Hepatitis C Virus Spontaneous Clearance: Immunology and Genetic Variance

Abstract

Hepatitis C virus (HCV) infection is one of the most common chronic viral infections in the world. Approximately 80–90% of acutely infected individuals develop persistent infection, which is a major risk for liver cirrhosis and liver cancer. However, a small portion of patients (10–20%) clear the virus. Clinical outcomes of HCV infection are determined by the interplay between the host immune response, and viral and environmental factors. In regulating immune responses, cytokines play an indispensable role that controls the underlying pathogenesis and the resulting outcome of HCV infection. Cytokines themselves are manipulated by polymorphisms in their genes. In fact, the majority of genetic variants that apparently confer a significant risk for chronic HCV infection have been localized in genes involved in cytokine synthesis and the ultimate immune response. So far, treatment strategies for HCV infection have remained controversial. Genotyping of different polymorphisms will aid clinical decision making for both current standard and personalized care. Genotyping can potentially be useful for future integration of other agents, which provides an opportunity for clinicians to personalize treatment regimens for HCV patients. This review summarizes findings of different studies on host immune responses after HCV infection and the association between cytokine gene polymorphisms and the likelihood of HCV clearance.

Introduction

H epatitis C virus (HCV) is a small positive-stranded RNA virus in the Flaviviridae family that persists in infected individuals (62). It is one of the major causes of liver disease and a significant concern for public health worldwide. In 80–90% of patients, infection progresses to chronicity. These patients are subsequently at risk for liver-related morbidity and mortality (62). However, a minority of patients spontaneously clear the virus. Estimates of clearance rates have ranged from 10–20%, and the duration of time to clearance has been reported to be as long as 3 y in some cases (74). Yet the exact rate of resolution is not known due to difficulties in identifying individuals who have recovered from infection. Some factors that are plausibly associated with viral clearance in various studies include: ethnicity (66,72), icteric clinical presentation (72), absence of human immunodeficiency virus (HIV) infection (66), short incubation period (30), rapid decline in HCV RNA levels (25), specific human leukocyte antigen (HLA) type I and II alleles (27), and vigorous HCV multi-specific immune responses by CD4⁺ and CD8⁺ T cells (34,61).

The management of HCV infection has become a matter of debate in recent years. To date, viral factors (e.g., genotype), and host factors (e.g., age of acquisition and sex and alcohol consumption), are known to alter both the natural history of the disease and the treatment outcomes (11,18,54). Although treatment with the current standard of care, pegylated interferon-α2a or -α2b plus ribavirin may be curative, it is effective in only 40–50% of patients (69), and interferon (IFN)-α-based therapies are associated with significant adverse effects and costs. In order to avoid unnecessary treatment, accurate markers are needed to identify patients who may spontaneously clear the virus prior to therapy. Therefore, understanding virus-host interactions that enable a fraction of patients with acute infection to clear HCV is probably a key to the development of more effective treatment and prevention strategies.

Various studies have suggested that the polymorphic genetic compositions of human populations, genetic variations, and sustained and broad CD4⁺ and CD8⁺ T-cell responses (11,62) probably play a critical role in self-limited HCV infection. That is the reason so many recent studies have focused on the genetic polymorphisms of cytokines and their receptors (17,67). The capacity of individuals for cytokine production has a major genetic component. As the indispensability of cytokines and the cellular immune response in the pathogenesis and eradication of chronic HCV infection has become clearer, the importance of the host immune response has also been reinforced (15). Consequently, several researchers have studied adaptive and innate immunity features in HCV infection. These studies revealed that HCV clearance is associated with vigorous HCV multi-specific Th-1-type immune responses by CD4⁺ and CD8⁺ T cells. In contrast, a lack of such a response appears to predict the onset of chronic infection (58).

The clinical relevance of different findings for HCV patients and nucleotide polymorphisms suggest that cytokine genotypic testing might help guide treatment decisions. Genotypic testing could detect those who are most likely to respond and therefore spare them from futile therapy. However, it should be considered that these human cytokine genotypes may be confused with HCV genotypes, which also predict treatment response. Discussing “favorable genotype” or “high-risk genotype” at present has two distinct meanings. Understanding the genetic basis of host-limiting infection of HCV (clearance without any HCV-specific therapy) provides a direct approach to finding clues to the treatment of this chronic infection. While the incidence of newly-acquired hepatitis C infection has declined in some countries, HCV infection remains a high-priority problem that needs to be addressed by utilizing many different approaches.

