**3. Evidence that does not match the hygiene hypothesis**

In great contrast to T1D and MS, rheumatoid arthritis (RA) has been declining in prevalence in recent decades (Gabriel &Michaud, 2009). Moreover, its geographical distribution is very different from that of T1D or MS. That is, RA is generally evenly distributed in the world and does not have a "North-south gradient". (Shapira et al., 2010b). The geographical distribution of systemic sclerosis (SSc) seems to be contrast to those of T1D and MS. Within Europe, SSc is less frequent in the north than south (Shapira et al., 2010b). In the case of ankylosing spondylitis (AS), a genetic factor (HLA-B27) rather than environmental factors seems to explain the prevalence of the disease (Shapira et al., 2010b). Therefore, the geographical distribution and/or chronological transition of some autoimmune diseases cannot be explained by the hygiene hypothesis.

For atopic disorders, the sibling effect itself has been confirmed (Benn et al, 2004; Matheson et al., 2009; Cullinan et al., 2003). However, some authors suggest that infections in early life do not explain the observed sibling effect (Benn et al., 2004; Cullinan et al., 2003). In a report on the preventive effects of endotoxins against AD (Flohr et al., 2005), apparent infections in early life were shown to increase the risk of AD. In a meta-analysis of relationships between exposure to furry pets and asthma and allergic rhinitis, there were different effects in different species of animals. That is, exposure to dogs increases the risk but exposure to cats decreases it (Takkouche et al., 2008). Moreover, in a meta-analysis of interrelationships between vaccinations with bacterial products (BCG and pertussis vaccine) and the incidence of asthma, no statistical association was found (Balicer et al., 2007). For T1D, there was no association between routinely recorded infections in early life and subsequent risk of the disease (Cardwell et al., 2008a). In addition, T1D had inverse relationships with asthma (Cardwell et al., 2003). These finding do not match the hypothesis that infections and exposure to pathogens protect against both atopy and autoimmunity. A systematic review of mycobacterial infections and atopy (Obihara et al., 2007) found that the negative correlations between them were mainly based on cross-sectional studies. The authors, therefore, claimed that population-based prospective studies would be needed.

Regarding etiology of MS, two hypotheses have been proposed (Milo & Kahana, 2010). The "prevalence hypothesis" postulates that MS is caused by a pathogen common in highincidence areas. In contrast, according to the more accepted "poliomyelitis-hygiene hypothesis", certain infections in early childhood protect against MS whereas infections with the same infectious agents later in life (e.g. adolescence) cause the disease, as in the cases of poliomyelitis. This concept is different from that of the "original" hygiene hypothesis, in that people living in hygienic conditions (= non-infected people) are protected from the disease. Epstein-Barr virus (EBV) is one of the infectious agents suspected to be causative of MS. Individuals infected with EBV in early childhood have a lower risk of MS than those infected in adolescence and as a consequence, suffer from

Parasitic Helminths as Potential Therapeutic Agents Against Autoimmune Disorders 595

control group, suggesting that the parasite might protect against autoimmune liver diseases (Aoyama et al., 2007). Most recently, Mutapi et al. (2011) reported epidemiological study in Zimbabwe on the interrelationships between *Schistosoma haematobium* infection and autoreactive antibody levels. They found an inverse relationship between infection intensity and anti-nuclear antibody (ANA) levels in schistosome- endemic areas. According to the study,

Compared to studies on human helminth infections and autoimmunity, reports on the interrelationships between helminth infections and atopic disorders are much more common. However, as there is considerable variation in results among the reports, systematic reviews and meta-analyses are important. According to reviews of crosssectional studies on the relationships between current parasitic infections and athma and atopy (Leonardi-Bee et al., 2006; Flohr et al., 2009; Feary et al., 2011), both intestinal helminths (Hookworms, *Ascaris*, *Trichuris*, *Strongyloides*, *Enterobius*) and schistosomes significantly lowered reactivity in the skin prick test. In contrast, only hookworm infections lowered the risk of asthma significantly; odds ratio (OR) = 0.50 (Leonardi-Bee et al., 2006). It is worth noting that *Ascaris* infections heightened the risk of asthma (OR = 1.34). Other

