autoimmune disorders [1, 2, 12]. Many studies supported the abundance of specific bacterial types with CD patients such as *Clostridium*, *Prevotella* and *Actinomyces and* specific microbial virulence genes such as viruses, including rotavirus and reovirus [2, 12]. In addition, a recent study reported that infants who carrying a high-risk genotype characterized by a low number of Bifidobacterium; *B. longus* [24]. The presence of these microorganisms and changing their function lead to an increase the autoimmune and inflammatory diseases [12]. Therefore, a number of recent studies supported the role of these microorganisms in CD development as a secondary cause with remains the direct cause proved [2, 12]. On the other hand, some studies suggested that some microorganism such as *Helicobacter pylori* or cytomegalovirus) may delay CD development, but the mechanism still unknown [2]. Some epidemiological studies focused on modulating the intestinal microbiota but still, the evidence is limited [25]. Furthermore, smoking is another environmental factor that mentions in some old studies. Snook et al. mentioned that the prevalence of CD was higher among

smokers [26]. Lower economic status as well as and it is worthy to mention under the risk factors of CD because it is the inferior hygienic environment [27]. Overall, explaining the development of CD requires deep and accurate evidence related to the patient's characteristics, genetic and environmental factors together that is currently not fully understood.

#### **4. Classification of variants of CD**

#### **4.1 Potential CD**

Potential CD characterized by positive antibody (IgA EmA and anti-tTG) for CD with HLA-DQ2/HLA-DQ8, positive genetic markers, a normal intestinal mucosa, and few inflammation signs [28]. The intestinal mucosa in the potential CD is normal or inflamed slightly due to an increase in Els number [29]. Patients with potential CD could be asymptomatic or they may have extraintestinal symptoms [29]. Most children (80%) who suffer from the potential CD are asymptomatic and the other 20% have intestinal symptoms and extraintestinal signs such as delay in the anthropometrics [30, 31]. In CD adults, the symptomatic phenotype is common than in children and mostly extraintestinal symptoms [30–32]. Regarding the treatment, the current studies suggested that symptomatic potential patients only should follow GFD [30–32] . However, only a small percentage of patients with potential CD stick to the GFD and they suffer from villous atrophy [29, 30, 32]. The villous atrophy in the CD cases happen normally due to many causes as shown in **Figure 3**, but the restricted GFD reduce the probability of most causes and consequently reduce the small intestinal villi damage [33, 34].

#### **4.2 Seronegative CD**

A CD genetic test is also important because a negative outcome definitively rules out the disorder and leads doctors to look for other causes of villous atrophy. Morphology of the intestinal mucosal and serology testing is the basics tool for CD diagnosis [1]. The endoscopy is performed after the serological tests that including EmAs TG2-Ab assays [2]. The serological tests are very accurate and sensitive, its sensitivity arrived at (90–100%) and 100% specificity for coeliac CD [35]. EmA

**Figure 3.**

*Causes of small intestinal villous atrophy.*

#### *Celiac Disease DOI: http://dx.doi.org/10.5772/intechopen.97834*

testing has long been considered the gold-standard tool for detecting the autoantibodies of CD [2]. On the other hand, the serological tests are considered a subjective test, indirect immunofluorescence, expensive and low throughput. Whereas the operator-independent enzyme is more common and operated on automated instruments by linking the immunosorbent assay (ELISA) and radio binding assay for TG2-Abs. the last method depends on the TG2 antigen quality, this means some of these tests could reveal negative and false-positive results negative and falsepositive results. However, low TG2-Ab may be associated with other autoimmune diseases such as infectious disease and DMT1 [36]. Furthermore, approximately 10% of CD patients are seronegative, meaning they are undetectable by any of the existing serological methods [37]. After one year of GFD adherence, patients performed seronegative which assure improvement in the disease symptoms and the histology, because the diagnosis in the seronegative case depends on detection of small intestinal mucosa injury [37].

#### **5. Diagnosis and screening of celiac disease (CD)**

Currently, the clinically diagnosed cases are reported the epidemiological studies, but most research reported that there are heavily underestimated cases in every country [2]. In the high knowledge countries, the prevalence of CD is closer to the real estimation, whereas the other country still has the submerged CD iceberg. Working on increasing the diagnostic rate of CD is still a point of contention [3, 38, 39]. The current findings reported that there is growth in CD diagnosis. Mucosal changes detected by duodenal biopsy and serological test positivity (antitTG antibodies, anti-endomysium antibodies (EmA), and deamidated gliadin peptide (DGP) antibodies) are the gold standard, according to the most recently updated data. Intestinal biopsy is a critical assistant to assure a correct diagnosis because there is no antibody test that can provide perfect accuracy of sensitivity and specificity [1]. Commonly, the Pediatrics skipped the duodenal biopsy if they have high anti-tTG antibodies, positive EmA, HLA-DQ2/HLA-DQ8 and CD symptoms CD based on the recent recommendations of the European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) [1, 40]. However, not all the world countries followed ESPGHAN recommendations such as the USA due to weakness in anti-tTG assays [41]. Despite this, duodenal biopsy remains an important part of the diagnosis of adult patients with suspected CD. The common clinical signs and symptoms of CD have evolved from childhood malabsorption symptoms to milder multi-organ manifestations that can occur in both childhood and adulthood [2, 12]. Loose stools, stomach pain, and flatulence are the most common signs, although in certain instances, no gastrointestinal disorders could be discovered [2]. In addition, there are some common clinical symptoms among CD patients. For instant, 10% of CD adults have Dermatitis herpetiformis and the iron deficiency anemia is also common [2, 8, 12, 42, 43]. Moreover, CD could be asymptomatic in specific cases for those who are screening with the high-risk group such as first-degree relative CD, DMT1 and down syndrome [2, 41, 42]. The current gold standard of care stated that four out of five of the following conditions are sufficient to diagnose CD [1, 45]: observe the typical signs and symptoms which include diarrhea and malabsorption; positive antibody test; positive HLA-DQ2 and/or HLA-DQ8, villous atrophy and damage in the intestine and clinical improvement resulted from following GFD. Most of these rules are used by the physicians to identify the other CD forms. Absence of intestinal damage point guides them to the potential CD, absence of the antibody

positivity guids them to the seronegative CD, absence of the typical signs and symptoms guide them to a non-classic CD while absence of the response to GFD guide them to non-responsive CD [1].

#### **5.1 Hematologic and blood biochemistry tests**

CD could be suspectable by routine blood tests [46]. Low albumin, hemoglobin and micronutrients such as potassium, calcium, vitamin D and magnesium are associated with classical CD. Concerning iron and ferritin values, low ferritin and microcytic anemia values are very common among CD patients. Dimorphic anemia, macrocytic and non-macrocytic is not popular among patients with CD [47]. Numerous micronutrients deficiency was detected among CD that leads to important symptoms and diseases such as vitamin D3 deficiency that causes osteopenia and osteoporosis [48]. Even in the absence of other relevant symptoms, a cryptogenic increase in transaminases may signal the onset of CD. Transaminases return to normal range within 6–12 months after following a GFD [49]. Among the adults, Corazza et al. reported that the blood smear has the ability to identify changes in the membrane and cytoplasm of red blood cells, whereas, Nomarski phase contrast microscopy could detect the pitted red cells [50]. However, autoimmune diseases and their complications such as refractory CD, ulcerative jejunoileitis, and lymphoma are associated with macroscopically apparent or even functional hyposplenism, which is a predisposing factor for the development of infectious diseases caused by encapsulated bacteria such as Meningococcus and Pneumococcus [51].

#### **5.2 Serology tests**

A CD genetic test is also important because a negative outcome definitively rules out the disorder and leads doctors to look for other causes of villous atrophy [1]. Morphology of the intestinal mucosal and serology testing is the basics tool for CD diagnosis [1]. The endoscopy is performed after the serological tests that including EmAs TG2-Ab assays [2]. The serological tests are very accurate and sensitive, its sensitivity arrived at (90–100%) and 100% specificity for coeliac CD [35]. EmA testing has long been considered the gold-standard tool for detecting the autoantibodies of CD [2]. On the other hand, the serological tests are considered a subjective test, indirect immunofluorescence, expensive and low throughput. Whereas the operator-independent enzyme is more common and operated on automated instruments by linking the immunosorbent assay (ELISA) and radio binding assay for TG2-Abs. the last method depends on the TG2 antigen quality, this means some of these tests could reveal negative and false-positive results negative and false-positive results. However, low TG2-Ab may be associated with other autoimmune diseases such as infectious disease and DMT1 [36]. Furthermore, approximately 10% of CD patients are seronegative, meaning they are undetectable by any of the existing serological methods [37]. After one year of GFD adherence, patients performed seronegative which assure improvement in the disease symptoms and the histology, because the diagnosis in the seronegative case depends on detection of small intestinal mucosa injury [37].

The diagnosis of CD is not simple as its overlaps with different conditions of villous atrophy such as *Giardia lamblia* (parasitic infections), Crohn's disease, autoimmune enteropathy, immunodeficiency, HIV enteropathy, tropical sprue, and Whipple disease [1, 52, 53]. However, CD patients with seronegative are associated more with autoimmune diseases compared with classical CD patients. This

#### *Celiac Disease DOI: http://dx.doi.org/10.5772/intechopen.97834*

association the morbidity due to the late diagnosis of a specific condition [52]. Deamidated Gliadin Peptide (DGP) antibodies test is the recent test that suggested in the CD diagnosis for cases the not detected by the EmA and TG2-Ab tests, but this method is not popular in the clinical practice [54]. In addition, number of commercial point-of-care rapid tests are available currently. For anti-DGPs and TG2-Abs detection [55]. Although there is little data on the performance of rapid tests, they provide immediate results in a primary care setting and may be useful in resource-constrained settings [55].

#### **5.3 Duodenal biopsy**

The duodenal biopsy still a cornerstone in the Morphological evaluation in CD confirmation and Histology still the gold standard choice of CD diagnosis [52]. With the addition of mild villous atrophy and minimal lesions as potential expressions of gluten-related intestinal injury, the histological requirements for CD have drastically modified [56]. Currently, four biopsies on the second duodenal portion are recommended and additional two biopsies at the bulb [57]. Based on Marsh classification (modified by Oberhüber), the different types of lesions of the intestinal mucosa are classified into five stages. This classification is used as a reference for CD diagnosis in all CD centres [58]. Lesion's types one and two described high IELs (with or without crypt hyperplasia) and standard villi. The potential CD is characterized by minimal intestinal lesions and positive anti-tTG and EmA. Moreover, this case (minimal intestinal lesions) is consistent with other causes such as allergies of some types of food as cow milk proteins, Crohn's disease, lymphocytic colitis. In lymphocytic enteritis, IEL cytometric pattern is more precise than subepithelial deposits of anti-TG2 IgA for CD diagnosis [59]. For CD patients, the recent evidence approved that the normal cut-off of IEL is ≥25 lymphocytes over 100 epithelial cells. The typical lesion (type 3) of CD demonstrates villous atrophy with a change in the villi-to-crypt ratio and an increase in IEL. Type three lesions are divided into three subdivided based on the severity of the atrophy as following: 3a means mild atrophy, 3b means partial atrophy, 3c means subtotal atrophy [58]. The diagnostic algorithm for CD diagnosis was illustrated by [1] in **Figures 4** and **5**.

#### **Figure 4.**

*The diagnostic algorithm for celiac disease diagnosis. Figure source [1].*

**Figure 5.**

*The diagnostic algorithm for seronegative villous atrophy. Figure source [1].*

#### **6. Complications and consequences of CD**

Most evidence suggests that having a late diagnosis of CD and/or not adhering to a strict GFD increase the mortality rate than the general population [60]. The most common complications of CD among elderly patients include Hyposplenism, Refractory CD, Hyposplenism and ulcerative jejunoileitis [1]. Around 30% of the CD adults have hyposplenism and it is associated with bacterial infections and other autoimmune diseases [61]. The refractory CD is associated with malabsorption, BMI reduction and diarrhea but it is from a small percentage of the CD cases (around 1–1.5%). Refractory CD is classified into two categories (type 1 and type 2). However, both categories are depending on the symptoms, improvement level and the CD patients response after following GFD [62]. Commonly the diagnosis of refractory CD takes place after one year of GFD by the negative serology tests [62].

Furthermore, the CD is correlated with malabsorption which resulted from villous atrophy [2]. Nutrition malabsorption leads to multiple deficiencies in the macronutrients such as the calories and micronutrients such as the vitamins and the minerals deficiencies [63, 64]. Following the GFD, on the other hand, was linked to adverse effects such as improvements in food delivery, insufficient fortification of gluten-free foods items, and individual dietary habits. The most common nutritional consequences of CD include Iron deficiency, Vitamin B12 deficiency, Vitamin D & calcium deficiencies, Folic acid deficiency, micronutrient and mineral deficiencies [63, 64]. All these nutrients deficiencies are linked with the traditional symptoms as diarrhea and BMI fluctuation [2]. Few studies have been assessed the nutritional value of the GFD, but most studies concluded that GFD has an imbalance of nutrients and could be associated with vitamins and minerals deficiencies such as calcium and vitamin and non-starch polysaccharides [2, 63, 64]. Therefore, the ideal treatment contains balanced GFD and the nutrients deficiencies should be diagnosed and treated by dietary supplement intake [63, 64].

#### **7. Celiac disease (CD) prevention and management**

#### **7.1 Prevention**

Inconsistent results were published regarding the prevention intervention for CD. The old study suggested starting early feeding wheat during breastfeeding to the infant [65]. In contrast, other intervention and cohort studies concluded that early wheat feeding practice did not prevent CD [3, 17]. Moreover, a number of systematic reviews and meta-analyses were approved that the infant practice such as duration of the breastfeeding not associated with CD prevention [15, 66]. Another Swedish cohort data reported that increasing the gluten consumption above five grams daily during the first two years is associated with an increase in the risk of CD among the study population [67]. From another side of view, some studies link intestinal infection and increase the CD risk factor [27, 68]. Therefore, the association between CD prevention effect and the environmental practice still needs further evaluation. Overall, the primary strategies of CD are the early and the correct diagnosis by screening diagnosis or by case finding [2]. Whereas secondary prevention reducing the symptom and the complications by following the recommended treatment [3, 17].