Here we will give an overview of the effect of functionally-relevant polymorphisms of HLA, IFN-γ, interleukin-10 (IL-10), IL-18, and IL-28B genes on HCV spontaneous clearance.

Genomic polymorphisms

HLA

The major histocompatibility complex (MHC) plays a critical role in the immune response, as the products of these genes present viral antigen to cytotoxic and helper T cells. Several immunogenetic studies have demonstrated the role of HLA variation in the outcome of HCV infection (64,65,68). The HLA loci display an unprecedented degree of diversity in the human genome, presumably an evolutionary adaptation to immune pressure from various infectious agents.

Studies of humans and animal models of HCV infection have demonstrated that HCV elicits innate immune responses early after infection. However, the virus can persist in the face of the innate immune response. Indeed, viral clearance occurs only in the presence of antiviral CD4⁺ and CD8⁺ T-cell responses (60). To elicit an adaptive cellular immune response, HCV antigens are processed into peptides that bind HLA molecules as a first step in the initiation of the immune response. These are then presented on the cell surface of either antigen-presenting cells or on infected cells such as hepatocytes. CD8⁺ and CD4⁺ T cells can recognize the complex of HLA class I peptides or class II peptides, and act as either effector T cells, helper T cells, or regulatory T cells, through direct killing, cell contact, or secreting cytokines, respectively (9,50,62). In HCV infection, recognition and elimination of infected cells by cytotoxic T lymphocytes (CTLs) require the presentation of specific HCV antigens on the membrane of hepatocytes in the context of HLA-A, -B, and -C antigens. In chronic HCV infection, HLA class I-restricted CTL responses may be responsible for controlling viral replication, and may be directly responsible for the histological injury sustained in chronic infection, rather than a direct hepatotoxic effect of the virus.

Several associations between HLA class I alleles and the self-limited course of HCV infection have been described in a large number of studies worldwide (Table 1). These studies vary significantly in design and cohort characteristics, which may account for some contradictory findings. However, there is general agreement that certain HLA class I alleles are associated with protection. For example, in a large study by Thio et al. in Caucasians and black Americans (64), HLA-A1101 and HLA-B57 were significantly more frequent in individuals with well-documented HCV clearance compared to chronically-infected patients. HLA-B57 was also found to be protective in a West African population (10). A correlation of HLA-B57 and a higher rate of viral clearance has been proven by Kim et al. They also found that HLA-B57-mediated antiviral immunity is associated with control of both HIV-1 and HCV, which suggests a common shared mechanism of a successful immune response against persistent viruses (31).

Table 1.

Association Between Human Leukocyte Antigen HLA Class I and II Alleles and HCV Spontaneous Clearance

HLA class I	Reference
HLA‐A03	McKiernan et al. (41)
HLA‐A1101	Thio et al. (64)
HLA‐B27	McKiernan et al. (41)
HLA‐B35	Ksiaa et al. (33)
HLA‐B57	Thio et al. (64), Chuang et al. 2007 (10)
HLA‐Cw05	Wang et al. (73)
HLA‐Cw01	McKiernan et al. (41)
HLA‐Cw0102	Thio et al. (64)

HLA class II
DQA1^*0103	Hohler et al. (26)
DQA1^*03	Cramp et al. (12)
DQA1^*0501	Yoon et al. (78)
DQB1^*03	Wang et al. (73), Harris et al. (22)
DQB1^*0301	Alric et al. (2), Cramp et al.(12), Minton et al. (42), Mangia et al. (37), Thursz et al. (68), Alric et al. (1), Thio et al. (64), Higashi et al. (24)
DQB1^*0303	Higashi et al. (24)
DQB1^*05	Wang et al. (73)
DRB1^*01	Barrett et al. (6), Wang et al. (73)
DRB1^*0101	McKiernan et al. (41), Thio et al. (64)
DRB1^*04	Cramp et al. (12)
DRB1^*08	Ksiaa et al. (33)
DRB1^*1101	Scotto et al. (53), Yenigun and Durupinar (77)
DRB1^*15	McKiernan et al. (41)
DRB3^*02	Harris et al. (22)

Importantly, the strongest association between any HLA allele and viral clearance has been observed for HLA-B27 (41,44). The mechanisms behind the protective role of HLA-B27 in different viral infections are less understood. Possible explanations include molecular mimicry between foreign and self-peptides presented by HLA-B27, the high binding capacity of HLA-B27, the unusual tendency of HLA-B27 to misfold, or the involvement of non-canonical forms of HLA-B27 such as heavy chain homodimers, leading to increased natural killer activation or linkage disequilibrium with other immune response genes (36,56).

