Hepatitis C virus (HCV) is a serious health problem worldwide that causes chronic infection in up to 85% of cases (1). Egypt has the highest prevalence in the world (2). HCV is the sole member of the genus Hepacivirus and can be grouped into 11 genotypes with distinct geographical distributions (3,4). It is a single-stranded RNA virus that encodes a single polypeptide chain. The polyprotein precursor is proteolytically cleaved into 4 structural proteins (C, E1, E2, and p7) followed by 6 nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (5). The NS2/3 protease is a cysteine protease required for HCV polyprotein processing and is responsible for the cleavage between NS2 and NS3, which is required for viral replication in vivo (6,7).
NS2 upregulates viral translation (8) and inhibits host cell apoptosis, which provides a cellular environment that is advantageous for viral replication without the elimination of the host cell (1). Moreover, it is required for NS5A activation (9), which in turn inhibits interferon-signaling pathway and interferon-stimulated gene activation, thus counteracting host cell antiviral response, and promotes viral translation and replication (10). NS3 functions both as a protease, cleaving mature NS proteins from the polyprotein, and an RNA helicase, which is believed to be required for the RNA replication (11). This demonstrates that all of the consequences of the action of NS2/3 and cleavage between NS2 and NS3 are in favor of hepatitis C viral replication and infection establishment in the host. The HCV NS2/3 protease shares no obvious sequence homology to any known proteases in the animal kingdom and would therefore make an attractive target for antiviral therapy (1).
Cystatin C is a nonglycated low-molecular-weight protein of 13.3 kDa, which belongs to the superfamily of cystatins. Its basic function is to inhibit and regulate the activity of cysteine proteases (12). Cystatin C displays the strongest inhibitory activity of all of the cystatins toward cysteine proteases and has a widespread distribution in human tissues and body fluids (13). Cystatin C has also been shown to exhibit antiviral functions, as suggested from experiments done with virus-infected cell lines (14). Cystatin C has been extensively studied in renal diseases as a marker of kidney function (15,16) and cardiovascular diseases (17,18), yet the results are controversial (19,20). In addition, elevated serum cystatin C in patients with chronic liver disease has been reported (21), but reports about its correlation with disease severity are contradictory (22,23). The relation of cystatin C levels, as a cysteine protease inhibitor, to the viral load in chronic HCV infection has never been addressed before.
The present study was undertaken to study the relation of cystatin C serum levels and HCV viremia in treatment-naïve children with chronic hepatitis C. It demonstrates that cystatin C may be one of the important therapeutic opportunities against HCV infection.
Inclusion criteria were children with treatment-naïve chronic HCV infection, ages 3 to 18 years and body mass index <95th percentile. Of 64 children with chronic HCV infection attending the outpatient and the inpatient of the Department of Pediatric Hepatology, National Liver Institute, Menofiya University, Egypt, between February 2010 and February 2011, only 27 were enrolled in the study after exclusion of those with ongoing or a history of antiviral therapy, those with cirrhosis or renal or cardiac impairment, or those who have any evidence of infection. Those with combined liver disease (eg, HCV combined with hepatitis B virus, autoimmune hepatitis, diabetes, thalassemia, or other metabolic disorder) or those receiving corticosteroids were also excluded from the study. Another group of 25 apparently healthy children were included in the study and served as controls. Signed informed consent was obtained from the parents of all of the patients and controls before enrollment in the study. The study was approved by the research ethics committee of the National Liver Institute, Menofiya University.
Three blood samples were collected from the patients. The first sample was allowed to clot naturally in the test tube; serum was separated and divided into small aliquots and stored immediately at −80°C until time of use for liver function tests, viral markers, kidney functions (urea and creatinine), C-reactive protein (CRP), and cystatin C assays. A second sample was added to ethylenediaminetetraacetic acid for complete blood count (CBC) and tested immediately. A third sample was added to sodium citrate for prothrombin time and tested just before performing liver biopsy. A serum sample was collected from controls for liver transaminases (alanine transaminase [ALT], aspartate transaminase [AST]), kidney functions, CRP, anti-HCV antibody (Ab), and cystatin C assay.
Etiological Diagnosis and Group Allocation
Diagnosis of chronic hepatitis C was based on the presence of serum anti-HCV Ab and persistently positive HCV-RNA for >6 months (24,25), negative hepatitis B viral markers, and absence of any associated liver disease, supported by the histopathological feature of HCV infection in liver biopsy. To eliminate the possibility of any renal complication, kidney functions and abdominal ultrasound screening for patients were included. Control groups were defined by apparently healthy individuals with no signs or symptoms of liver disease or any other diseases, normal liver transaminases, normal kidney functions, negative anti-HCV Ab, and negative CRP.