#### **7.2 CD management**

To date, the only effective treatment for CD is consuming gluten-free food such as fruit, vegetables, legumes, and gluten-free cereals. According to Codex Alimentarius (Codex Standard 118-1979) the recommended amount of gluten for CD patients does not exceed 20 mg/kg of gluten [69]. The researchers now are trying to improve the quality of gluten-free products by developing new analysis methods for gluten detection [3]. The safety of the gluten-free diet is an important aspect for CD patients because the contaminated products could have an illegal amount of gluten. In addition, the tolerable level varies among the CD patients.

The compliance rate of CD patients to the GFD is very poor. In general, children have more adherence to the GFD than adults because they are diagnosed in early childhood [3]. Patients compliance is affected by the age, gender, socioeconomic status of the patients [70]. Many studies were conducted on the CD patients compliance to the GFD to improve adherence [70–73]. However, some of these studies result was based on the symptoms diagnosis after following GFD and other studies used Anti-TG2 serology test to check the compliance rate. In general, the antibody titers decrease gradually after following a strict GFD, sometimes it takes a longer time in the case if it was high at diagnosis [70]. However, the best test is performing duodenal biopsies to check the compliance and the adherence of the CD patients. Comino et al. reported that detection of the gluten immunogenic peptides (GIP) in patient's feces urine was used as a biomarker for the patients gluten intake and consequently to the GFD adherence [74]. However, the last method is non-invasive as duodenal biopsies and it is relatively simple but it has weakness point in the relation of the antibody levels and with the dietary assessment questionnaires [74].

Following GFD is the only treatment to date and efficacious for most patients but following strict GFD is not easy. On the other hand, Most CD patients prefer any non-dietary treatments like vaccination, supplements, or medications [75].

Therefore, studies are making a great effort to find alternative treatments for CD patients [12]. A number of studies reported the availability of proteolytic enzymes (glutenases) of microbial or plant origins that could degrade gluten proteins quickly in similar conditions to the human stomach (low pH). These glutenases enzymes are promising drugs to eliminate the immunogenic capacity of dietary gluten due to their effectiveness in cleaving proline-and glutamine-rich gluten sequences [76–78]. Vitro and pre-clinical studies indicated that the glutenase leads to reduce the number of gluten epitopes in wheat-containing food [76–78]. Bethune believes that using glutenases as an oral enzymatic therapy for CD is a good idea [79]. These enzymes can be produced by the germination of wheat and it could be used to eliminate residual gluten from the food, but it is not enough to use as oral supplements [80]. Alternative treatment for CD patients is still being researched in vitro and in vivo, either as an add-on therapy to the GFD, as a rescue therapy after accidental gluten exposure, or as a substitute for the GFD [3].

#### **8. Gluten-free diet (GFD)**

The recommended and the documented treatment for CD patients, to date, is lifelong strict adherence to GFD [3, 8, 9, 44, 81–83]. Catassi et al. revealed that consuming 50 mg of gluten for three months could destroy the small intestine [84]. However, there is no documented recommended dose for the threshold dose for CD especially for the children [12]. While good adherence to the GFD leads to better intestinal healing and consequently improves the symptoms compared with patients who consumed gluten-contaminated food [12]. Using GFD led to avoid the gluten peptide sources located in the wheat, rye, barley and all crossbreeds of these cereals [85]. Wheat varieties such as kamut, einkorn, spelt also should be avoided because it is derived from wheat and many cereals are still in debate because it is contaminated by the wheat as oat [12]. However, wheat is the main component of the main food items such as bread and pasta. In addition, it is used in food processing as a thickening and stabilizing agent. Therefore, it exists in many food products. Following GFD leads to improve the symptoms which are considered as the first key sign of GFD adherence [86]. In addition, the small intestinal histology assessment and the inflammation tests, serology tests and dietary history assessment are the clinical checkpoints that are used to confirm the GFD adherence [87]. However, patients who are following GFD notes improvements in all the previous symptoms. Furthermore, some studies reported that following restricted GFD among CD patients is associated positively with the intelligence and education level [88]. In contrast, untreated patients and poor adherence to the GFD could lead to intestinal mucosa destruction and consequently suffer from nutrients deficiencies such as iron-deficiency anemia, malnutrition, malabsorption such as calcium and vitamin D, bone disorders [64]. Improvement the symptoms, malabsorption adjustment and health status recovery could be observed gradually after following GFD [89]. On the other hand, following GFD is a common trend worldwide as some peoples consider omitting wheat from the diet to support the health status [90]. Furthermore, patients with gluten intolerance that caused or Non-celiac gluten sensitivity follow GFD without proof of CD [91].

WHO codex [92] FDA define the gluten-free term in (2013) that food contains <20 parts per million (ppm) of gluten which equivalent to 20 mg of gluten per kg of food [12]. The starch of gluten contains little amounts of residual gluten, therefore, most factories nowadays are trying to purify the starch based on the standard to meet the Codex requirements in the US [12]. In other countries such as Australia and New Zealand, the guidelines are different. The rule is zero gluten in gluten-free food [12]. Currently, the most common analysis method to measuring the gluten in the food is R5 ELISA (Mendez) [93]. Despite the current use of this assay, it is not accurate to detect the contaminated product such as the oats and the barely as they

#### *Celiac Disease DOI: http://dx.doi.org/10.5772/intechopen.97834*

contain different -molecular-weight peptides unlike that found in the wheat [94]. Therefore, improve more accurate analysis methods to detect the gluten in the food is still under enhancement [12].

Overall, following GFD is the ideal treatment for CD patients, but this diet includes a number of considerations as it contains a high amount of carbohydrates and a lower amount of fibers compared to the normal diet [12]. The ideal GFD should include balanced nutrients besides deity supplements if needed in the case of the nutrients deficiencies [63, 64]. The nutrient recommendations (based on age and gender) revealed that the carbohydrates should cover 55% of total calories with adequate dietary fiber (20–35 g daily). Around 25–30% or less of the total caloric should come from the monounsaturated and polyunsaturated fatty acids. In addition, five servings of fruit or vegetables daily at least are also recommended [63, 64].

### **9. Follow-up CD patients**

Consult a dietitian is strongly recommended for CD patients after confirming the diagnosis [63, 64]. The dietitian can help the patient how to balance the GFD to improve the symptoms through six main principles that have been recommended for CD patients by NIH guidelines [95]. As well as follow of the CD patients is very important to confirm the patent's adherence to GFD and to check the consequences and the complications [2]. Analysis of the serum antibodies used to detect the GFD although other tests such as serological testing are also recommended [2]. The only reliable tool now is repeating the biopsy during the GFD but it is challenging [96].

#### **10. Conclusions**

Based on the most recent research, the prevalence of CD is increasing over time CD and it is starting to become emerge in some countries such as Africa and Asia. Many factors were suggested by a large number of studies that include mainly; Genetic predisposition, exposure to gluten, loss of intestinal barrier function, a proinflammatory innate immune response triggered by gluten; inappropriate adaptive immune response is the main reason for CD. The imbalanced gut microbiome has an indirect cause that could enhance disease development. Economic status and smoking are considered as other risk factors that could be related to CD. To date, there is still a gap in the scientific evidence regards the relationship between all the suggested factors that could lead to the CD. Therefore, more data should be collected to estimate the prevalence of CD in the world. In addition, further research is needed to identify the factors that influence the progression of CD autoimmunity to mucosal damage, as well as the biomarkers that predict this progression, so that preventive measures and non-dietary treatments can be developed. Following GFD is the effective treatment until now. Raise awareness level of CD regards the early diagnostic, effective diet, and other related issues are the most appropriate policy to be implemented to improve the diagnostic rate of CD.

#### **11. Recommendations**

• Biopsy remains the main and the essential tool for CD diagnosis and the serology cannot substitute for biopsy in the diagnosis of adult CD.


### **Conflict of interest**

The authors declare no conflict of interest.

#### **Acronyms and abbreviations**


### **Author details**

Nour Amin Elsahoryi Department of Nutrition, Faculty of Pharmacy and Medical Sciences, The University of Petra, Amman, Jordan

\*Address all correspondence to: nour.elsahoryi@uop.edu.jo

© 2021 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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#### *Celiac Disease DOI: http://dx.doi.org/10.5772/intechopen.97834*

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#### **Chapter 11**

## Sialoendoscopy in Juvenile Recurrent Parotitis That Could Be Primary Pediatric Sjogren's Syndrome

*Brigida Iorio, Roberto De Luca, Gianpaolo Tartaro and Giuseppe Colella*

#### **Abstract**

Parotid swelling often is encountered in the pediatric population, essentially acute and self-limiting, which usually represents viral or bacterial infections. Less common etiologies include juvenile recurrent parotitis (JRP) or pneumoparotid or anatomic abnormalities. Sjögren's syndrome is common in JRP (40% almost). Levels of suspicion for an autoimmune disorder should be maintained for children affected by JRP, particulary in bilateral glands involvement in order to optimize diagnoses and facilitate treatment. Cytological examination of saliva, which is normally in children is acellular, shows granulocytes, lymphocytes, and in some cases 50% of bacteria. Sialoendoscopy typically shows whitish ductal walls and the presence of stenosis without evidence of solid obstructions and/or mucous membranes. Sialoendoscopic treatment can improve symptoms thanks to local anti-inflammatory therapy and sialoendoscopic washing.

**Keywords:** juvenile recurrent parotitis (JRP), Sjögren's syndrome (SS), sialoendoscopy, pediatric parotitis, sialoadenitis

#### **1. Introduction**

Juvenile recurrent parotitis (JRP) was firstly described by Rose in 1953 [1]. The prevalence of JRP in children is 10 times lower than in adults, yet it is the second leading cause of inflammatory salivary gland disease in children after mumps, and the differential diagnosis between them is difficult in young children [2].

JRP is defined when a minimum of 2 and maximum of 30 episodes per year of painful parotid inflammation occur, usually associated with fever, swelling, and erythema of the overlying skin gland [3, 4].

Sjögren's syndrome (SS) is an idiopathic systemic autoimmune disease affecting exocrine glands with classic symptom complex of dry eyes (xerophthalmia) and dry mouth (xerostomia). Extraglandular involvement in SS may affect many systems such as renal, central nervous system, and vascular and hematological ones. Primary SS (pSS) occurs with a prevalence of 0.1-0.4% in the Caucasian population with a female predominance in the fourth and fifth decades. Conversely, in

childhood and early adulthood SS is an extremely rare autoimmune condition (only 147 cases described in literature) [5, 6].

We hypothesized a higher prevalence of pSS in our young patients and we adopted a protocol with clinical, laboratory, and endoscopic examination. Sialoendoscopy has gained an increasing role in both diagnosis and treatment of JRP in a pediatric age. After its introduction in 1991 for the treatment of salivary disorders in adults, sialoendoscopy has also been validated as a safe technique in JRP treatment by Nahieli and Marchal [2, 7].

#### **2. Clinical findings**

#### **2.1 JRP**

JRP clinical symptoms include intermittent, usually unilateral swelling of the parotid gland, which occurs suddenly (over minutes or hours) and may persist for days or weeks [8]. The first episode typically occurs in scholar age, between 3 and 6 years, more often in males [9].

The etiopathology of JRP remains obscure; many factors have been suggested for the development of JRP including retrograde infection of the duct, viral or bacterial infection, autoimmune disease [10], disrupted enzyme activity [11], dental occlusion disorders [8, 12, 13], hypogammaglobulinemia, immunoglobulin A deficiency, and immunoglobulin G3 deficiency [14, 15]. These theories subtend a phagocyte dysfunction and humoral immunodeficiency. Another hypothesis considers JRP as mucosa-associated lymphoid tissue disorder, hyperplastic cells surrounding the ducts in a manner similar to chronic inflammatory disorders. The recurrent nonsuppurative ductal inflammation in JRP leads to a squamous ductal metaplasia, progressive parotid atrophy, and insufficient salivary outflow throughout the ductal system [16].

Histologically, there are intraductal cystic dilatations of peripheral ducts with periductal lymphocytic infiltration, called sialectasis [2]. The ecstatic ducts are usually 1–2 mm in diameter and typically have a white appearance of the ductal layer without the healthy blood vessel coverage, when compared with a normal gland [9]. This aspect is believed to be the characteristic of JRP.

The diagnosis is based on the clinical history, and clinical examination shows parotidomegaly with or without mucopurulent salivary secretion from the Stensen's duct with papilla hyperemia. Diagnosis is confirmed by ultrasound and sialography [17, 18]. Some studies also describe the use of magnetic resonance (MR), MR-sialography, characterized by T1-weighted hypointensity and T2-weighted hyperintensity; MR and MR-sialography, and CT and sialo-CT are reserved for special cases in which expansionary diseases may be suspected [19].

Thanks to reproducible, safe, and economic rule salivary gland ultrasonography in the most used imaging, especially in childrens. Choi [20], Blatt [21], and Xie [17] proposed classification on sialographic images: Glandular homogeneity and the presence of hypoechogenic areas were evaluated and graded (range 0–3).

Grades 0–1 were considered to correspond to normal/nonspecific changes and grades 2–3 to correspond to pathologic changes.

Treatments in acute are based on antibiotics and anti-inflammatory corticosteroid therapy; rule of low-dose preventive corticosteroid was not confirmed. Chronic management intercurrent periods are based on massage, warmth, chewing gum use, and sialogogues. Sialography reduces frequency of acute episodes but also sialoendoscopy is useful in diagnostic and treatment of JRP. Many authors underline a reduction of episodes [2, 22, 23]. In summary, JRP is a clinical condition *Sialoendoscopy in Juvenile Recurrent Parotitis That Could Be Primary Pediatric Sjogren's Syndrome DOI: http://dx.doi.org/10.5772/intechopen.94193*

characterized by parotid gland recurrent episodes of pain and swelling, usually accompanied by fever and malaise determined by inflammation. This condition affects infants and children between 3 and 6 years old, with a clear preference for the male, and usually disappears at puberty. It is associated with not obstructive parotid gland sialectasis. Although the affected gland demonstrates distal duct sialectasis, it seems there is evidence of obstruction in most cases. Symptoms are generally unilateral, when bilaterals are always marked on one side. The number of occurrences individually varies but is more commonly repeated every 3–4 months. Some recent studies (Houghton et al.) suggest that some symptoms, including recurrent conjunctivitis and mumps, if added to the diagnostic criteria encoded by AECG greatly increase the sensitivity of the latter in the diagnosis of pSS in the child. In the pSS population, misclassification is especially problematic at disease onset and early in disease course, when classic symptoms and signs are often not manifested [24].