The data regarding associations of other HLA class I alleles and HCV infection is less consistent. As an example, HLA-B35 was found to be a protective allele in a Tunisian study population (33), but was associated with viral persistence in a Korean study (78). In addition, several other HLA class I alleles have been associated with viral clearance (Table 1).

CD4⁺ T cells are activated through the binding of the TCR to peptides presented by HLA class II molecules (HLA-DP, -DQ, and -DR in humans). The peptide binding region of the HLA class II molecules is formed by both the alpha and the beta chain. Thus both gene loci may contribute to differences in peptide binding, and one individual may have four different types of HLA-DQ and HLA-DR molecules. Comparable to HLA class I molecules, different HLA class II molecules bind to different viral peptides, which may modulate the antiviral T-cell response, depending on the genetic background of the individual.

Several associations between HLA class II alleles and the outcome of HCV infection have been identified (Table 1). DQB1*0301 was associated with clearance of HCV infection in numerous studies conducted in populations throughout the world (2,3,22,42,53,68,77). Interestingly, DQB1*0301 is in close linkage disequilibrium (non-random association between polymorphisms at different loci) with DRB1*1101, and DRB1*1101 is also associated with HCV clearance in several studies (1,12,22,24,37,42,64,68). It is assumed that the DQB1*0301 and DRB1*1101 alleles may present HCV epitopes more effectively to CD4⁺ Th cells than others, resulting in a vigorous proliferative response to HCV, and likely to disease recovery (20,21,29).

Wang et al. demonstrated that DRB1*01, DQB1*03, and DQB1*05 are associated with viral clearance in Midwestern Americans. Their results reveal an ethnically and geographically different distribution of HLA genes that determine the outcome of HCV infection (73). Alric et al. extend their findings by showing that DQB1*02 is present less frequently in subjects with spontaneous viral clearance compared to those who are chronically infected. Their study also showed that spontaneous HCV clearance is favored by female gender and class II antigens (1). It seems that a number of other genes, both within and outside the MHC, are able to influence the nature and magnitude of the immune response, and may as well play a role in the clearance of HCV. Clearly, additional studies are needed to address the immunological mechanisms that determine the protective or detrimental effects of HLA alleles in HCV infection. Differences in studies illustrate the potential limitations in performing association studies in homogenous cohorts. As a result, there is a need for testing these associations across viral genotypes and HLA phenotypes to identify the most relevant epitopes as a path to understanding the mechanisms of HCV clearance and vaccine development.

IFN-γ

IFN-γ is a multifunctional cytokine that is produced by effector T and natural killer cells. It determines the development of Th-1 cells and is critical for host defense against a variety of intracellular pathogens, including HCV. IFN-γ efficiently inhibits HCV replication in the replicon system in vitro (16), and the intrahepatic level of IFN-γ appears to be associated with viral clearance in a chimpanzee model (75). The human IFN-γ gene on chromosome 12q24.1 spans ∼5.4 kb and contains four exons that encode a 146-aa protein. Several polymorphisms within the IFN-γ noncoding regions, such as +874A/T, CA repeat microsatellite, and +179T/G, have been implicated in several autoimmune and chronic inflammatory conditions (7,47). However, none of them has been shown to be related to HCV infection. Huang et al. performed a study to identify potential markers of the IFN-γ gene associated with the response to IFN-γ-based therapy and spontaneous recovery in HCV infection. This study presents compelling evidence that a previously unnoticed functional variant in the proximal promoter region of the IFN-γ gene affects IFN-induced or spontaneous recovery from HCV infection. This relationship is in accord with the well-documented role of IFN-γ in the immune response to hepatitis C, and supports the hypothesis that host genetic factors play a major role in disease outcome and treatment response of HCV infection. The G allele at −764 confers a stronger induction of the IFN-γ gene and favors viral clearance and a response to exogenous IFN-γ-based therapy. This study reports a polymorphism variant in the IFN-γ promoter, −764C/G, which is functionally important in regulating IFN-γ gene expression in vitro. The G allele is significantly associated with viral clearance and treatment response (28). This variant is more common in Caucasian-Americans, consistent with the findings that spontaneous recovery is more common in Caucasians (66). The −764G variant has a higher binding affinity to the NF-κB motif, resulting in higher levels of promoter activity, and thus perhaps explaining the association with HCV recovery and treatment response (28). Overall, IFN-γ shows low-level associations with HCV spontaneous clearance, and to some extent sustained viral response to treatment (28,63).