Certain relevant clinical values, including CBC, liver function tests, kidney function tests, CRP, and prothrombin time, were performed for every analyzed sample. Viral markers were performed using enzyme-linked immunosorbent assay according to the manufacturer's instructions: HCV Ab (Innogenetics, Ghent, Belgium), hepatitis B virus surface antigen, hepatitis B virus core immunoglobulin M, and immunoglobulin G Abs (Dia Sorin, Saluggia, Italy). Real-time polymerase chain reaction for HCV-RNA was performed for patients only using COBAS Ampliprep/COBAS TaqMan (Roche Molecular Systems, Branchburg, NJ; detection limit is 15 IU/mL). According to the viral load, viremia was classified arbitrarily into low (15–2 × 105 IU/mL), moderate (>2 × 105–2 × 106 IU/mL), and high viremia (>2 × 106 IU/mL). Serum cystatin C assays were performed for all of the patients and controls using an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN). Genotyping/subtyping was done by restriction fragment length polymorphism using restriction enzymes HaeIII, RsaI, MvaI, and HinfI on polymerase chain reaction–amplified 5′-untranslated region (5′-UTR).
Liver Biopsy and Histological Evaluation
Liver biopsy was performed using a true-cut needle. Specimens were fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin, Masson's trichrome, reticulin, and Perl stains. Hepatic necroinflammatory activity and liver fibrosis were evaluated according to Ishak staging and grading score (26). Necroinflammatory activity was classified into mild (score 1–5), moderate (score 6–8), and severe (score 9–18). Fibrosis was classified into mild (stage 1), moderate (stages 2–3), and severe fibrosis or cirrhosis (stages 4–6) (27).
Values were expressed as mean ± standard deviation or number (percentage) of individuals with a condition. For quantitative data, statistical significance was tested by either the Kruskal-Wallis nonparametric test or the Mann-Whitney U test. For qualitative data, significance was tested by the χ2 test. Correlation was tested by the Spearman test. Results were considered significant if P value is ≤0.05. Statistical analysis was done using the SPSS statistical package version 13 (SPSS Inc, Chicago, IL) on an IBM-compatible computer.
Study Population Characteristics
The study included 27 children with chronic HCV infection: 13 (48.1%) girls and 14 (51.9%) boys. Their mean age was 11.66 ± 3.99 ranging from 3 to 17 years. Genotype 4a was found in 23 patients, whereas HCV genotype could not be determined in 4 patients. Mode of HCV infection was surgery in 10 (37%), blood transfusion in 6 (22.2%), male circumcision in 5 (18.5%), vertical transmission in 3 (11.1%), dental procedures in 1 (3.7%), and unknown in 2 (7.4%) children. A second group of 25 apparently healthy children were included as controls: 8 (32%) girls and 17 (68%) boys. Their mean age was 10.16 ± 3.61 ranging from 4 to 16 years. Both groups were age and sex matched (P = 0.149 and 0.27, respectively). Both groups had normal serum urea (26.02 ± 6.71 vs 23.14 ± 5.24 mg/dL; P = 0.338) and creatinine (0.57 ± 0.19 vs 0.58 ± 0.19 mg/dL; P = 0.903) with no significant difference between both groups. ALT (40.37 ± 23.42 vs 20.8 ± 6.01 U/L) and AST (43.74 ± 26.75 vs 27.24 ± 6.31 U/L) values of patients with chronic hepatitis C were significantly higher than that of the controls (P = 0.0002 and 0.002, respectively).
Cystatin C Levels Differed Significantly in Patients and Controls and Correlated Negatively With HCV Viremia
Serum cystatin C levels were significantly higher in patients than in the controls (1.4 ± 0.47 vs 0.99 ± 0.49 mg/L; P = 0.006). Moreover, cystatin C was significantly higher in those with low viremia (1.55 ± 0.41) than in those with moderate viremia (0.99 ± 0.43) and controls (0.99 ± 0.49; P = 0.013 and P = 0.0004, respectively); however, there was no statistical significant difference between those with moderate viremia and controls (P = 0.909) (Fig. 1). To investigate the parameters related to different cystatin C levels in patients with low and those with moderate viremia, the demographic, laboratory, and histopathological parameters were compared between both groups. With the exception of albumin (P = 0.016), all of the parameters were comparable with no statistically significant difference (Table 1). The correlation of cystatin C with age, laboratory, and histopathological parameters was tested (Table 2). No significant correlation was found between age and cystatin C in both HCV and control groups (P = 0.714 and 0.517, respectively). In the HCV group, there was no significant correlation with any of the studied parameters except for a significant positive correlation with serum albumin (P = 0.008) and a greatly significant negative correlation with HCV viremia (P < 0.0001) (Fig. 2).