#### **2.2 Juvenile Sjögren's syndrome**

Juvenile Sjögren's syndrome is extremely rare; the average presentation time is around 10 years and affects 77% of girls. Juvenile Sjögren's syndrome begins with major salivary gland swelling but can involve in 50% of patient many organ systems with neurologic, dermatologic, musculoskeletal, vascular, gastrointestinal, respiratory manifestations. There are no criteria for Juvenile Sjögren's syndrome and the use of adult criteria has not been validated. Recently, ultrasound criteria were developed compared with promising results both with salivary gland biopsy and with seroprevalence of antibodies anti-Ro/SSA and anti-La/SSB. Homogenity and the presence of hypoechogenic zones and cystic areas were evaluated by ultrasound of the parotid and submandibular glands on a scale from 0 to 3 where the degree 0–1 corresponds to normal and degrees 2 and 3 to the pathological changes [25]. Primary SS is a difficult diagnosis in childhood because of different presentation of symptoms. There are specific laboratory findings as lymphocytic infiltration of exocrine glands, hypergammaglobulinemia, anti-Ro/SSA, and anti-La/SSB antibodies but the manifestations of oral dryness are rarer and appear later, mainly concerning dry and cracked lips and tongue depapillation [26].

There are several proposed sets of diagnostic criteria for adult pSS. The revised European Community Study Group classification criteria proposed by the American-European consensus group (AECG) [24] have been validated for adults and include six items: ocular symptoms, oral symptoms, evidence of keratoconjunctivitis sicca, focal sialoadenitis by minor salivary gland biopsy, instrumental evidence of salivary gland involvement, and presence of SSA or SSB autoantibodies. In adults, the presence of four of the criteria, with the exclusion of patients who have negative autoantibodies or minor salivary gland biopsy, was found to have a sensitivity of 89.5% and specificity of 95.2%. The proposed diagnostic criteria for Sjogren's syndrome in adults (formulated by the European Community Study Group and later revisited by the American-European Consensus Group Criteria) cannot be applied for the diagnosis in children because they have an unacceptably low sensitivity. Further, the criteria for the diagnosis of juvenile pSS suggested by Bartunkova et al. [27] were not validated because pediatric patients rarely have sicca syndrome or xerophthalmia at presentation (almost always present in the adult with pSS) and autoantibodies often appear late in the course of the disease but often present with parotid symptomatology (mainly intended as swelling and pain) as evidenced by studies carried out by Baszis et al. and by our clinical experience. According to our opinion, levels of suspicion for an autoimmune disorder should be maintained for children affected by recurrent parotitis, particularly bilateral involvement in order to timely recognize this disease and to facilitate treatment and screening for complications.

Treatment of pSS is based on anti-inflammatory and immunosuppressive drugs especially in patients with muscle and joint pain; salivary substitutes and cholinergic stimulators can be applied locally through oral dry [26].

#### **2.3 Clinical experience**

We reported our experience in the sialoendoscopic management of JRP that represents a valid and effective treatment.

We enrolled 16 consecutive patients aged between 5 and 17 years (mean 7.5), 10 males and 6 females, referred to Multidisciplinary Department of Medical and Dental Specialities, Division of Oral and Maxillofacial Surgery (Università degli Studi della Campania Luigi Vanvitelli, Naples, Italy) in the last 5 years. They were subjected to clinical, serological, microbiological and ultrasound screening for excluding tumors or infectious diseases. We have therefore included children with remitting unilateral or bilateral swelling of the parotid region that may last from a few days to months; with the presence or absence of autoantibodies suggestive of autoimmune disease, we analyzed typical SS antibodies (SS-A, SS-B, anti-dsDNA, anti-Sm, anti-RNP), antinuclear antibody (ANA), rheumatoid factor (RF). Ultrasound images were characterized by areas of ectasia and hypoechoic spots or sialoendoscopic features of whitish ductal walls, ectasia, and stenosis. Seven of 16 patients, 4 males and 3 females, with a mean age of 9.57 years, reported SS clinical and laboratories signs.

Exclusion criteria were the presence of viral markers, neoformations detectable with methods of imaging, the acclaimed presence of autoimmune disease, sarcoidosis, graft-versus-host disease (GVHD), past head and neck radiation, and known human immunodeficiency virus infection or hepatitis C infection.

Patients thus selected were diagnosed with JRP and were treated with sialoendoscopy (intraductal wash of saline solution and steroids) or other anti-inflammatory drugs (**Figures 1** and **2**).

From the formulation of diagnosis, patients were subjected to careful follow-up checks with 6, 12, and 18 months during which they proceeded to repeat initial serological screening if the endoscopic or systemic therapy became ineffective.

The endoscopic-assisted procedures were performed in the ambulatory operating suite. After detection of the impaired gland, a local anesthesia with lidocaine 2% to the orifice region and a gradual dilatation of the duct orifice were performed, (thanks to increasing diameter lacrimal probes from 0000 to 0 size) and also with a standard salivary dilator. In this way, we reached 1.3 mm diameter, matching the outer diameter of the sialoendoscope diagnostic unit (Erlangen Sialoendoscope— Karl Storz). The larger (1.6 mm diameter) scopes were introduced as needed. The diagnostic unit 0.8 mm was introduced into the duct and was advanced forward, until reaching the ductal system and thanks to continuous lavage with isotonic saline solution. The plaques were washed out, and any structures were dilated. Mucous plugs and debris were removed with irrigation or with a forceps if necessary. At the end of the procedure, the ductal system was irrigated with a steroid- solution of Besamethasone, under direct vision while withdrawing the scope in order to treat the inflammation of the ductal epithelium (sialodochitis) and to promote the dilatation of ductal structures. In the post-operatory, we prescribed Rovamycine 3000 U.I. every 12 hours for one week. The follow-up period was at least 12 months to 5 years. In all 16 patients, sialoendoscopic treatment was effective. We reported only 2 patients with a recurrence at 5 and 3 years, respectively. Seven of 16 patients (43,75%) presented SS features. In contrast to other cases of JRP resolution, the symptoms were not immediate, but an average of three sialoendoscopic treatments; the patients identified had the following characteristics (**Table 1**).

*Sialoendoscopy in Juvenile Recurrent Parotitis That Could Be Primary Pediatric Sjogren's Syndrome DOI: http://dx.doi.org/10.5772/intechopen.94193*

**Figure 1.** *Sialoendoscopic treatment in young patient.*

**Figure 2.** *Sialoendoscopic image of duct.*

pSS is probably underdiagnosed in childhood, as the mode of presentation can be large and are not rare long delays in diagnosis. And likely that some patients diagnosed with PSS in adulthood have experienced the onset of symptoms during


**Table 1.** *Clinical, ultrasound, endoscopic, and laboratory features of patients with suspected SS.*

*Inflammation in the 21st Century*

*Sialoendoscopy in Juvenile Recurrent Parotitis That Could Be Primary Pediatric Sjogren's Syndrome DOI: http://dx.doi.org/10.5772/intechopen.94193*


**Table 2.**

*Presentation differences in adults and children.*

childhood. In fact, the clinical presentation of pSS in childhood may differ from the clinical presentation in adulthood (**Table 2**). The cases of pediatric PSS are reported to have a higher incidence of recurrent parotitis and a lower incidence of xerostomia and xerophthalmia. We found dry eyes in four patients. Laboratory and pathological findings in children with SS are positive antinuclear antibody (ANA) of 80% and autoantibodies to nuclear antigens Ro/SSA and La/SSB2 of only 70–75% [27]. In our experience, we have detected the presence of specific antibodies in only two patients.

In pediatric recurrent parotitis, laboratory evaluation and sialoendoscopy may be very helpful. A combination of positive ANA, RF, SS-A, and SS-B; hypergammaglobulinemia; elevated amylase (parotid or pancreatic); elevated ESR and suggestive chronic inflammatory endoscopic patterns are suspicious for SS (however, the presence of anti-double-stranded DNA antibodies or hypocomplementemia raises the concern for other systemic autoimmune disorders, such as systemic lupus erythematosus, SLE, or another connective tissue disease) [5].

#### **3. Conclusion**

In summary, parotitis is a frequently encountered pediatric problem. Although infection, recurrent juvenile parotitis, and anatomic abnormalities are more common etiologies, primary pediatric SS should be considered when encountering a patient with recurrent parotitis, especially no responsive to sialoendoscopic treatment. We have to consider pSS when JRP is assessed, despite the rareness of condition. Typical sign of sialectasis is the same in JRP and in pSS as ultrasonography ones. Antibodies are not in all patients: anti-Ro/SSA 29% seronegativity and anti-La/SSB 33%; similarly, eye dryness is present in just over half of patients.

These patients typically exhibit a distinctive laboratory and autoantibody profile and will benefit from early referral to a pediatric rheumatologist for treatment and monitoring for disease complications. Accurate diagnosis of pSS in the pediatric population is difficult. Recurrent parotitis should alert the clinician to the possibility of pSS especially if it does not respond to treatment with anti-inflammatory therapy and sialoendoscopic washing. The proposed pediatric criteria lack sensitivity and clinical utility. Until validated diagnostic criteria are available, clinical acumen will prevail as the gold standard. Reid et al.'s theory of ascending infections from the upper respiratory tract would merit a multicenter study of prevalence in patients undergoing adenotonsillectomy that should slow down the frequency of the disease. Our intention is to verify this deduction in the near future. Prospective multicenter studies are needed to further characterize pSS in the pediatric population, and to better define and develop appropriate classification criteria.

Consideration should be given to inclusion of specific obligatory criteria such as the presence of SSA or SSB autoantibodies or classic histopathology changes on minor salivary gland biopsy. The development of an international database would enable epidemiologic and clinical study.

In the majority of our patients, it is suggested that sialoendoscopy can offer a minimally invasive and gland-preserving approach to obstructive salivary glands diseases; this technique is proven to be safe, suitable in children under local anesthesia and able to improve swelling, pain, and social life.

### **Conflict of interest**

The authors declare no conflict of interest. Neither author nor any member of their families received any material or financial gain or personal advancement in the production of this manuscript. We have read the Helsinki Declaration and have followed the guidelines in this investigation. For each patients, we have written parent's contents.

### **Notes/thanks/other declarations**

Thanks to our young patients.

### **Author details**

Brigida Iorio1 \*, Roberto De Luca1 , Gianpaolo Tartaro2 and Giuseppe Colella2

1 Maxillofacial Surgery Unit, Department of Medical, Surgical and Dental Speciality, Campania University "Luigi Vanvitelli" Naples, and Department of Neuroscience, Reproductive and Odontostomatologic Sciences, University of Naples Federico II, Naples, Italy

2 Maxillofacial Surgery Unit, Department of Medical, Surgical and Dental Speciality, Campania University "Luigi Vanvitelli" Naples, Italy

\*Address all correspondence to: brigida.iorio@libero.it; brigida.iorio82@gmail.com

© 2021 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.

*DOI: http://dx.doi.org/10.5772/intechopen.94193 Sialoendoscopy in Juvenile Recurrent Parotitis That Could Be Primary Pediatric Sjogren's Syndrome*

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#### **Chapter 12**

## Inflammation in the Pathogenesis of Rheumatoid Arthritis and in Experimental Arthritis: Evaluation of Combinations of Carnosic Acid and Extract of *Rhodiola rosea* L. with Methotrexate

*Silvester Ponist, Katarina Pruzinska and Katarina Bauerova*

#### **Abstract**

The host immune response generates the pro-inflammatory immune response as a protective measure against invading pathogens, allergens, and/or trauma. However, dysregulated and chronic inflammation may result in secondary damage to tissues and immune pathology to the host. Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease which primarily involves synovial inflammation, joint pain, immobility, and stiffness. Increased infiltration of inflammatory immune cells and fibroblast-like synoviocytes into joints, form pannus and small blood vessels that lead to synovium and cartilage destruction. In this chapter we will focus on the role of inflammatory cytokines (IL-1β, IL-6 and IL-17), chemokine monocyte chemotactic protein-1 and matrix metalloproteinase-9 in the pathogenesis of experimental arthritis in animals and in human RA. Further, we will be discussing about methotrexate's (cornerstone of anti-rheumatic therapy) immune suppressing activity, anti-inflammatory properties of carnosic acid and extract of *Rhodiola rosea* L., and their innovative combination treatments with methotrexate in rat adjuvant arthritis.

**Keywords:** arthritis, IL-1β, IL-6, IL-17, monocyte chemotactic protein-1, matrix metalloproteinase-9, carnosic acid, *Rhodiola rosea* L

#### **1. Introduction**

Inflammation is an inherent defensive mechanism against damage of tissues, infection and is quickly stopped in physiological state of organism. In chronic diseases, the inflammation continues and is able to cause substantial organ and tissue damage. A lot of evidence showed that pathological inflammatory response is closely related with different chronic diseases, particularly autoimmune ones, such as systemic lupus erythematosus, rheumatoid arthritis (RA), inflammatory bowel disease, diabetes, and gout [1–3]. Although the key feature of inflammatory dysregulation in many chronic diseases has been supported by plenty of studies,

the pathogenesis of this dysregulation in the autoimmune diseases is not well understood yet. Knowledge about the signaling and mechanism of regulation of inflammation will bring noticeable clinical benefits for the therapy of autoimmune disease.

In this chapter we will present our preliminary results from new original combination treatments of methotrexate with carnosic acid and with extract of *Rhodiola rosea* L and discuss about the role of IL-1β, IL-6 and IL-17, chemokine monocyte chemotactic protein-1 and matrix metalloproteinase-9 in the pathogenesis of experimental arthritis in animals and in human RA.