IL-10

IL-10 plays an important role in the regulation of cellular immune responses and in the suppression of proinflammatory cytokine secretion in a number of different cell types. This cytokine is a synthesis inhibitory factor that balances the Th-1 and Th-2 immune response, and is involved in many aspects of human disease. In patients chronically infected with HCV, the production of inappropriate amounts of cytokines, such as IL-10 and TNF-α, have been reported to be associated with HCV clearance, fibrogenesis, and even resistance to interferon therapy (71,76). Cacciarelli et al. (8) reported elevated serum levels of IL-2, IL-4, and IL-10 in a proportion of patients with untreated chronic HCV infection. These in vivo results have received in vitro confirmation by the demonstration of a long-lasting increase of IL-10 production by CD4⁺ T cells following HCV infection of the cells (13). Various studies have demonstrated a striking difference between individuals in their ability to produce IL-10 after in vitro stimulation, and these differences have been ascribed to polymorphisms within the genomic regulatory regions, or within signal sequences of the respective genes (47,70). Several polymorphic sites within the promoter region of the IL-10 gene have been described; the combination of three single nucleotide polymorphisms at positions 1117, 854, and 627 from the transcription start site produces three different haplotypes (GCC, ACC, and ATA), which are associated with differential IL-10 expression. The ATA haplotype might be responsible for diminished IL-10 expression, while the GCC haplotype correlates with high levels (14,70). By using appropriate control groups, Mangia et al. found that inheritance of the −1117A, −854T, and −627A alleles, combined as an extended haplotype of the IL-10 gene, was significantly associated with spontaneous eradication of the HCV infection (38). The capacity for IL-10 production among individuals has a major genetic determinant, as polymorphisms in the IL-10 gene promoter have resulted in the demonstration of differing rates of IL-10 mRNA synthesis in vitro (14). The observed associations between ATA haplotype and favorable course of HCV infection may reflect diminished IL-10 expression, which may favor viral clearance. The findings of Mangia et al. (38) raise the possibility that the genetically controlled variability of IL-10 between individuals may account for some of the differences in the clinical outcome of HCV-infected patients: subjects who are genetically low producers of IL-10 might be more prone to HCV RNA clearance. Also, several studies have examined the relevance of functional IL-10 gene polymorphisms in hepatitis C. Carriers of an IL-10 high-producer genotype (−1082G/G) have been shown to clear the virus (35,45). Interestingly, the associated IL-10RB gene encodes a receptor chain for IL-10-related cytokines, including IFN-λ, which has recently been associated with HCV clearance (17,40,49,67). Knowledge of these polymorphisms may have implications for clinical practice and may be helpful in treatment management.

IL-18

IL-18, a proinflammatory cytokine, is an important regulator of the innate and acquired immune responses. IL-18 is involved in both Th-1 and Th-2 immune responses, depending on the context of the immunological milieu. In the presence of IL-12, IL-18 stimulates IFN-γ expression, promoting the Th-1-mediated immune response, whereas without IL-12, IL-18 stimulates Th-2 responses. IL-18 is significantly upregulated in HCV chronically-infected persons compared to healthy persons or asymptomatic carriers. High levels of IL-18 are correlated with hepatic injury (52), indicating a key role of IL-18 in HCV pathogenesis. By comparing patients who either had persistent HCV infection or had cleared HCV infection, An et al. (4) demonstrated that two functional promoter variants (−607 C/A and −137 G/C) in the IL-18 gene were associated with HCV clearance in African-American patients. A haplotype (AC) carrying both −607A and −137C was strongly associated with HCV clearance. These findings point to a critical role of IL-18 in determining the outcome of HCV infection, and suggest a common role of these IL-18 SNPs in viral hepatitis. These two alleles have been associated with lower IL-18 expression levels, suggesting that higher levels of IL-18 promote persistence of HCV infection, and lower levels of IL-18 production may help to protect hepatocytes from the persistent inflammation induced by HCV. Zhou et al. (79) demonstrated that the promoter sequence with haplotype AG exhibits lower promoter activity than that with CG, and that −607A was associated with lower serum IL-18 levels than −607C (79). Monocytes from −137C carriers produce a lower amount of IL-18 than −137G carriers (5). Giedraitis et al. (19) found that haplotypes CG and AG had higher transcription activity than haplotype AC. Based on these promoter activity studies, the more common alleles, −607A and −137C or haplotype AC, are associated with HCV clearance (19). If these results are validated, IL-18 could be considered as a target for therapeutics.