Cystatin C Levels Did Not Differ Significantly With Histopathological Changes or With Severity of Liver Disease
All of the patients underwent liver biopsy. Two of them had mild steatosis. Activity ranged from A2 to A10 and fibrosis ranged from F1 to F4. None of the patients had cirrhosis. We compared cystatin C levels in patients with mild, moderate, and severe fibrosis (1.49 ± 0.46, 1.33 ± 0.52, and 1.59 ± 0.02 mg/L, respectively) and activity (1.43 ± 0.38, 1.31 ± 0.58, and 1.77 ± 0.03 mg/L, respectively). In addition, we compared the levels in patients with and without hepatic steatosis (1.74 ± 0.78 vs 1.37 ± 0.46 mg/L, respectively). There was no significant difference in serum cystatin C levels between patients with different stages of hepatic fibrosis (P = 0.717), different grades of activity (P = 0.365), or presence or absence of steatosis (P = 0.406). To look at the relation between cystatin C and the severity of liver disease, patients were regrouped according to the level of transaminases into those with normal versus elevated ALT or AST. There was no significant difference in cystatin C levels between those with normal and those with elevated ALT (1.41 ± 0.47 vs 1.39 ± 0.52 U/L; P = 0.657). Similarly, there was no significant difference between those with normal and those with elevated AST (1.33 ± 0.56 vs 1.49 ± 0.36 U/L; P = 0.306).
The aim of the present study was to evaluate the serum cystatin C level in children with chronic HCV infection and its relation to the level of HCV viremia. To the best of our knowledge, this is the first study to be performed in this concern.
Our results showed that serum cystatin C was significantly higher in patients with HCV than in controls. This finding may be supported by that reported by Chu et al (22), in which average cystatin C was significantly higher in patients with HCV than that in controls. Similarly, Takeuchi et al (21) reported the greatly significant elevation (P < 0.0001) of serum cystatin C in patients with chronic hepatitis compared with healthy controls.
The present study showed that there was no significant difference in cystatin C level according to different stages of fibrosis, grades of activity, or the presence or absence of steatosis (P > 0.05). In addition, cystatin C was not correlated with any CBC parameters or liver functions, except for serum albumin, which directly correlated with cystatin C (P = 0.023). Apart from albumin and contrary to our results, Takeuchi et al (21) reported that significant correlations were observed among cystatin C levels and total bilirubin levels, albumin levels, and platelet counts. Serum cystatin C concentrations correlated well with histological stages despite the lack of correlation with histological grades. Moreover, Chu et al (22) revealed a direct relation between cystatin C and the severity of liver diseases. Such a relation was not found in our study. This may result from the difference in population characteristics in these studies because they were adults with chronic hepatitis, cirrhosis, and hepatocellular carcinoma, whereas ours was performed in children and none had cirrhosis.
Interestingly, our results showed a greatly significant negative correlation between serum cystatin C and HCV viremia (P < 0.0001). This would be of utmost importance, implicating a possible role of cystatin C as an inhibitor of hepatitis C viral replication. Cystatin C has been reported to have an antiviral effect on poliovirus–, herpes simplex virus–, and coronavirus–infected cell cultures (28,29), and salivary cystatins were able to inhibit herpes simplex virus replication (30). One of the proposed mechanisms of cystatin C as an antiviral is the anticysteine protease function against HCV NS2/3 protease. Another possible mechanism is the immunomodulatory role of cystatin C leading to the augmentation of the TH1 response (14), which is protective particularly against intracellular pathogens (31).
Cystatin C is mainly extracellular, whereas HCV is found both extracellular in the serum and intracellular, such as hepatocytes (32) and peripheral mononuclear cells (33). This raises the question of whether cystatin C can antagonize intracellular HCV. The answer to this question is the study performed by Ekstrom et al (34), in which they demonstrated that human cell lines internalized fluorophore-conjugated cystatin C when exposed to physiological concentrations. Intracellular cystatin C increased, reaching 4 to 6 times the baseline level. Moreover, the observation that recombinant cystatin C can be internalized in the cells of several tissues following intraperitoneal injection could lead to a mechanism allowing the protein to increase the total intracellular cysteine protease inhibitory activity significantly (35).
When we compared cystatin C levels in patients with low viremia and in those with moderate viremia with the controls, the levels were significantly higher in those with low viremia than in those with moderate viremia (P = 0.013) and controls (P = 0.0004), whereas there was no significant difference between the levels in those with moderate viremia and controls (P = 0.909); that is to say that the patients with low viremia could upregulate cystatin C levels from those present in healthy controls, which in turn shared in the antiviral action against HCV as reflected by the low viral load. Those who could not upregulate their serum levels of cystatin experienced a higher viral load (moderate viremia). El-Araby et al (36) demonstrated that the only determinant factor for response to antiviral therapy in children with chronic hepatitis C was the pretreatment level of viremia in which lower viral loads were significantly associated with rapid and sustained virological responses. We postulate that higher serum cystatin C would increase the chances for lower viral loads, which in turn increase the chances for either the spontaneous clearance of the virus or successful antiviral therapy.
In conclusion, our study demonstrated that higher serum cystatin C levels in children with chronic HCV were associated with lower HCV viremia and those with higher viral load failed to upregulate the basal serum levels that were found in healthy controls. The limitation in our study is the small number of patients, but the greatly significant correlation may reflect a possible inhibitory effect of cystatin C on HCV replication through inhibiting its NS2/3 (cysteine protease) enzyme and is tempting for further in vitro and in vivo studies for cystatin C as a promising adjuvant therapy for HCV infection.
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Keywords:Copyright 2012 by ESPGHAN and NASPGHAN
cystatin C; cysteine protease; genotype 4; hepatitis C virus; viral load