#### **2. Cytokines involved in rheumatoid arthritis**

To fully understand a complex disease like a RA, animal models are indispensable due to their ability to mimic the conditions and demonstrate the similarity to the human RA. Rodent models are essential for further knowledge of the pathogenic processes of RA in humans and therefore are important in the process of testing new and already existing drugs for their efficiency and safety. There are many animal models used for the research of RA, but each model varies in the similarities to the human RA. The most frequently used animal models are collageninduced arthritis and adjuvant-induced arthritis models. Less often are used animal models with proteoglycan-induced arthritis and streptococcal cell wall-induced arthritis [4].

The adjuvant-induced arthritis (AIA) model has been used widely for testing novel drugs for inflammatory arthritis and for studies of the disease pathogenesis. After administering an injection with complete adjuvant, it was possible to induce polyarthritis [5]. AA is inducible in susceptible rat strains, for example, Lewis rat strain, by a single subcutaneous injection of heat-killed *Mycobacterium tuberculosis* H37Ra in oil. Following the induction, the inflammation begins in 8–10 days, the symptoms are the most apparent on the 15th or 16th day, and then undergo spontaneous recovery. Autoimmune inflammation of the paws starts with the infiltration of mononuclear cells, mostly lymphocytes, macrophages, and monocytes [6]. The severity of the RA could lead to chronic malformation of affected joints, together with ankylosis. Adjuvant-induced arthritis exhibit similar symptoms to human RA, such as joint swelling, invasion of lymphocytes, and destruction of cartilage [4].

The difference between AIA in rats and human RA seems to be in the rapid onset of the erosive polyarthritis in the AIA model, Rheumatoid Factor is not present, the disease seems to have a monophasic course. There is also an involvement of axial skeleton seen in the model of AIA, affected gastrointestinal, genitourinary tract and skin, periostitis, ankylosis, and extra-articular manifestations not typical of RA [7]. Inflamed joints of rats with AIA contain activated T-cells. T-cells infiltrating joints originate from several compartments, such as the spleen, Peyer's patches, lymph nodes, and T-cell pool that recirculates [8]. Specific antigen heat shock protein (Hsp65) has been shown to activate the immune response, with peptide 180–186 being the responsible epitope [9]. The cytokines that are expressed in the joint during the early stages of inflammation include IL-17, IFN, and TNF-α, as well as cytokines implicated in macrophage stimulation. Increased levels of IL-4, IL-6, monocyte chemotactic protein 1 (MCP-1), and TGF-β can be observed as inflammation progresses in the joint. TNF-α, IL-1β, IL-21, and IL-17 all contribute to the pathology of this disorder [8]. The main source of the irreversible tissue damage is in an area rich in macrophages, called the pannus, which is located at the junction of the synovium lining of the joint capsule together with the cartilage and a bone. Pannus cells migrate over the cartilage and into the subchondral bone, subsequently

#### *Inflammation in the Pathogenesis of Rheumatoid Arthritis and in Experimental Arthritis… DOI: http://dx.doi.org/10.5772/intechopen.99073*

causing the erosion of these tissues [10]. The activity of matrix metalloproteinases (MMPs) seems to be the reason for the irreversible destruction of the cartilage seen in RA. MMPs are enzymes produced as a response to proinflammatory cytokines as IL-1 and TNFα by activated macrophages and fibroblasts [11]. MMPs can be further divided into three main groups. Collagenase MMP-1 (interstitial) and MMP-8 (neutrophil), whose major substrates are collagen forms I, II, and III, belong to the first group. The second group consists of the gelatinase/type IV collagenases such as MMP-2, the 72kD gelatinase A, and 92-kD gelatinase B (MMP-9). The main function of these matrix metalloproteinases from the second group is to degrade gelatin and collagen type IV in the basement membrane. Group 3 consists of the stromelysins, stromelysin 1 (MMP-3), stromelysin 2 (MMP-10), and pump-1 (MMP-7). These stromelysins have activity against a range spectrum of substrates, mainly proteoglycans, fibronectin, laminin, and some collagens [11]. During arthritis, especially MMP-1 and MMP-3 play an important role in the pathophysiology of the disease, and what is worse, the destruction of the connective tissue they cause is largely irreversible [12–14]. Fibroblasts from a healthy organism produce very low levels of both enzymes [12–14]. On the other hand, during RA and osteoarthritis levels of these enzymes rapidly increase in response to various stimuli [12–14]. Potent inducers of collagenases and stromelysins could be cytokines such as IL-1α and IL-1β, epidermal growth factor (EGF), platelet-derived growth factor, and tumor necrosis factor α. Inducers of these two enzymes could also be crystals of monosodium urate monohydrate, debris phagocytosis, and formulation of multinucleated giant cells. In an environment of stimulated synovial fibroblast cells, which resembles proliferating rheumatoid synovial tissue, collagenase and stromelysin becomes major gene product of these synovial fibroblasts [14]. Patients with RA and OA also have higher levels of collagenase and stromelysin in cartilage and the synovial fluid, especially patients with RA [15, 16]. The level of enzymatic activity is increased concordantly with the severity of the disease [17]. Apart from MMPs, there are other enzymes synthesized by cells within cartilage and bone as well as infiltrating inflammatory cells. These enzymes include aspartic, serine, and cysteine endopeptidases such as cathepsin B, which are capable of cleaving and therefore destructing the main components of cartilage and bone (such as proteoglycan and collagen type I, II, IX, X, and XI) [18].

#### **2.1 Interleukin-1β**

Interleukin-1β (IL-1β) is a cytokine belonging to the same family of cytokines as IL-1α, yet they show different features and are produced by two different genes [19]. IL-1β is mainly produced by macrophages as an inactive precursor (pro-IL-1β) and then cleaved by cysteine protease caspase-I into its mature form (IL-1β) [20]. The major distinction between IL-1β and IL-1α is that pro-IL-1β is biologically inactive, while pro-IL-1α and mature IL-1α can bind to their receptors and therefore stimulate cellular responses. Most IL-1α also stays coupled with the plasma membrane and stimulates cells by direct cell–cell interaction, which can induce its functions [21]. IL-1β is produced by blood monocytes, tissue macrophages, and dendritic cells by direct cellular contact with stimulated T-lymphocytes, a mechanism related to chronic inflammation [22]. IL-1β mRNA requires an extra signal for synthesis so transcription of IL-1β is a rate-limiting step of its synthesis. The extra signal to induce the production of IL-1β can be a microbial product or cytokines as TNF-α, IL-1α, IL-18, or IL-1β itself [23]. By binding to the same receptors as IL-1α and IL-1β, yet not inducing any consequent cellular responses, IL-1 receptor antagonist (IL-I Ra) acts as a naturally occurring inhibitor [24]. IL-1β seems to be not present in healthy individuals, or its levels

are hard to detect by standard assays. Such low levels are needed to be maintained due to the potency of IL-1β to induce inflammatory responses [25]. During RA, serum levels of IL-1β are higher in patients with RA compared to healthy individuals, and the concentrations of IL-1β increase during the acute phase of the disease [26].

#### **2.2 Interleukin-6**

IL-6 has been suggested to be a major player in the pathological changes during RA because of the broad spectrum of activities IL-6 participates in. Il-6 is recognized as an endogenous pyrogen [27], and also as an inducer of acute phase response genes [28]. IL-6 stimulates B- and T-cells activity and promotes proliferation of plasmablast into mature immunoglobulin-producing plasma cells [29]. IL-6 acts stimulatory on the immune system's cells, vascular endothelial cells, synovial fibroblasts, and osteoclasts upon coupling with its soluble IL-6 receptor (sIL-6Rα). Activated sIL-6Rα complex stimulates the production of a subset of chemokines by endothelial cells and subsequently upregulates the expression of adhesion molecules, resulting in direct recruitment of leukocytes to the sites of inflammation [30]. Apart from that, by having stimulatory effects on synovial fibroblast and osteoclast activation, IL-6 contributes to the formation of synovial pannus and bone resorption in inflamed joints [31, 32]. Interestingly, patients with various forms of arthritis have high levels of IL-6 in serum and synovial fluids, but on the other hand, their structural cells from joints (chondrocytes, fibroblasts, synoviocytes, and endothelial cells) lack expression of IL-6R [33]. These cells are also not responsive to IL-6 itself. The complex of IL-6 bound to its receptor might, therefore, represents the mechanism behind the action of IL-6 during arthritis. In a synovial fluid of RA patients, it has been shown that an increase in sIL-6Rα correlates with the extent of the joint destruction which coincides with more advanced stages of RA [32].

#### **2.3 Interleukin-17**

IL-17 is another cytokine possibly contributing to the pathogenesis of RA. IL-17 is produced by CD4+ CD45RO+ memory T cells in synovium during RA, upon activation with phorbolmyristate acetate/ionomycin or CD3/CD28 Abs [34, 35]. IL-17A is relatively homologous to IL-17F (~50%) with which it can form heterodimers (IL-17A/F). Activated human CD4+ T cells produce IL-17A/F heterodimers along with IL-17A and IL-17F homodimers [36]. The signaling is based on the coupling of IL-1A and IL-1F to a multimeric receptor composed of two subunits IL-17RA and IL-17RC [37]. Cytokines from the IL-17 family activate pro-inflammatory pathways through activating NF-κB or inducing signaling through MAPK and the C/EBP transcription factors. It seems IL-17A signaling intends to activate a gene expression of an innate-type inflammatory effector program that mediates potent inflammation and plays a critical role in a defense of a host [38]. It has been shown that IL-17 can trigger the production of IL-6, IL-8, GM-CSF, and also prostaglandin E2 (PGE2), a strong mediator of inflammation, in human synoviocytes [34, 35, 39]. Additionally, IL-17 showed stimulating effect on granulopoiesis in a murine model [40], on osteoclastogenesis [41], up-regulated synthesis of NO in cultured human cartilage [42], stimulated the synthesis of proinflammatory mediators as TNF-α, IL-1β, IL-10, IL-12, stromelysin, and IL-1Ra in human peripheral blood macrophages [43]. Furthermore, levels of IL-17 in synovial fluid and serum from RA patients are high in contrast to OA patients [44].

*Inflammation in the Pathogenesis of Rheumatoid Arthritis and in Experimental Arthritis… DOI: http://dx.doi.org/10.5772/intechopen.99073*

#### **2.4 Monocyte chemoattractant protein-1**

The rheumatoid synovial environment suggests a possible role for leukocyte chemoattractant molecules such as chemokines. Chemokines form a superfamily consisting of low molecular weight peptides (7–15 kDa) with conserved fourcysteine motif and consist of at least two subfamilies: first are the C-X-C (α) chemokines which all majorly attract neutrophils. Here belong IL-8, melanoma growth stimulating activity, and epithelial neutrophil-activating peptide 78. Secondly, C-C (β) chemokines are RANTES (regulated upon activation normal T cell expressed and secreted), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α), which chiefly recruit T cells and monocytes [45]. Many of the cells present in RA joints, such as endothelial cells, macrophages, fibroblasts, and lymphocytes can release chemokines. In the pathogenesis of RA, members of both subclasses of chemokines have been implicated. The production of MCP-1 is enhanced in human RA patients compared to osteoarthritis patients [46]. In the murine model of collagen-induced arthritis the earliest detectable levels of MIP-1α, MCP-1, and MIP-2 expression were observed 4 weeks after the initial collagen challenge [47].

#### **2.5 Matrix metalloproteinase 9**

Degradation of articular cartilage is important feature of RA and is caused by elevated activity of proteolytic enzymes [48]. In RA, synovial fibroblasts are extensively producing the matrix-degrading enzymes [49] known as matrix metalloproteinases (MMPs). MMPs are a zinc-dependent peptidases, which are degrading the components of extracellular matrix. MMPs are the key proteases associated with the degradation and invasion through anatomical barriers [50]. The MMP-9 (gelatinase B) and MMP-2 (gelatinase A), are very important in the degradation of collagen by cleaving the denatured collagen, produced by collagenases. Moreover, these MMPs degrade other substrates, such as collagen I and II [51] and aggrecan, which is abundant in cartilage [50].

MMP-9 has a posttranscriptional regulation on multiple levels. Its activity is inhibited in tissues by inhibitors of metalloproteinase (TIMP-1 to TIMP-4) with strongest binding between TIMP-1 and MMP-9 [52]. MMPs (including MMP-9) are produced and secreted in latent soluble form of enzyme, which needs activation extra-celullarly. In tissues the mast cell-derived tryptase and chymase are effective activators of MMPs [53, 54]. Regulation of MMPs is situated at the level of their transcription. Expression of MMPs is modulated by different stimuli including also cytokines [55] and growth factors [56].

MMP-9 was first discovered in neutrophils [57]. MMP-9 is also present in other leukocytes including T cells, macrophages, and eosinophils [58]. MMP-9 cleaves IL-8 and increases its activity as a chemoattractant for neutrophil more than 10-fold according to acute and chronic inflammatory processes [59]. The evidence is now growing that along with the storage of serine proteases, mast cells are secreting significant amount of MMPs such as MMP-9 [60, 61]. Although there is limited evidence for the expression of MMP-9 in mast cells in rheumatoid synovium [62], its regulation in RA is poorly understood. MMP-9 expression in rheumatoid synovial mast cells is via its regulation by TNF-α and IFN-γ in cord blood-derived human mast cell and the human mast cell line-1 (HMC-1). MMP-9 is not a product which is permanently stored in mast cells, but this enzyme is secreted under inflammatory conditions. MMP-9 may help in the migration of mast cell progenitors to inflammatory sites and could also promote the local damage of tissues [63]. In RA, MMP-9 is markedly elevated in serum and joint synovial fluid and positively correlates

with disease progression and severity [64]. MMP-9 knockout mice show decreased severity of antibody-induced arthritis [65].