IL-28B

Type III interferons include three members: IFN-λ1, IFN-λ2, and IFN-λ3, and the genes encoding these molecules are clustered on human chromosome 19q13 (32,55). IL-28B (IFN-λ3) produces an antiviral state by triggering a cascade through the JAK-STAT pathway that upregulates the IFN-stimulated genes (ISGs). The effects of IL-28B are similar to those of IFN-α and IFN-β; however, IL-28B binds to a distinct receptor that may upregulate a different set of ISGs (39). Thus, even though both IFN-α and IL-28B signal through the JAK-STAT pathway, IL-28B SNP apparently has a distinct effect on the immune response to HCV. In addition to inducing ISG expression, IL-28B may activate alternate antiviral pathways, such as the adaptive immune response, which may be more important in HCV. This is supported by a study in which IL-28B, when used as a vaccine adjuvant, significantly decreased splenic regulatory T cells, increased splenic and peripheral blood CD8⁺ T cells, and led to increased antigen-specific perforin induction and degranulation (43). It is also plausible that the IL-28B and IFN-α pathways are synergistic, and that synergism more strongly enhances the immune response to HCV.

One of the most recent breakthroughs in hepatitis C treatment has been the finding that polymorphisms located near the IL-28B gene (encoding for IFN-λ3) can influence disease outcomes. In particular, their predictive value in spontaneous and treatment-induced viral clearance has been recognized. Since the first four studies were published in 2009 and early 2010 (17,49,57,59), which reported evidence for this association, additional works have been conducted clarifying the role of IL-28B polymorphisms in several aspects of hepatitis C. Different studies have shown that a SNP on chromosome 19q13, rs12979860, is associated with spontaneous and treatment-induced clearance of HCV, and is one of the strongest known genetic associations with any chronic viral infection to date. The evaluation of IL-28B polymorphisms in a uniquely homogenous cohort performed by Tillmann et al. has for the first time demonstrated that genetic variation in IL-28B is inter-related with the clinical presentation of acute hepatitis C infection. The results reported by the Tillmann group confirmed that the IL-28B rs12979860 polymorphism is highly associated with spontaneous viral clearance (69). In the same Genome Wide Associated Study (GWAS) related to treatment response, Ge et al. found that the rs12979860 C allele was significantly more common in a random multiethnic population than in an HCV-infected control cohort (17). This raised the question whether the C allele affords some protection against the development of chronic HCV infection. Thomas et al. (67) subsequently evaluated the SNP rs12979860 to assess the association with spontaneous clearance. They investigated an ethnically diverse population from six different clinical and study cohorts, with either spontaneous clearance of acute HCV infection or the development of persistent infection. Across all cohorts, those with the C/C genotype were three times more likely to clear HCV acutely than those with non-C/C (i.e., heterozygotes [C/T]), and homozygotes for the minor allele T/T. Thomas et al. (67) found that the polymorphism at the IL-28B gene demonstrates a similar effect on spontaneous clearance across ethnic groups. In accord with this observation, Rauch et al. (49) conducted a GWAS on a Caucasian cohort of patients who had spontaneously cleared HCV and compared them to patients with chronic hepatitis C. They extended results of previous studies by finding that only the IL-28B gene locus was associated with clearance, with a minor advantage conferred to heterozygotes (49).