The evidence obtained in cross-sectional studies is indirect. Direct evidence of ameliorating effects of helminths can be obtained by intervention studies. According to the review literature above (Flohr et al., 2009), in some intervention studies, allergic skin sensitization increased after de-worming treatment. This finding was reproducibly observed in independent studies in Venezuela (Lynch et al., 1993), Gabon (van den Biggelaar et al., 2004) and Vietnam (Flohr et al., 2010). However, in a study in Ecuador (Cooper et al., 2006), there was no increase in the prevalence of atopy or clinical allergies after de-worming treatment. Furthermore, in the study in Vietnam (Flohr et al., 2010), the clinical symptoms of allergy did not worsen in the treated group despite the increased sensitization. Remarkably, a study in Venezuela (Lynch et al., 1997) showed a clinical *improvement* in asthma after regular antihelminthic treatment. Taken together with cross-sectional studies of allergies in helminthinfected individuals, the overall results could be summarized as follows: 1) In general, parasitic helminths suppress skin sensitization. 2) However, parasitic helminths do not always suppress clinical allergies and can sometimes worsen allergic symptoms. Regarding the timing of helminth infections, a study in Brazil is especially noteworthy (Rodrigues et al., 2008). In the study, heavy infections with *T. trichiura* in early childhood were shown to reduce reactivity to allergens in later childhood. Even if the child was not infected at the time of the skin test, this protective effect was observed. Cooper et al. (2009) summarized studies on helminth infections and clinical allergies in a review, in which he stated that different helminths have different effects on allergies depending on the timing of exposure. According to the review, *Trichuris* and hookworms are protective, whereas *Ascaris* and *Toxocara* may be risk factors in certain situations. Further studies, especially de-worming intervention studies or therapeutic clinical trials using helminths, may be necessary to

Until several years ago, major autoimmune diseases such as MS, T1D, RA and CD and animal models thereof had been classified as Th1-dependent diseases. Recently, however,

anti-helminthic treatment significantly increased ANA levels.

geohelminths had no significant effects on the risk of asthma.

establish a general view of the anti-allergic effects of helminths.

**6. T cell subsets and autoimmunity** 

**5. Influence of helminth infections on human atopic disorders** 

infectious mononucleosis (IM). Additionally, non-infected people are known to have the lowest risk of MS (Ascheiro & Munger, 2010), suggesting the plausibility of the poliomyelitis-hygiene hypothesis. In addition to the causal relationship betwen EBV and MS, a number of infectious agents (HCV, rotavirus, Coxsackie virus, HHV-6, *Helicobacter pylori*, *Trypanosoma cruzi* etc.) were reported to have an association with autoimmunity (Kivity et al., 2009; Getts & Miller 2010).

Collectively, numerous reports seem to contradict the hygiene hypothesis. In the 99th Dahlem Conference on Infection, Dr. Matricardi tried to reconcile the conflicting evidence (Matricardi, 2010). For instance, infections with orofecal pathogens like hepatitis A virus (HAV) were shown to be inversely correlated with atopy whereas infections of airborne viruses (measles virus, mumps virus, rubella virus etc.) were not (Matricardi et al., 1997; Matricardi et al., 2000). He proposes gastrointestinal infections that stimulate immunological changes in gut-associated lymphoid tissue (GALT) to be important for protective effects against atopic diseases (Matricardi, 2010, Matricardi et al., 2000). Likewise, to explain why some microorganisms protect against immunological disorders and others do not, a refined version of the hygiene hypothesis, the "old friends hypothesis" has been proposed (Rook, 2009). According to this hypothesis, immunological disorders develop if "old friends" (=microorganisms present in humans for a long time on the evolutionary time scale) are expelled from the body. Parasitic helminths and saprophytic bacteria are representative "old friends". To avoid having to fight the host's immune systems long term, they become relatively harmless and continuously activate regulatory dendritic cells (DCs) and regulatory T cells (Treg) at background levels. Consequently, the presence of "old friends" renders hosts resistant to immune dysregulation. This hypothesis seems very attractive, especially to explain immunoregulatory mechanisms by parasitic helminths.

### **4. Influence of helminth infections on human autoimmune disorders**

There have been a few papers on the interrelationships between helminth infections and autoimmune diseases in humans. Correale and colleagues have reported the influence of helminth infections on MS. In a prospective, double-cohort study, they found that MS patients infected with helminths (*Hymenolepis nana*, *Trichuris trichiura*, *Ascaris lumbricoides*, *Strongyloides stercolaris* or *Enterobius vermicularis*) showed fewer exacerbations, reduced disability scores, and lower MRI activity compared with uninfected MS patients (Correale & Farez, 2007). Furthermore, the infected MS patients showed an increase in myelin basic protein (MBP)-specific IL-10 and TGF-β - producing cells and a decrease in IL-12 and IFN-γ producing cells. B cells from helminth-infected MS patients tend to produce high levels of IL-10, brain-derived neurotropic factor (BDNF), and nerve growth factor (NGF) (Correale et al., 2008). More importantly, some MS patients who received anti-parasite treatment, due to an exacerbation of helminth-derived symptoms, showed an increase in MS activity (Correale & Farez, 2011). The authors also showed that both B cells and DCs from intestinal helminthinfected MS patients had increased expression of TLR2 and some of immunological changes observed in helminth Ag-exposed cells were TLR2-dependent (Correale & Farez, 2009). Collectively, these results suggest that parasitic helminths have anti-MS activity in humans. Fleming & Cook (2006) summarized the relationship between prevalence of *T. trichiura* and MS at the country / regional level. According to the data, MS is prevalent only in *T. trichiura*-free countries. Regarding other autoimmune diseases, in a case-control study in Okinawa (Japan), *S. stercolaris* was less frequent in the autoimmune liver disease group than