#### **3. Innovative combination treatments of methotrexate with natural compounds in experimental arthritis**

Current drugs for rheumatoid arthritis (RA) are: corticosteroids, diseasemodifying anti-rheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), and biological response modifiers [66]. However, these antirheumatics have several adverse effects. NSAIDs are dangerous to patients due to the adverse effects such as bleeding of upper gastrointestinal tract, liver, and kidney adverse reactions [67]. Moreover, cognitive disorders, headaches, allergic reactions often force the patients to stop the treatment. This behavior is greatly limiting the use of NSAIDs. The long-term administration of corticosteroids can induce hypersplenism, hypertension, infection, osteoporosis and fractures [68]. DMARDs often cause diarrhea, rashes, vomiting, decreased white blood cell levels, and impaired kidney and liver functions [69]. Biological agents with high target specificity and less side effects are the new agents for therapy of RA [70]. However, these biological agents are expensive and not available for many patients [71]. Thus, development of novel anti-rheumatic drugs and strategies for RA therapy is a high priority. The combination treatments of low-dose methotrexate (MTX) with natural substances, which have the potential to improve the efficacy and to reduce adverse side effects of drugs, could be one possible direction in these strategies for RA therapy. Extract or phytochemical selected for combination therapy with MTX is expected to have anti-inflammatory and antioxidant activity to treat the inflammation and oxidative stress, occurring during RA development. Many chronic diseases with inflammatory pathology are abundant in elderly population. The widely administered anti-inflammatory drugs have many side effects and are expensive (biologic drugs). Alternative option are natural extracts and substances used in traditional medicine. These natural products offer a possibility to identify the bioactive compounds and for the development of new inflammatory drugs. Traditional remedies and phytochemicals are being used for the treatment of inflammatory and other disorders since ancient times [72] and with proper scientific research background can be more extensively used for treatment also in the present.

#### **3.1 Methotrexate**

MTX is still for decades a primary antirheumatic drug and the cornerstone of the RA treatment. MTX has an acceptable safety profile, efficacy, and low cost as well as many years of clinical experience make it the gold standard of RA treatment and the key drug for combination with different biological drugs [73]. MTX is usually effective in RA treatment and patients are usually administered for several years with MTX, thus information about long-term safety is very important. However, administration of MTX is in some cases limited because of its toxic adverse effects. During long treatment period by MTX, often adverse reactions occur such as mucous ulceration, cytopenia, nausea, liver damage and serious infections. Some studies showed that due to toxic manifestations, the interruption of MTX treatment in RA patients is in the range from 10–37% [74].

Despite the introduction of numerous biologic agents for the treatment of RA, low-dose MTX therapy remains still the gold standard in the RA therapy. MTX is generally the first-line drug for the treatment of RA, psoriatic arthritis, and it enhances the effect of most biologic agents in RA. Methotrexate inhibits

#### *Inflammation in the Pathogenesis of Rheumatoid Arthritis and in Experimental Arthritis… DOI: http://dx.doi.org/10.5772/intechopen.99073*

polyglutamates inhibit aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase (ATIC), leading to intracellular accumulation of AICAR and increased adenosine release; adenosine binds to cell surface receptors and suppresses many inflammatory and immune reactions [75].

The activity of MTX has also been studied in monocyte cell lines. Different from fibroblast like synoviocytes and T-lymphocytes, monocytes trigger apoptosis as a response to MTX treatment. Moreover, MTX activates a dose-dependent elevation in the expression of inflammatory cytokines, such as TNF, IL-1 and IL-6, in monocytic cell lines [76]. Adenosine (AS) via its receptors regulates monocyte activity, and hence MTX may influence monocytes indirectly by increasing AS release by other immune cells. AS binds to its A1 receptor on peripheral blood monocytes and activates the formation of giant cells with multiple nuclei [77]. Moreover, the binding of AS to A2a receptors and A3 receptors on monocytes decreases the production and release of IL-6 and TNF and initiates the transformation of inflammatory M1 phenotype of monocytes to anti-inflammatory M2 phenotype.

Macrophages with M2 phenotype have are responsible for termination of inflammation, clearing the apoptotic cells and support wound healing by secreting profibrotic and angiogenic cytokines. Adenosine, binding on A2a receptors, inhibits the production of inflammatory cytokines and promotes the expression of anti-inflammatory mediators such as vascular endothelial growth factor and IL-10 [78]. A2a receptor stimulation triggers a switching from an M1 (pro-inflammatory phenotype) to a modified macrophage M2 phenotype [79]. One way by which A2a receptor binding affects macrophage function is by stimulating the expression of the NR4A - orphan nuclear receptor, which is inhibiting the activation of NFκBdependent nuclear gene expression [80]. A2b receptor also induces the switching from a M1 macrophage phenotype to a M2 phenotype [81]. Cultivating synovial fibroblasts and T cells from RA patients triggered T cell TNF-α, IL-17, and IFNγ expression, which resulted in increased fibroblast IL-6, IL8 and IL-15 expression [82]. Methotrexate inhibited the upregulation of IL-6, IL8 and IL-15 by stimulated RA synovial fibroblasts. MTX also decreased IFNγ and IL-17 expression in T cells co-cultured with RA synovial fibroblasts (**Table 1**).

#### **3.2 Combination of methotrexate and carnosic acid**

In our previous study, we have selected the carnosic acid for combination with methotrexate for its anti-inflammatory and antioxidative properties, to reduce the development of rat adjuvant arthritis.

#### *3.2.1 Carnosic acid*

Carnosic acid (CA) was discovered first by Linde in *Salvia officinalis* L. [83]. Carnosic acid (C20H28O4, **Figure 1**), is a phenolic diterpene that belongs to the terpene class of secondary metabolites [84], is localized in rosemary leaves, more precisely in chloroplasts of trichome cells. CA and carnosol have been reported to display beneficial effects against acute and chronic inflammation, cardiovascular diseases, obesity, and cancer [85, 86], inhibition of prostaglandin synthesis [87], skin inflammation [88], inhibition of NF-κB [89], inhibition of 5-lipoxygenase [90] and antioxidant activity *in vivo* [91].

CA prevented cartilage degeneration though induction of hemeoxygenase-1 (HO-1) in cell culture with human chondrocytes. The results showed that CA increased enzyme levels in a dose-dependent manner. Moreover, it was able to restore HO-1 levels under IL-1β treatment, which specifically inhibits the antioxidant effects of this enzyme. CA induced HO-1 and miR-140 expression in human


**Table 1.**

*Immune regulatory action of low dose MTX in the RA synovial tissue (according to Miranda-Carús et al. [82]).*

**Figure 1.**

*Chemical structure of carnosic acid.*

articular chondrocytes, thus cartilage degeneration was attenuated by CA treatment [92]. The activation of macrophages triggered by exogenous infection or endogenous stress stimuli is thought to be implicated in the pathogenesis of various inflammatory diseases. In a study of Wang et al. [93], authors applied an integrated approach based on unbiased proteomics and bioinformatics analysis to elucidate the anti-inflammatory property of CA. CA significantly inhibited the increase of NO and TNF-α, downregulated cyclooxygenase-2 (COX-2) protein expression and decreased the transcriptional level of inflammatory genes including NOS-2, TNF-α, COX-2, in LPS-stimulated RAW264.7 macrophages. The liquid chromatographybased assessment showed CA negatively regulated 217 proteins elicited by lipopolysaccharide (LPS), which are responsible for multiple inflammatory pathways including nuclear factor (NF)-κB, MAPK and FoxO signaling. A following analysis showed that CA effectively inhibited ERK/JNK/p38 MAPKs, IKKβ/IκB-α/NF-κB and FoxO1/3 signaling. These results illustrate the ability of CA to regulate the inflammatory signaling triggered by LPS [93].

In another study by de Oliveira [94] authors have found that activation of cell antioxidant defense is mediated via transcription factor nuclear factor erythroid 2-related factor (Nrf2). Therefore, authors investigated whether CA is able to block paraquat (PQ )-induced inflammatory alterations in SH-SY5Y neuroblastoma cells. CA reduced the PQ-induced changes on the levels of TNF-α, IL-1β, and COX-2 via

#### *Inflammation in the Pathogenesis of Rheumatoid Arthritis and in Experimental Arthritis… DOI: http://dx.doi.org/10.5772/intechopen.99073*

signaling responsible for the activation of the Nrf2/HO-1 pathway. Furthermore, they observed a crosstalk between the Nrf2/HO-1 signaling pathway and the activation of the nuclear factor-κB [94]. Two Rosemary extracts and their main components - CA and carnosol affected the cell migration. Monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) were determined by Western blot and gelatin zymography, respectively, in RAW 264.7 macrophages and vascular smooth muscle cells (VSMCs). MMP-9 and MCP-1 levels were significantly diminished with methanol extract (RM), n-hexane fraction (RH), and CA in RAW 264.7 macrophages. RM, RH, CA, and carnosol suppressed TNF-α induced VSMC migration by inhibiting MMP-9 expression. Rosemary, especially its CA component, has potential anti-atherosclerotic effects related to cell migration [95].

Liu and colleagues [96] studied the anti-inflammatory activity of CA on destruction of osteoclasts, fibroblast-like synoviocytes in the collagen-induced arthritis model. Abovementioned *in vitro* and *in vivo* experiments showed that CA inhibited the expression of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α, IL-17, IL-8 and MMP-3, and suppressed the secretion of RANKL. Moreover, authors determined that CA reduced osteoclastogenesis and resorption of the bone *in vitro* and had therapeutic protective activity against joint damage *in vivo*. Further results showed that CA inhibited RANKL-induced activations of MAPKs (JNK and p38) and NF-κB resulting in the suppressing of NFATc1 [96].

#### *3.2.2 Effect of the combination therapy of methotrexate and carnosic acid in rat adjuvant arthritis*

In this section we will present our preliminary results from combination therapy of methotrexate (MTX) and carnosic acid in rat adjuvant arthritis.

Hind paw volume (HPV) was significantly increased on days 14, 21 and 28 during the development of AA. CA in monotherapy was without a significant effect on this parameter. The administration of methotrexate in sub-therapeutic dose markedly reduced HPV on days 14 and 21, but not on day 28. The combination of MTX and CA was more effective in decreasing the HPV on days 14, 21 and 28 than MTX in monotherapy. The most effective reduction of HPV was on day 21 (**Table 2**).

MCP-1 is responsible for recruiting monocytes on the sites of inflammation, and it is involved in the pathogenesis of human [46] and also in experimental arthritis [47]. AA caused a significant increase in the levels of MCP-1 on days 14, 21 and 28. Neither CA nor MTX administered in monotherapy were able to significantly reduce the elevated MCP-1 levels on days 14, 21 and 28. On day 21, only the combination of MTX and CA significantly decreased the level of MCP-1 in plasma of AA animals (**Table 3**).

#### **3.3 Combination of methotrexate and ethanol extract of** *Rhodiola rosea*

*Rhodiola rosea* L. is known as an adaptogen and has been confirmed to possess protective effects against inflammatory diseases, including cardiovascular diseases, neurodegenerative diseases, diabetes, sepsis, and cancer [97]. Less is known about the anti-inflammatory activity of Rhodiola extract in the experimental arthritis, thus we decided to select this extract for our study in monotherapy and in combination with methotrexate.

#### *3.3.1* Rhodiola rosea *L.*

In this section we will focus on the anti-inflammatory effect of *Rhodiola rosea* L. (RhR). RhR has been found to possess anti-inflammatory properties in diseases


*CO: healthy control animals, AA: untreated arthritic animals, AA-CA: arthritic animals treated with carnosic acid, AA-MTX: arthritic animals treated with methotrexate, AA-CA-MTX: arthritic animals treated combination of methotrexate and carnosic acid.*

*Values are expressed as average ± standard error of mean, statistical significance was calculated using ANOVA-Tukey–Kramer post hoc test.*

*\* p < 0.05.*

*\*\*p < 0.01. \*\*\*p < 0.001 vs. CO. + p < 0.05. ++p < 0.01. +++p < 0.001 vs. AA. # p < 0.05 vs AA-MTX.*

#### **Table 2.**

*Effect of carnosic acid, methotrexate and their combination on hind paw swelling.*


*CO: healthy control animals, AA: untreated arthritic animals, AA-CA: arthritic animals treated with carnosic acid, AA-MTX: arthritic animals treated with methotrexate, AA-CA-MTX: arthritic animals treated combination of methotrexate and carnosic acid.*

*Values are expressed as average ± standard error of mean, statistical significance was calculated using ANOVA-Tukey–Kramer post hoc test.*

*\* p < 0.05.*

*\*\*p < 0.01.*

*\*\*\*p < 0.001 vs. CO. + p < 0.05 vs AA.*

#### **Table 3.**

*Effect of carnosic acid, methotrexate and their combination on levels of monocyte chemotactic protein-1 in blood plasma.*

such as sepsis, endotoxemia, asthma *in vivo* and *in vitro*. Pu et al. [97] have found that seven compounds (Ferulic acid, Kaempferol, Salidroside, Tyrosol, Catechin, Gallic acid and Caffeic acid phenethyl ester) isolated from RhR showed protective activity against LPS-induced sepsis in mice via decreasing TNF-α, nitric oxide and lactate dehydrogenase [97]. By many scientists, salidroside (SAL) was reported to possess protective ability in many disease models through particularly regulating different inflammatory mediators.

SAL decreased the inflammatory injury via reducing inflammatory cytokines (IL-1β, TNFα, IL-6), small molecules (mainly nitric oxide), chemokines (monocyte chemo-attractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α) and COX-2 in animal models, such as LPS induced endotoxemia in mice [98], LPS induced murine acute lung injury [99], ovalbumin induced asthma in

#### *Inflammation in the Pathogenesis of Rheumatoid Arthritis and in Experimental Arthritis… DOI: http://dx.doi.org/10.5772/intechopen.99073*

mice [100], and ethanol triggered acute gastric ulceration [101]. Further *in vitro* experiment confirmed the protective effects of SAL in neuro-inflammation. In murine microglial BV2 cells treated by LPS, Lee et al. showed that the main compounds of RhR (salidroside and rosarin) reduced the production of nitric oxide and inflammatory cytokines such as IL-6, IL-1β, and TNF-α via the NF-κB and MAPK signaling pathways [102]. Another *in vitro* study showed that SAL may inhibit the synthesis of inflammatory mediators. Authors found that in mice macrophages (J774.1 and RAW264.7) activated by LPS, SAL pre-treatment can reduce the levels of IL-1β, TNFα, IL-6, NO and MCP-1 via NF-κB pathway [103]. Further experiment showed that the mechanism might also be associated with down regulation of STAT3 and JAK2, and with translocation of STAT3 in nucleus [99]. STAT3 belongs to STAT (Signal Transducers and Activators of Transcription) family and has a key role in inflammatory processes. Many cytokines bind to GP130, which is a IL-6-type cytokines receptor, and activate Janus kinases (JAKs), what leads to the phosphorylation of STAT3. The phosphorylated STAT3 is translocated into the nucleus and regulates the expression of different target genes including also pro-inflammatory mediators [104].

