Interleukin-28B polymorphisms and interferon gamma inducible protein-10 serum levels in seronegative occult hepatitis C virus infection.

Polymorphisms upstream interleukin (IL)-28B gene and serum levels of interferon gamma inducible protein-10 (IP-10) are associated with spontaneous and treatment-induced hepatitis C virus (HCV) clearance. Patients with seronegative occult HCV infection are anti-HCV and serum HCV-RNA negative but have viral RNA in liver and abnormal values of liver enzymes. We examined if the rs12979860 polymorphism of IL-28B and serum IP-10 levels differ between chronic and seronegative occult CV infection. IL-28B polymorphism was determined with allele specific TaqMan probes in total DNA isolated from peripheral blood mononuclear cells and IP-10 by an enzyme-linked immunosorbent assay in serum from 99 patients with seronegative occult HCV infection and 130 untreated patients with chronic hepatitis C. IL-28B genotypes were also determined in 54 healthy volunteers. Prevalence of the IL-28B CC genotype was significantly higher in seronegative occult HCV infection (52/99; 52.5%) than in chronic hepatitis C (32/130; 24.6%, P < 0.0001) or healthy controls (19/54: 32.5%, P = 0.039). Among patients with seronegative occult HCV infection, HCV-RNA load in liver was significantly lower in those with the IL-28B CC genotype than in those with CT + TT genotypes (2.8 × 10(5) ± 5.8 × 10(4) vs. 4.1 × 10(5) ± 5.9 × 10(4) copies/µg of total RNA respectively; P = 0.023). Mean serum IP-10 levels were significantly lower in patients with seronegative occult HCV infection than in patients with chronic hepatitis C (160.8 ± 17.9 vs. 288.7 ± 13.3 pg/ml respectively; P < 0.0001). These findings suggest that the host immune response plays an important role in seronegative occult HCV infection in comparison with chronic hepatitis C.

Detection of hepatitis C virus (HCV) core-specific antibody suggests occult HCV infection among blood donors.

BACKGROUND: Blood transfusion safety is based on reliable donor screening for transmissible infections such as the hepatitis C virus (HCV) infection. STUDY DESIGN AND METHODS: A novel HCV core-specific antibody was assayed on random single donations from 2007 first-time blood donors who tested negative for anti-HCV and HCV RNA on routine screening. Sample collection broke the code between donations and donors for ethical reasons. RESULTS: Forty-two donations (2.1%) displayed reactivity in the novel test. The specificity of the reactivity was evaluated by a peptide inhibition assay, and testing against additional nonoverlapping HCV core peptide epitopes and other HCV antigens was performed on these samples. Six donations (14.3%; 0.30% from the total) were considered to contain anti-HCV after such supplemental testing. HCV RNA detection was also performed in peripheral blood mononuclear cells (PBMNCs) and serum or plasma samples from reactive donors after virus concentration by ultracentrifugation. HCV RNA tested negative in all PBMNCs samples, and a very low amount of viral genome was detected in serum or plasma concentrates from three anti-HCV core-reactive donors (7.1%) but not among concentrates from 100 randomly selected nonreactive donors. Sequencing of these polymerase chain reaction products revealed differences between the isolates that excluded partially sample contamination from a common source. CONCLUSION: These findings argue in favor of an ongoing occult HCV infection among these blood donors and account for some rather low, but perhaps not negligible, infection risk for such donations. Future studies involving larger samples of donations from traceable donors would enlighten the significance of these findings for the viral safety of the blood supply.

Efficacy of immunotherapy with TG4040, peg-interferon, and ribavirin in a Phase 2 study of patients with chronic HCV infection.

BACKGROUND & AIMS: TG4040 is a modified vaccinia Ankara (MVA) virus that expresses the hepatitis C virus (HCV) proteins NS3, NS4, and NS5B. We performed a phase II open-label study to determine the efficacy, safety, and immunotherapeutic properties of TG4040 in combination with pegylated interferon α-2a and ribavirin (PEG-IFNα/RBV) in patients with chronic HCV infection. METHODS: Treatment-naive patients with HCV genotype 1 infection were assigned randomly to 1 of the following groups: PEG-IFNα/RBV for 48 weeks (group A, n = 31), PEG-IFNα/RBV for 4 weeks followed by PEG-IFNα/RBV for 44 weeks with 6 injections of TG4040 (group B, n = 63), or TG4040 for 12 weeks (7 injections) followed by PEG-IFNα/RBV for 48 weeks with 6 injections of TG4040 (group C, n = 59). The primary end point was complete early virologic response (cEVR), defined as HCV-RNA level less than 10 IU/mL after 12 weeks of PEG-IFNα/RBV treatment. RESULTS: In group C, 64.2% of evaluable patients achieved cEVR, compared with 30.0% in group A and 45.9% in group B (P = .0003 for group C vs A). A higher percentage of patients achieved a sustained virologic response 24 weeks after therapy ended in group C (58.2%) than in groups A (48.4%) or B (50.8%). HCV- and MVA-specific T-cell responses were observed predominantly in group C. As expected, most patients given injections of TG4040 developed anti-MVA antibodies. The combination of TG4040 and PEG-IFNα/RBV was reasonably well tolerated. However, PEG-IFNα-associated thrombocytopenia developed in 3 patients who carried the class II HLA allele DRB01*04. CONCLUSIONS: A higher percentage of patients with chronic HCV infection who received immunotherapy with TG4040 followed by TG4040 and PEG-IFNα/RBV achieved a cEVR compared with patients who received only PEG-IFNα/RBV therapy. These findings show that immunotherapies that activate T cells are effective in patients with chronic HCV infection. ClinicalTrials.gov number, NCT01055821.