infectious mononucleosis (IM). Additionally, non-infected people are known to have the lowest risk of MS (Ascheiro & Munger, 2010), suggesting the plausibility of the poliomyelitis-hygiene hypothesis. In addition to the causal relationship betwen EBV and MS, a number of infectious agents (HCV, rotavirus, Coxsackie virus, HHV-6, *Helicobacter pylori*, *Trypanosoma cruzi* etc.) were reported to have an association with autoimmunity

Collectively, numerous reports seem to contradict the hygiene hypothesis. In the 99th Dahlem Conference on Infection, Dr. Matricardi tried to reconcile the conflicting evidence (Matricardi, 2010). For instance, infections with orofecal pathogens like hepatitis A virus (HAV) were shown to be inversely correlated with atopy whereas infections of airborne viruses (measles virus, mumps virus, rubella virus etc.) were not (Matricardi et al., 1997; Matricardi et al., 2000). He proposes gastrointestinal infections that stimulate immunological changes in gut-associated lymphoid tissue (GALT) to be important for protective effects against atopic diseases (Matricardi, 2010, Matricardi et al., 2000). Likewise, to explain why some microorganisms protect against immunological disorders and others do not, a refined version of the hygiene hypothesis, the "old friends hypothesis" has been proposed (Rook, 2009). According to this hypothesis, immunological disorders develop if "old friends" (=microorganisms present in humans for a long time on the evolutionary time scale) are expelled from the body. Parasitic helminths and saprophytic bacteria are representative "old friends". To avoid having to fight the host's immune systems long term, they become relatively harmless and continuously activate regulatory dendritic cells (DCs) and regulatory T cells (Treg) at background levels. Consequently, the presence of "old friends" renders hosts resistant to immune dysregulation. This hypothesis seems very attractive,

especially to explain immunoregulatory mechanisms by parasitic helminths.

**4. Influence of helminth infections on human autoimmune disorders** 

There have been a few papers on the interrelationships between helminth infections and autoimmune diseases in humans. Correale and colleagues have reported the influence of helminth infections on MS. In a prospective, double-cohort study, they found that MS patients infected with helminths (*Hymenolepis nana*, *Trichuris trichiura*, *Ascaris lumbricoides*, *Strongyloides stercolaris* or *Enterobius vermicularis*) showed fewer exacerbations, reduced disability scores, and lower MRI activity compared with uninfected MS patients (Correale & Farez, 2007). Furthermore, the infected MS patients showed an increase in myelin basic protein (MBP)-specific IL-10 and TGF-β - producing cells and a decrease in IL-12 and IFN-γ producing cells. B cells from helminth-infected MS patients tend to produce high levels of IL-10, brain-derived neurotropic factor (BDNF), and nerve growth factor (NGF) (Correale et al., 2008). More importantly, some MS patients who received anti-parasite treatment, due to an exacerbation of helminth-derived symptoms, showed an increase in MS activity (Correale & Farez, 2011). The authors also showed that both B cells and DCs from intestinal helminthinfected MS patients had increased expression of TLR2 and some of immunological changes observed in helminth Ag-exposed cells were TLR2-dependent (Correale & Farez, 2009). Collectively, these results suggest that parasitic helminths have anti-MS activity in humans. Fleming & Cook (2006) summarized the relationship between prevalence of *T. trichiura* and MS at the country / regional level. According to the data, MS is prevalent only in *T. trichiura*-free countries. Regarding other autoimmune diseases, in a case-control study in Okinawa (Japan), *S. stercolaris* was less frequent in the autoimmune liver disease group than

(Kivity et al., 2009; Getts & Miller 2010).

control group, suggesting that the parasite might protect against autoimmune liver diseases (Aoyama et al., 2007). Most recently, Mutapi et al. (2011) reported epidemiological study in Zimbabwe on the interrelationships between *Schistosoma haematobium* infection and autoreactive antibody levels. They found an inverse relationship between infection intensity and anti-nuclear antibody (ANA) levels in schistosome- endemic areas. According to the study, anti-helminthic treatment significantly increased ANA levels.