Rao et al. (48) studied the role of IL-28B polymorphism and gender as possible predictive factors for spontaneous clearance of HCV in a Chinese population. The association of protective IL-28B genotypes with HCV clearance was confirmed, and gender was found to be an independent predictive factor for the resolution of HCV (women spontaneously cleared the virus more often than men). Compared to previous studies primarily indicating a strong association between rs8099917 and rs12979860 and HCV clearance, the genotyping of eight SNPs investigated by Rao et al. showed that in the Chinese population four different SNPs were involved, and rs12979860 was not among them (48). Spontaneous and treatment-induced HCV clearance has also been analyzed in infected children (23,51). One of the aims of studies in this regard was to evaluate the correlation between IL-28B polymorphisms and HCV clearance in children, as it was already established in infected adults. Ruiz Estremera et al. (51) studied the role of IL-28B polymorphisms in mother-to-child transmission, and in spontaneous clearance of HCV in infected children as well. Concerning the vertical transmission, the SNP rs12979860 was found not to be associated with a high risk of vertical transmission, but the CC genotype was revealed to be an important factor for spontaneous clearance of HCV in children. Payer et al. (46) studied the prevalence of different IL-28B genotypes and IP-10 levels in patients with acute hepatitis C (AHC), whether or not they achieved spontaneous clearance of the infection. The study confirmed that the protective genotypes (rs8099917 TT and rs12979860 CC) were more common in patients who experienced spontaneous clearance compared to those who did not clear HCV. IP-10 levels were lower in patients who spontaneously resolved the infection; thus when looked at cumulatively, IP-10 levels and IL-28B polymorphisms may help identify AHC patients who need early IFN treatment.

The mechanism by which SNPs affect IL-28B function has not been elucidated. Whether IFN-λ may serve as an alternative treatment modality for HCV infection is under investigation. Clinical studies assessing the safety and efficacy of HCV treatment with exogenous IFN-λ3 are currently underway. Early results suggest that IFN-λ3 treatment inhibits HCV replication and is associated with a limited adverse-effect profile. However, hepatotoxicity in both healthy volunteers and HCV-infected patients has been described. It is now a priority to determine the mechanisms via which IL-28B promotes viral defense, and the full range of viruses affected by these mechanisms. IL-28 analyses are likely to be included in future treatment studies. Furthermore, there may be implications for the clinical utility of these tests to identify patients with a low likelihood of response to standard therapy who may be candidates for early treatment with specifically-targeted anti-HCV drugs.

Conclusions

HCV has evolved an extensive array of mechanisms to evade innate and adaptive immune defenses. These strategies include viral factors that directly target mediators of intracellular antiviral defenses, as well as disruptions of multiple key nodes of cellular and humoral immunity. Although understanding the molecular details of elimination by the immune system has expanded, the relative contribution of these potential mechanisms to the clinical persistence of infection remains to be defined. The lack of appropriate model systems remains a significant obstacle. Spontaneous HCV clearance is difficult to assess without documented history of acute infection or liver biopsy, and long, accurate follow-up is needed to ensure that viral elimination is real. These latter inconsistencies may be in part due to small sample sizes, ethnic differences, differing experimental conditions, and the involvement of healthy individuals as controls instead of true controls (i.e., HCV-exposed individuals who have cleared the virus). Since genetic polymorphisms are ethnic-related, additional studies with patients from various ethnic cohorts, as well as patients with various degrees of hepatic disease, are needed to confirm associations and to determine the role of cytokine genotypes/phenotypes in the progression of HCV. Comprehensive studies are likewise needed to provide a better understanding of the host immune response to HCV.

However, identification of spontaneous clearance mechanisms of HCV is leading to an increasing array of potential therapeutic strategies, which may shift the balance back towards host control of HCV. This knowledge can then be utilized in developing new treatment options, including manipulation of the cytokine network, an approach that is currently being applied to several autoimmune diseases with some success. Clearly, before these tests are utilized in a clinical setting, further studies are mandatory.

A complex interplay between various genes is likely to modulate the anti-HCV response rather than a single allele. In addition to providing new insights into cell biology and immunology of the host, we hope that our research ultimately translates into improved outcomes for patients who suffer from this significant cause of liver disease. Further advances in our understanding of the role of host factors in the pathogenesis of this disease, and identifying prognostic indices, are key elements in finding new therapies that may help us develop evidence-based patient management strategies.

Footnotes

Acknowledgment

We would like to express our gratitude to Dr. Sadaf Ghajarieh Sepanlou from the Digestive Disease Research Institute of Iran for her constructive comments.

Author Disclosure Statement

No competing financial interests exist.

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