Osteoarthritis (OA) is the most common disease, which seriously affects the daily life of the elderly. Currently, no drug therapy has been shown to explicitly block the progression of OA. The study by Gao et al. [105] showed that salidroside could significantly promote the proliferation of chondrocytes in OA rats induced by an anterior cruciate ligament transection and renew the OA-induced changes of cartilage. Salidroside increased the levels of aggrecan and collagen II and reduced the MMP-13 level. Moreover, salidroside reduced Th-17 cells and the levels of IKBα and p65, and IL-17, while elevated the count of CD4 + IL-10+ cells and IL-10. The reduction of IL-17 levels further diminished the dissociation of IKBα to p65, what resulted in the reduction of the release of VCAM-1 and TNF-α. Salidroside decreases the cartilage degradation via promoting proliferation of chondrocytes, reducing collagen fibrosis, and regulating the inflammatory processes and immune responses through NF-κB pathway in anterior cruciate ligament transection-induced OA in rats [105]. Another study involving chondrocytes by Wu et al. [106] showed that salidroside suppressed IL-1β-induced apoptosis in chondrocytes. Salidroside stimulated proliferation of chondrocytes, reduced IL-1ß-triggered inflammation and apoptosis, and scavenged NO and reactive oxygen species generated by chondrocytes. Salidroside upregulated the level of B-cell lymphoma 2 protein and downregulated the level of apoptosis regulator Bax. Salidroside also inhibited the production of caspase 3/9 and suppressed the phosphorylation of phosphoinositide-3-kinases (PI3K) and protein kinase B (AKT). These results indicate that salidroside prevents osteoarthritis by its anti-inflammatory, anti-apoptotic and pro-proliferating activities by suppressing the PI3K/AKT pathway [106].

#### *3.3.2 Effect of the combination therapy of methotrexate and extract of* Rhodiola rosea *in rat adjuvant arthritis*

Hind paw volume (HPV) was significantly increased on days 14 and day 21 during the development of AA. Administration of *Rhodiola rosea* ethanol extract (RS) in monotherapy markedly decreased HPV on day 14, but it had no effect on HPV on day 21. MTX and the combination of MTX with RS administered in monotherapy significantly decreased the HPV on days 14 and 21 (**Table 4**).

AA caused significant increase in the levels of IL-6 on days 14 and 21. Administration of MTX in monotherapy significantly decreased the plasmatic level of IL-6 only on day 14. Administration of RS in monotherapy had no effect on


*CO: healthy control animals, AA: untreated arthritic animals, AA-RS: arthritic animals treated with extract of Rhodiola rosea, AA-MTX: arthritic animals treated with methotrexate, AA-RS-MTX: arthritic animals treated combination of methotrexate and extract of Rhodiola rosea.*

*Values are expressed as average ± standard error of mean, statistical significance was calculated using ANOVA-Tukey–Kramer post hoc test*

*\*\*\*p < 0.001 vs. CO.*

*++p < 0.01.*

*+++p < 0.001 vs. AA.*

#### **Table 4.**

*Effect of* Rhodiola rosea *ethanol extract, methotrexate and their combination on hind paw swelling.*


*CO: healthy control animals, AA: untreated arthritic animals, AA-RS: arthritic animals treated with extract of Rhodiola rosea, AA-MTX: arthritic animals treated with methotrexate, AA-RS-MTX: arthritic animals treated combination of methotrexate and extract of Rhodiola rosea.*

*Values are expressed as average ± standard error of mean, statistical significance was calculated using ANOVA-Tukey–Kramer post hoc test.*

*\* p < 0.05 vs. CO.*

*+ p < 0.05, vs. AA.*

#### **Table 5.**

*Effect of* Rhodiola rosea *ethanol extract, methotrexate and their combination on levels of IL-6 in blood plasma.*

levels of IL-6. However, the combination treatment of MTX and RS significantly decreased the levels of IL-6 on both measured days (**Table 5**).

#### **4. Conclusions**

Animal models of rheumatoid arthritis (RA) are used widely in research on pathogenesis of inflammatory arthritis and in the testing of potential anti-arthritic agents. In this chapter we highlighted the importance of inflammatory mediators IL-1β, IL-6, IL-17, MCP-1 and MMP-9 in experimental arthritis and RA. We have demonstrated, that MTX is a therapeutic standard for human arthritis as well as for adjuvant arthritis in rats, which make this model suitable for studying the pharmacotherapy of RA. Our preliminary results with combination treatments of MTX with carnosic acid and *Rhodiola rosea* ethanol extract showed, that these combinations are more effective in reducing hind paw volume, and the levels of MCP-1 and IL-6 than MTX in monotherapy. Thus, natural compounds with anti-inflammatory activities could be also a perspective candidate for combination treatments with MTX to treat human autoimmune diseases.

*Inflammation in the Pathogenesis of Rheumatoid Arthritis and in Experimental Arthritis… DOI: http://dx.doi.org/10.5772/intechopen.99073*

#### **Acknowledgements**

This work was supported by grants: VEGA 2/0115/19, VEGA 2/0136/20, APVV-15-0308, APVV SK-PT-18-0022 and SAS-BAS project: Anti-inflammatory effect of astaxanthin, sulforaphane and crocus sativus extract evaluated in two rodent models of age related diseases.

### **Conflict of interest**

Authors have no conflict of interests.

### **Notes/thanks/other declarations**

Authors thanks to colleagues Jana Urgosova, Danica Mihalova, MSc. Martin Chrastina, MSc., Frantisek Drafi, PharmDr., PhD. and Karol Svik, MSc., PhD for their support and assistance in performing experiments of rat adjuvant arthritis.

### **Author details**

Silvester Ponist1,3, Katarina Pruzinska1,2 and Katarina Bauerova1 \*

1 Center of Experimental Medicine, Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovak Republic

2 Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Slovak Republic

3 Slovak Medical University in Bratislava, Bratislava, Slovak Republic

\*Address all correspondence to: katarina.bauerova@savba.sk

© 2021 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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[95] Chae IG, Yu MH, Im NK, Jung YT, Lee J, Chun KS, Lee IS. Effect of Rosemarinus officinalis L. on MMP-9, MCP-1 levels, and cell migration in RAW 264.7 and smooth muscle cells. J Med Food. 2012;15:879-886. DOI: 10.1089/jmf.2012.2162.

[96] Liu M, Zhou X, Zhou L, Liu Z, Yuan J, Cheng J, Zhao J, Wu L, Li H, *Inflammation in the Pathogenesis of Rheumatoid Arthritis and in Experimental Arthritis… DOI: http://dx.doi.org/10.5772/intechopen.99073*

Qiu H, Xu J. Carnosic acid inhibits inflammation response and joint destruction on osteoclasts, fibroblastlike synoviocytes, and collagen-induced arthritis rats. J Cell Physiol. 2018; 233:6291-6303. DOI: 10.1002/jcp.26517.

[97] Pu WL, Zhang MY, Bai RY, Sun LK, Li WH, Yu YL, Zhang Y, Song L, Wang ZX, Peng YF, Shi H, Zhou K, Li TX. Anti-inflammatory effects of *Rhodiola rosea* L.: A review. Biomed Pharmacother. 2020;121:109552. DOI: 10.1016/j.biopha.2019.109552.

[98] Guan S, Feng H, Song B, Guo W, Xiong Y, Huang G, Zhong W, Huo M, Chen N, Lu J, Deng X. Salidroside attenuates LPS-induced proinflammatory cytokine responses and improves survival in murine endotoxemia. Int Immunopharmacol. 2011;11:2194-2209. DOI: 10.1016/j. intimp.2011.09.018.

[99] Qi Z, Qi S, Ling L, Lv J, Feng Z. Salidroside attenuates inflammatory response via suppressing JAK2-STAT3 pathway activation and preventing STAT3 transfer into nucleus. Int Immunopharmacol. 2016;35:265-271. DOI: 10.1016/j.intimp.2016.04.004.

[100] Wang J, Jin RG, Xiao L, Wang QJ, Yan TH. Anti-asthma effects of synthetic salidroside through regulation of Th1/Th2 balance. Chin J Nat Med. 2014;12:500-4. DOI: 10.1016/ S1875-5364(14)60078-9.

[101] Chang X, Luo F, Jiang W, Zhu L, Gao J, He H, Wei T, Gong S, Yan T. Protective activity of salidroside against ethanol-induced gastric ulcer via the MAPK/NF-κB pathway in vivo and in vitro. Int Immunopharmacol. 2015;28:604-615. DOI: 10.1016/j. intimp.2015.07.031.

[102] Lee Y, Jung JC, Jang S, Kim J, Ali Z, Khan IA, Oh S. Anti-Inflammatory and Neuroprotective Effects of Constituents Isolated from *Rhodiola rosea*. Evid Based Complement Alternat Med. 2013;2013:514049. DOI: 10.1155/2013/514049.

[103] Huang Q, Hu XL. [Effects of salidroside on the secretion of inflammatory mediators induced by lipopolysaccharide in murine macrophage cell line J774.1]. Sheng Li Xue Bao. 2017;69:41-46.

[104] Hillmer EJ, Zhang H, Li HS, Watowich SS. STAT3 signaling in immunity. Cytokine Growth Factor Rev. 2016;31:1-15. DOI: 10.1016/j. cytogfr.2016.05.001.

[105] Gao H, Peng L, Li C, Ji Q, Li P. Salidroside Alleviates Cartilage Degeneration Through NF-κB Pathway in Osteoarthritis Rats. Drug Des Devel Ther. 2020;14:1445-1454. DOI: 10.2147/ DDDT.S242862

[106] Wu M, Hu R, Wang J, An Y, Lu L, Long C, Yan L. Salidroside Suppresses IL-1β-Induced Apoptosis in Chondrocytes via Phosphatidylinositol 3-Kinases (PI3K)/Akt Signaling Inhibition. Med Sci Monit. 2019;25:5833-5840.

#### **Chapter 13**

## Antiphospholipid Syndrome and Pregnancy-Diagnosis, Complications and Management: An Overview

*Panagiotis Tsikouras, Christina Tsiggalou, Anastasia Bothou, Aggeliki Gerede, Ifigenia Apostolou, Fotini Gaitatzi, Anna Chalkidou, Xanthoula Anthoulaki, Spyridon Michalopoulos, Georgios Dragoutsos, Ioannis Tsirkas, Irini Babageorgaka, Theopi Nalbanti, Natalia Sachnova, Alexios Alexiou, Konstantinos Nikolettos, Christos Tsalikidis, Stefanos Zervoudis, Panagiotis Peitsidis and Nikolaos Nikolettos*

#### **Abstract**

Antiphospholipid syndrome which is also known as APS is an autoimmune disease which represents an acquired form of thrombophilia. The etiology of APS remains unknown. This disorder occurs when the immune system mistakenly attacks some of the normal human proteins and manifests itself as recurrent arterial or venous thrombosis and it could emerge after abortions or in recurrent pregnancy loss. In APS, the body produces the wrong antibodies against phospholipid-binding proteins, that is present in the blood and plays an important role in coagulation. Antibodies are specific proteins that usually target and neutralize the body's invaders, such as viruses and bacteria. When antibodies attack phospholipid-binding proteins, blood clots abnormally. Specifically, it could cause blood clots in veins or arteries leading to stroke and various pregnancy complications such as: endometrial death, miscarriage, preeclampsia, intrauterine growth restriction and prematurity. APS is divided into primary and secondary, which is associated with autoimmune diseases and more often with systemic lupus erythematosus (SLE), while antibodies against cardiolipin are detected in many other conditions (infections, malignancies, drugs, etc.). The symptoms of APS, in addition to arterial and/or venous thrombosis and pregnancy complications, are multisystemic and the differential diagnosis of the primary APS from the secondary, in the context of SLE, is of particular clinical interest and is subject of this literature review.

**Keywords:** antiphospholipid syndrome, pregnancy, management neonatal outcome

#### **1. Introduction**

APS characterized by thrombosis of the arteries, veins and microvessels and/or with pregnancy morbidity, with persistently elevated antiphospholipid antibody (aPLs) titers. The syndrome was first described in 1983 by Professor G. Hughes, at Hammersmith Hospital (Hughes Syndrome) [1, 2]. APS is a prothrombotic condition (with related complications such as deep vein thrombosis, pulmonary embolism, etc.), belonging to autoimmune diseases of unknown cause, and is strongly associated with pregnancy [1–3]. Generally, autoimmune diseases namely Sjorgen's syndrome, Spondylanthritis, rheumatoid arthtritis RA have an incidence of 5–8% in the general population organoid or systemic, and represent the 2nd cause of hospitalization in Internal Medicine Departments and the 3rd cause of morbidity/ mortality [1–4]. APS occurs as primary in the absence of findings of other autoimmune diseases or as secondary in 36% of cases in the context of another autoimmune disease (SEL, Sjogren's disease, inflammatory bowel disease, etc.), while it is present in about 5% of patients with subclinical SEL or coexists with another underlying systemic autoimmune disease in 6% of cases [5, 6].

#### **2. Epidemiology**

The incidence of the syndrome is increased with age. During pregnancy, aPLs detection ranges from 0 to 11%, with an average incidence of about 2%. On the contrary, the syndrome is detected in up to 37% of the patients with systemic erythematosus lupus (SEL). In the nonpregnancy setting, venous thromboses are more common than arterial ones and can be diagnosed by imaging techniques and/ or histologic evidence [5–7].

#### **3. Frequency**

According to literature APS is the cause for 1 out of 5 Deep Vein Thrombosis ('DVTs'), 1 out of 5 cases of SLE (arterial stroke) in young patients (age <45 years) and 1 out of 5 miscarriages. Especially for Obstetrics Hughes Syndrome is currently recognized as the leading cause for the miscarriages [5–7]. In addition, APS is diagnosed as the underlying diagnosis in a still unknown percentage of cases previously misdiagnosed as migraine, Alzheimer's disease, and Multiple MS. It is estimated that the true incidence of the syndrome can be up to 1–2% or more in the general population [5–7]. Correlations-Percentages The mean age of APS onset is >30 years, with a female to male ratio of 5:1 and relapses usually take place at the same or similar area of the body. There is no apparent racial preference, but an increased incidence of SEL is reported in African Americans and Spaniards. Patients with SEL have positive aPLs in a percentage of 15–35%, but only about 50% of these patients will develop APS symptoms [5–7].

#### **4. Clinical subtypes**

Special categories of APS are described usually correlated to the target-organ or the severity of the manifestations. The following are included: Generalized APS, Arterial APS, APS and heart, APS and kidneys, Cerebral APS, Pediatric APS, Neonatal APS, Catastrophic CAPS and Obstetric APS [5–7].

*Antiphospholipid Syndrome and Pregnancy-Diagnosis, Complications and Management… DOI: http://dx.doi.org/10.5772/intechopen.99283*

#### **5. Catastrophic APS**

The 0.8% of the cases is characterized as Catastrophic Antiphospholipid Syndrome (CAPS). It is a very rare and severe form of APS. There are diffuse clots in the small vessels throughout the body. Sometimes it can appear as the first manifestation of APS and even without clinical or serological confirmation of SEL. Early diagnosis is necessary and immediate start of an aggressive treatment is inevitable [5, 7–9]. CAPS is caused when at least 3 different systems are affected at intervals of days or weeks, with multiple thrombosis in large and small vessels. The organs that are usually affected are: a) kidneys, b) lungs, c) heart, d) small-large vessels with consequences as peripheral limb ischemia, stroke, myocardial infarction, thrombosis of blood vessels of abdominal organs with a mortality rate of 50% [5, 7–9]. The observed thrombocytopenia is usually mild between 100-150x109 /L but severe thrombocytopenia can be also observed. The prevalence of the syndrome in the general population ranges between 2% and 4% meaning 40–50 patients in 100,000 based on the criteria (mean age of diagnosis 34 years) and 7: 1 in the SEL/ APS combination [5, 7–9].

#### **6. Therapeutic interventions for catastrophic APS**

Anticoagulant therapy + corticosteroids.

Anticoagulant therapy + corticosteroids + plasmapheresis.

Anticoagulant therapy + corticosteroids + IV γ-globin.

Anticoagulant therapy + corticosteroids + plasmapheresis + IV γ-globulin.

Diffuse intravascular coagulation is not usually seen in primary or secondary APS but it occurs in about 25% of patients with catastrophic APS. It is estimated that more than 53% of cases are related to the primary syndrome. It is estimated that approximately 10% of patients with primary APS will be diagnosed with another autoimmune, disorder, such as SEL, at some point in their lives which is estimated to coexist in up to 37% of patients with APS. Patients with primary APS and a female/male ratio incidence of 3.5: 1 should not be classified as SEL patients, as they are two different disease entities [5, 7–9]. Also, aPLs found in a variety of rheumatic and autoimmune diseases should not be confused with APS. The latter includes autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, rheumatoid arthritis and cutaneous manifestations such as edema. Also, the same antiphospholipid antibodies are detected either temporarily or permanently after various infections, such as hepatitis A, hepatitis B, acquired immune deficiency (AIDS), mumps, toxoplasmosis, erythema etc. The use of certain medicines, such as hydralazine, procainamide, and amoxicillin, phenothiazines may cause a serious differential diagnosis with APS, which requires immediate diagnostic and therapeutic treatment to reduce the increased maternal risk, and in particular perinatal morbidity and mortality due to placental infarction [5, 7–9].

#### **7. Pathophysiology**

Dysfunction of the vascular epithelium as well as oxidative damage and modifications of phospholipid-bound proteins that interfere with the regulation of coagulation are possible. It occurs when the immune system mistakenly attacks some of the normal proteins in the blood. The mechanism by which aPLs causes thrombosis is not fully understood. APLs consist of a heterogeneous group of

#### *Inflammation in the 21st Century*

autoantibodies that react primarily with plasma proteins associated with negatively charged phospholipids (epitope formation). Moreover, also react directly with both phospholipid-binding proteins and phospholipids [10–12].

However, aPLs have been shown to target not only cell membrane phospholipids, but also plasma proteins. Initially aPLs are directed directly against negative cell membrane phospholipids and autoantibodies are directed against plasma proteins with high affinity for these anionic phospholipids [10–14]. Phospholipids are like building blocks of cell membranes and the appearance of aPLs is due to the "projection" of their anions to the extracellular space [10–14]. This phenomenon is usually happening due to various causes, such as trauma, ischemia, inflammation, infections, or drug interactions. They were first observed in 1906 in 1–5% of the normal population.

The prevailing theory is that APS initially causes a disorder in cell apoptosis procedure resulting in the exposure of cell membrane phospholipids and their subsequent binding to various plasma proteins, such as β2-glycoprotein I. This binding leads, through activation, to generation of intracellular mediators (such as nuclear factor kappa B and mammalian target of rapamycin), and the formation of a phospholipid-protein complex resulting in the discovery of a new epitope, which then becomes target of autoantibodies [10–14].

Recent studies suggest that oxidized β2-glycoprotein I is capable of binding to and subsequently activating dendritic cells in a similar pathway to the one induced by activation via the Toll-like receptor 4 (TLR-4), resulting in its induction and the production of autoantibodies.

A total of 4 types of aPLs have been isolated [14–18].


Types of anti-b2 GPI antibodies [14–18]. There are several types of anti-β2 glycoprotein I antibodies, but not all of them are harmful. Those that target specific β-2 glycoprotein epitopes, such as the epitope in the N-terminal I domain of the molecule, are associated with the onset of clinical manifestations of the syndrome [14–18].

#### **8. Correlation between APS clinical events and the type of anti-β2GPI antibodies**

Patients with triple positivity in aPLs: have higher anti-β2GPI – domain I antibody titles, compared to patients with single or double positivity. Almost all *Antiphospholipid Syndrome and Pregnancy-Diagnosis, Complications and Management… DOI: http://dx.doi.org/10.5772/intechopen.99283*

anti-β2GPI – domain I IgG antibodies tested positive after 12 weeks, in contrast to innocent transient aPLs, which appear to be immune to infections. Although antiβ2GPI-Domain 1 (β2GPI-D1) IgG antibodies have been associated with thrombosis and pregnancy morbidity in APS patients, these antibodies are found in only one third of the patients [14–18].

#### **9. Cell β2GPI/anti-β2GPI antibody receptors**

Annexin A2 and Toll-like receptor (TLR) 4 are receptors for the entire β2GPI molecule. However, the absence of both of these receptors does not lead to the complete inhibition of the binding of the anti-β2GPI antibody, a finding that supports the view that there are additional surface adhesion molecules that play a role. TLR1, TLR2 and TLR6 are potential cell receptors [14–18].

#### **10. Association of APS with pathogenic intestinal bacteria**

Β2GPI binds to LPS via domain V. Presence of a large amount of LPS probably increases the vascular distribution of APLs, by increasing the expression of TLR2 and especially TLR4. Because the main source of LPS in healthy people is the gut, it is possible that the presence of gut microbes in the gut increases them. Administration of intestinal flora-specific antimicrobial therapy to experimental animals showed a reduction in the thrombotic manifestations of APS [14–18].

#### **11. The role of the complement in the pathogenesis of APS**

Immediate evidence: In experimental models with C6-deficient rats and C5-depleted mice, it was shown that the addition of monoclonal anti-domain I MBB2 did not induce thrombosis or miscarriage. C4d and C3b sections have also been found in deposits on the placenta of women with APS [14–18].

Indirect evidence: In-vivo studies of the efficacy of drugs with C5 action. The non-complement-fixing anti-domain I monoclonal MBB2 [DELTA] Ch2 and the complement inhibitor C5-inhibitor rEV576 prevented the thrombotic complications of APS in vivo in experimental animals.

#### **12. APL action mechanisms**

APLs activate endothelial cells resulting in the expression of adhesion molecules (such as intercellular cell adhesion molecule-1 ICAM-1, vascular cell adhesion molecule-1 VCAM-1, E-selectin), and ultimately the overproduction of tissue factor TF.

APLs activate monocytes and cause increased tissue factor expression.

APLs activate platelets resulting in increased glycoprotein 2b-3a expression and thromboxane A2 synthesis. It has also recently been shown that aPLs induce the release of NETs from endothelial cells and these in turn further activate platelets. Their presence is enhanced by other conditions such as neoplasms, myelodysplastic syndromes, paraproteinemias, etc. [14–18].

These proteins (aPLs) normally bind to the phospholipid components of membranes and protect against activation of coagulation mechanisms. Autoantibodies displace these "protective" proteins and promote the formation of clots in the cells of the vascular endothelium resulting in arterial and venous thrombosis.

In particular, aPLs antibodies include antibodies to Lupus Anticoagulant (LA), antibodies to cardiolipin (aCL), antibodies to β2GPI (αβ2GPI) and antibodies to other phospholipid-binding proteins and other phospholipids. LA, aCL and αβ2GPI antibodies are important in the diagnosis of antiphospholipid syndrome [12–18].

#### **13. The most likely explanation for the pathogenesis of APS**

They occur in people with a genetic predisposition after accidental exposure to infectious agents or in a rheumatic disease environment such as SEL. It is the "second hit" theory required for the full development of the syndrome. Of course, not all people with antiphospholipid antibodies have thrombosis or obstetric complications.

Τhe "two-hit theory" has been proposed, i.e. that in the manifestation of the disease only the presence of antiphospholipid antibodies is not sufficient but a second thrombotic risk factor must coexist (e.g. age, hypertension, infection, inflammation, diabetes mellitus, obesity, smoking, pregnancy and obstetrics, surgery, etc.) [14–18].

Regarding the pathophysiology of obstetric complications, trophoblast and perishable blood vessel thrombosis is at the heart of this process. Various mechanisms have been proposed that lead to this result:


#### **14. Diagnostic criteria**

"APL profile" includes: the type of autoantibodies and the presence of 2–3 types of autoantibodies. The title of aPLs is mid-high instead of low and their persistent positivity is certified by multiple tests. APS is characterized by the following lab test results [19–22].

*Antiphospholipid Syndrome and Pregnancy-Diagnosis, Complications and Management… DOI: http://dx.doi.org/10.5772/intechopen.99283*

Persistent presence of antiphospholipid antibodies (at least 2 positive results during a period of >12 weeks) including lupus anticoagulant, anticardiolipin antibodies and b2-glycoprotein 1<sup>α</sup> antibodies [19–22].

Risk factors: pregnancy, labour, contraceptives, malignancy, infection (E-coli, Shigella, Salmonella, Streptococcus, Staphylococcus etc), injury, surgical procedures, operations, fractures, drugs, no compliance to the anticoagulant therapy and many other factors [19–22].

#### **15. Laboratory findings**

Usually there is severe thrombocytopenia, positive direct Coombs, microangiopathic hemolytic anemia, DIC findings in some patients and the presence of a heterogeneous group of antiphospholipid antibodies.

Laboratory confirmation is done with clotting assays for the detection of lupus anticoagulant and with solid phase assays-Elisa for the detection of anti-cardiolipin and anti-β2 glycoprotein antibodies [19–24].

1.The positive ACA IgG, Wolf Anticoagulant, ANA, ds-DNA, ENA (Ro, La) etc. are a heterogeneous group of IgG/IgM antibodies directed against plasma proteins involved in coagulation cataract activation. They are attached to PLs of the outer membrane of cells. While these antibodies cause thrombosis in vivo, in vitro prolong phospholipid-dependent coagulation tests. Detection of lupus anticoagulant (coagulation tests – 3-step procedure) prolongs the phospholipid-dependent coagulation time. Lupus anticoagulant is considered positive when it is detected in at least one of the two methods with the following diagnostic criteria – steps:

#### **15.1 The screening test**

	- Activated partial thromboplastin time (aPTT),
	- Russel snake venom dilution time (dRVVT)
	- Confirmation of the presence of coagulation inhibitor and not the absence of coagulation factor mixing test: Proof that the prolongation is due to the presence of coagulation inhibitor.

#### **15.2 Anti-cardiolipin antibodies (aCL) (IgG-IgM)**

Presence of antibodies against cardiolipin (aCL) – IgG moderately to strongly positive (>15–20GPL) – IgM(> 15 – 20MPL) moderate to strongly positive, but with the simultaneous presence of LA [19–24].

#### *Inflammation in the 21st Century*

They are directed against epitopes resulting from modulatory changes in the β2GPI (domains-V) molecule after binding to cardiolipin. They are directed directly against cardiolipin (CL). They are associated with infections, syphilis, in healthy individuals and are not related to APS [19–24].

#### **15.3 Antiβ2 GPI antibodies (IgG-IgM)**

Antibodies against β2GPI (anti-β2GPI), β2-glycoproteinI (β2 GPI) natural anticoagulant plasma protein associated with negatively charged molecules: phospholipids, heparin, lipoproteins (oxLDL), activated PLTs/Ecs, apoptotic cell membranes.

They turn directly against β2-GPI and are a heterogeneous population that recognizes epitopes in different regions (I-V) of the protein.

Antigen: purified human β2GPI with tight adhesion to plates for low avidity binding of antibodies and detection of domain I in situ.

Anti-LA/β2GPIs are positively associated with a particularly increased risk of thromboembolic complications and detection with new methods offers new opportunities for risk assessment [19–22].

ELISA: αCL/β2GPI detects various antibody specificity es with relatively low clinical utility – their exclusion from the criteria has been widely discussed, they are still a diagnostic criterion but current screening guidelines should be followed faithfully [19–22].

aβ2GPI antibodies: correlate very well with the clinical manifestations of APS – They are positive in patients with thrombosis who have negative LA and aCLs.

If aPL title > 40 GPL or IU at least 2 times in 12 week interval then APS is diagnosed. The most important are Abs against β2GPI and against prothrombin which show LA activity. Lupus anticoagulant is part of the APS antibody spectrum, reacting in the liquid phase. In contrast, other aPLs such as anti-CL and anti-β2 glycoprotein (β2-GPI-1) antibodies are detected by solid-phase immunoassay. For the APS diagnosis it is necessary to use both solid phase methods and coagulation tests for LA [19–24].

#### **16. Other aPLs**

Prothrombin antibodies: They have good association with LA, use in seronegative APS.

Antibodies to Annexin V, II Associated with thrombosis in APS.

Proteins against protein C and S: Lower sensitivity and specificity than aCLs/IgG.

Antibodies against vimentin (anti-Vim/CL: Positive (55%) in seronegative SN-APS, their use needs documentation.

The diagnosis of the syndrome should be avoided when a period of <12 weeks or > 5 years separates the clinical from the laboratory characteristics (regardless of the most presented first) [24–26].

#### **17. Secondary antibodies in APS**


*Antiphospholipid Syndrome and Pregnancy-Diagnosis, Complications and Management… DOI: http://dx.doi.org/10.5772/intechopen.99283*


APLs are a very heterogeneous family of antibodies and more than 30 different antibodies have been reported in patients with APS called 'antibody burst'. Their positivity does not offer much in the absence of clinical findings although confirmation is necessary for the duration of the symptoms. Ultimately the history and clinical picture determine the treatment [24–28].


Positive antinuclear antibodies are often found at low titers, without necessarily being associated with the presence of SLE [19–26].

Coagulation methods for detecting anticoagulant lupus are affected by oral anticoagulant therapy (coumarin, newer anticoagulants), but also by therapeutic doses of standard heparin. For this reason, testing should be done before starting or after cessation of these drugs. In contrast, administration of low molecular weight heparin in prophylactic doses of aspirin or clopidogrel does not appear to significantly affect the detection of anticoagulant lupus.

### **18. APS-pregnacy**

#### **18.1 Clinical findings**

APS is diagnosed in up to 40% of women with a history of miscarriage, intrauterine fetal death (> 18th week of gestation) or placental vascular disease, ie preeclampsia, intrauterine fetal growth retardation, placental abruption. However, in a percentage of 50–60% the causes remain unclear [26–34].

#### **18.2 Manifested clinically with**


#### **18.3 Obstetric antiphospholipid syndrome triggered in pregnancy**

The findings of some studies raise the suspicion of a subtype triggered by gestational APS, with a transient increase in antiphospholipid antibodies only during pregnancy.

#### **18.4 Pregnancy morbidity**

The morbidity of pregnancy is certified by the following parameters:

	- at least 1 unexplained fetal death in the 2nd or 3rd quarter (> 10 W)
	- at least 1 birth at gestational age <34 W, due to preeclampsia or placental insufficiency

According to the International Consensus Criteria of 2006, any of these antibodies, if tested positive in at least two laboratory tests at least 12 weeks apart, in combination with a clinical thrombosis or obstetric complication, leads to the diagnosis of APS.

*Antiphospholipid Syndrome and Pregnancy-Diagnosis, Complications and Management… DOI: http://dx.doi.org/10.5772/intechopen.99283*

There must be at least one laboratory and one clinical criterion for the diagnosis of primary APS. Antiphospholipid antibodies are detected by the enzyme-linked immunosorbent assay (ELISA) in which the phospholipid cardiolipin is used as the antigen. It is actually a complex of cardiolipin with a serum protein called β2-glycoprotein I (β2-GPI). The above protein plays an inhibitory role in blood clotting, and when the complex binds to antiphospholipid antibodies, its effectiveness as an anticoagulant decreases [26–42].

Laboratory findings usually show high titers of IgG or IgM antibodies against cardiolipin or lupus anticoagulant, which must be detected in the same patient in two different samples taken at least 6 weeks apart. The above mechanism is due to the frequent recurrent thrombi that affect both the large and small vessels of the arterial or venous limb. In addition, mild cytopenia occurs quite often, which usually subsides with the end of pregnancy.

#### **18.5 The diagnosis of the syndrome can be suspected in**

Arterial or venous thrombosis.

Presence of anticardiolipin antibodies, IgG, IgM, Anti-beta2-GPI and Lupus anticoagulant [26–42].

Adverse outcomes of pregnancy.

APS can be diagnosed, if one or more of the clinical criteria and one or more of the laboratory criteria are met.

Although a causal association between obstetric complications and antibody detection is difficult to identified, however the lupus anticoagulant is the major predictor of labor adverse events including both mother and fetus. Although spontaneous abortions before the 10th week are relatively common in the general population, it seems that this risk is higher in patients with the syndrome. Fetal demise due to insufficient blood flow is most probably caused by placental insufficiency triggered by placental infarctions [26–42].

This placental insufficiency is likely to be associated with delayed intrauterine fetal development, severe preeclampsia, premature rupture of the membranes, premature placental abruption, and preterm fetal death (preterm and preterm death) in the 20th week of pregnancy, which usually has the worst outcome, as well as the increased risk of premature ejaculation [26–42].

Regarding the relationship of aPLs with preeclampsia, their detection seems valuable only in cases of severe preeclampsia before the 34th week of pregnancy. For cases of severe intrauterine fetal growth retardation, there are studies that report its relationship to the presence of antibodies, while other studies do not seem to reach this conclusion.

#### **19. Discussion**

The risk of miscarriage in women with antiphospholipid antibodies is higher from the 10th week of pregnancy onwards. But also in women with a history of six miscarriages before the 10th week of pregnancy, antiphospholipid antibodies are detected in rates of 10 to 20% without the presence of other clinical manifestations [42–44].

Pregnancy complications in women with APS are due to decreased placental perfusion based on local thrombosis, which is probably caused by the interaction of aPL with annexin V of the trophoblast resulting in inhibition of its anticoagulant activity. Other manifestations of aPL include thrombocytopenia (40–50%), hemolytic anemia (14–23%), renal disease that has only recently been recognized as a consequence of APS, and Liveto redicularis [42–48]. Female patients with APS and kidney disease from antiphospholipid antibodies typically have high blood pressure, which is an additional serious risk to their pregnancy and can lead to the complications mentioned above [42–48].

#### **20. When will we check a patient for aPLs**


Finally, for the presence of antiphospholipid syndrome, women of reproductive age who have any of the following characteristics should be screened (other than those mentioned above): False positive test for syphilis, stroke and venous thrombosis without other predisposing prolongation of aPTT, SLE and autoimmune hemolytic anemia [48–58].

Of course, from the medical history of the pregnant woman should always be sought the episodes of venous thrombosis that are usually observed in the veins of the lower extremities, which are not necessarily accompanied by episodes of pulmonary embolism, but also in rarer localizations, such as the sphenoid sinuses of the skull and the small visceral vessels. Autoantibodies and microthrombotic mechanisms could affect the normal implantation, the trophoblasts' expansion and the development of effective fetoplacental circulation leading to abortions of the first trimester [48–58].

At older gestational ages endometrial death is attributed to massive placental thrombosis while the mechanisms associated with other complications (preeclampsia) are unknown. In terms of laboratory findings, moderate to high IgG or IgM antibodies to cardiolipin (20–50 GPL, 20–80 MPL respectively) or lupus anticoagulant should be detected [48–58].

Patients who present with clinical manifestations of APS could be permanently negative for the three main autoantibodies. Women with positive aPLs are more likely to have thromboembolic events, miscarriage or fetal death, intrauterine growth retardation, severe preeclampsia, and placental abruption. However, the presence of these antibodies cannot exclude the possibility of a successful

#### *Antiphospholipid Syndrome and Pregnancy-Diagnosis, Complications and Management… DOI: http://dx.doi.org/10.5772/intechopen.99283*

pregnancy and/or estimate the risk of potential complications. The existence of a burdensome obstetric or pathological history seems to play a more important role, since the reporting of thrombotic episodes, SLE or fetal death is associated with a 40% chance of premature birth and a greater than 30% chance of intrauterine growth retardation. From the beginning of pregnancy until the 20th week, visits should be made every 15 days and then every week until delivery [52–60].

Ultrasound examination of fetal development, but also the evaluation of the amount of amniotic fluid, should begin in the 16th week and be repeated every month unless there is a pathological finding. There is evidence that bilateral presence of notches in the uterine arteries, on Doppler screening at 24 weeks, can detect with satisfactory sensitivity those patients who develop preeclampsia and IUGR if they have a positive lupus anticoagulant. The umbilical artery test with Doppler from the 26th week until childbirth offers great help, while for the same period of time a weekly cardiotocographic test (non-stress test) should be performed, as well as an ultrasound control of the amount of amniotic fluid. The most commonly used regimen involves the administration of aspirin (80 mg daily) and heparin, either crystalline or low molecular weight, in prophylactic doses [60–64].

The patient's health condition before pregnancy will determine the resumption of therapy after childbirth. Thus, two to three days after delivery, women taking coumarin derivatives before pregnancy (due to a history of a thromboembolic event) should discontinue heparin (after an INR of 2–2.5) and resume taking these drugs. To decrease the possibility of a new thromboembolic event, women with a thrombotic history during the late pregnancy should be treated with prophylactic doses of heparin or coumarin derivatives for 6 weeks postpartum. For women without a thrombotic history, the anticoagulant therapy could be continued for the first five postpartum days at most. Compared to conventional heparin, less complications were related to low molecular weight heparins. Regarding the duration of the treatment, some recommend the prophylactic administration until the completion of the 37th week, then proposing induction of labor and others the administration until the automatic onset of labor with the simultaneous administration of vitamin K antagonists.

Hyperimmune γ-globin is no longer recommended because there is no clear evidence of improved perinatal outcome. Coumarin is not administered particularly in the 1st and 3rd trimesters as potential teratogens and due to easy passage through the placenta coagulation disorders in the fetus and because they are associated with greater maternal morbidity. Their administration is indicated only in rare cases of contraindication to heparin or aspirin [60–68].

Complications of anticoagulant therapy in pregnancy include embryopathy (nasal hypoplasia, spotted epiphyses), CNS abnormalities (Dandy-Walker syndrome, visual atrophy), fetal bleeding, hemorrhagic manifestations, skin allergies, thrombocytopenia and osteoporosis [60–72].

The basic principles of APS treatment include the systematic monitoring of the pregnant woman, the continuous assessment of the condition of the fetus, the administration of medication and the selection of the most appropriate time and manner of delivery. Despite the lack of large cross investigations, the usual therapeutic directions includes the corticosteroids, the aspirin, heparin, hyperimmune gamma globulin and coumarins.

Treatment should be applied only when the risk of complications is considered to be higher and after a thorough discussion with the pregnant woman. The prognostic factors of poor outcome are the title of anticardiolipin antibodies and the obstetric history [60–72].

Aspirin significantly reduces the risk of thrombosis by blocking platelet aggregation. It is considered safe during pregnancy. Until now, there are no final conclusions regarding the efficacy of the above therapy as monotherapy. Hydroxychloroquine inhibits aPL-B2GPI complexes on phospholipid surfaces, annexin A5, TF tissue factor, TLPs *Toll-like receptors*, statins such as pravastatin inhibition of *Nuclear factor kappa B* (*NF-*κ*B*) is protein transcription factor, *Anti*-*CD20* monoclonal antibodies (mAbs) resistant APS and aspirin are recommended to prevent and treat preeclampsia [60–72].

The choice of time and method of delivery depends mainly on the presence or absence of complications of the disease during pregnancy. In any case of intrauterine suffocation of the fetus, induction of labor is required with all the possible harmful consequences of prematurity. In asymptomatic forms of the disease and when there are no signs of fetal difficulty, childbirth is preferred as much as possible at the end of pregnancy.

Worldwide, the route of delivery has been the subject of intense controversy and it is not clear if vaginal delivery or cesarean (c-)section is safer for the mother. Thus, prelabour c-section or vaginal delivery should be guided by obstetric criteria [60–72].

APS (primary or secondary) is a chronic systemic autoimmune disorder that mainly affects young women of childbearing age. APL can impair trophoblast function and can cause implantation failure by not allowing fusion of the cytotrophoblast and can also developing small thrombophilia increase abortion rate. Natural killer cells attach to the cytotrophoblast of the embryo. However, the mechanisms by which such cells may or may not affect the embryo is not proven. Moreover, after the implantation, there is a slight inflammatory response. Patient with recurrent miscarriages and infertility develop less prominent reaction that may prevent the fetus from implantation [72–74].

#### **21. Conclusion**

The effect of the syndrome on pregnancy is accompanied by a multitude of serious complications that significantly increase the rates of maternal and especially perinatal morbidity and mortality.

There is an urgent need to create a new laboratory method, which will detect with great sensitivity and specificity all antiphospholipid antibodies and for this purpose large multicenter studies are already being done, the results of which are awaited.

#### **Acknowledgements**

I would like to warmly thank Ms. Dr. Lefkou Eleftheria and Associate Professor Panagiotis Skendros, who with their vast experience and valuable help and guidance on resolving various problems related to antiphospholipidemic syndrome contributed significantly to complete the background of the chapter.

*Antiphospholipid Syndrome and Pregnancy-Diagnosis, Complications and Management… DOI: http://dx.doi.org/10.5772/intechopen.99283*

#### **Author details**

Panagiotis Tsikouras1 \*, Christina Tsiggalou<sup>2</sup> , Anastasia Bothou1 , Aggeliki Gerede1 , Ifigenia Apostolou3 , Fotini Gaitatzi1 , Anna Chalkidou1 , Xanthoula Anthoulaki1 , Spyridon Michalopoulos1 , Georgios Dragoutsos1 , Ioannis Tsirkas1 , Irini Babageorgaka1 , Theopi Nalbanti1 , Natalia Sachnova1 , Alexios Alexiou1 , Konstantinos Nikolettos1 , Christos Tsalikidis4 , Stefanos Zervoudis4 , Panagiotis Peitsidis1,5 and Nikolaos Nikolettos1

1 Department of Obstetrics and Gynecology, Democritus University of Thrace Medical School, Alexandroupolis, Greece

2 Department of Laboratory, Democritus University of Thrace Medical School, Alexandroupolis, Greece

3 Neonatal Intensive Unit Care, University Hospital of Thrace Medical School, Alexandroupolis, Greece

4 Second Department of Surgery University Hospital of Thrace Medical School, Alexandroupolis, Greece

5 Technological Educational Institute of Athens and Rea Maternity Hospital, Athens, Greece

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

© 2021 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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### Section 6