High prevalence of occult hepatitis C virus infection in patients with primary and secondary glomerular nephropathies.

The association of hepatitis C virus (HCV) infection and glomerulonephritis is well known. However, the relationship between immune-mediated glomerulonephritis and occult HCV, characterized by the presence of HCV-RNA in liver or in peripheral blood mononuclear cells in the absence of serological markers, is unknown. We tested this in 113 anti-HCV-negative patients; 87 with immune-mediated glomerulonephritis and 26 controls with hereditary glomerular nephropathies. All patients were serum HCV-RNA negative by conventional real-time PCR. Significantly, occult HCV-RNA (detectable viral RNA in peripheral blood mononuclear cells or in serum after ultracentrifugation) was found in 34 of 87 patients with immune-mediated glomerulonephritis versus 1 of 26 control patients. The serum creatinine levels were significantly higher in patients with immune-mediated glomerulonephritis with than in those without occult HCV (1.5 versus 1.1 mg/dl, respectively). A multivariate analysis adjusted for gender showed a significantly increased risk of occult HCV in patients with immune-mediated glomerulonephritis versus the controls (odds ratio of 13.29). Progression to end-stage renal disease tended to be faster in patients with immune-mediated glomerulonephritis and occult HCV than in the negative cases. Thus, occult HCV is strongly associated with immune-mediated glomerulonephritis and may have a role in the progression of the disease.

Impact of isolated hepatitis C virus (HCV) core-specific antibody detection and viral RNA amplification among HCV-seronegative dialysis patients at risk for infection.

Amplification of hepatitis C virus (HCV) RNA from blood detected occult HCV infections in 30.9% of 210 HCV-seronegative dialysis patients with abnormal liver enzyme levels that had evaded standard HCV testing practices. Isolated HCV core-specific antibody detection identified three additional anti-HCV screening-negative patients lacking HCV RNA amplification in blood who were considered potentially infectious. Together, these findings may affect management of the dialysis setting.

Cardioprotective Mechanisms against Reperfusion Injury in Acute Myocardial Infarction: Targeting Angiotensin II Receptors.

Ischemia/reperfusion injury is a process associated with cardiologic interventions, such as percutaneous coronary angioplasty after an acute myocardial infarction. Blood flow restoration causes a quick burst of reactive oxygen species (ROS), which generates multiple organelle damage, leading to the activation of cell death pathways. Therefore, the intervention contributes to a greater necrotic zone, thus increasing the risk of cardiovascular complications. A major cardiovascular ROS source in this setting is the activation of multiple NADPH oxidases, which could result via the occupancy of type 1 angiotensin II receptors (AT1R); hence, the renin angiotensin system (RAS) is associated with the generation of ROS during reperfusion. In addition, ROS can promote the expression of NF-κΒ, a proinflammatory transcription factor. Recent studies have described an intracellular RAS pathway that is associated with increased intramitochondrial ROS through the action of isoform NOX4 of NADPH oxidase, thereby contributing to mitochondrial dysfunction. On the other hand, the angiotensin II/ angiotensin type 2 receptor (Ang II/AT2R) axis exerts its effects by counter-modulating the action of AT1R, by activating endothelial nitric oxide synthase (eNOS) and stimulating cardioprotective pathways such as akt. The aim of this review is to discuss the possible use of AT1R blockers to hamper both the Ang II/AT1R axis and the associated ROS burst. Moreover; we suggest that AT1R antagonist drugs should act synergistically with other cardioprotective agents, such as ascorbic acid, N-acetylcysteine and deferoxamine, leading to an enhanced reduction in the reperfusion injury. This therapy is currently being tested in our laboratory and has shown promising outcomes in experimental studies.

Long-term virological follow up of patients with occult hepatitis C virus infection.

BACKGROUND: Patients with occult hepatitis C virus (HCV) infection (HCV-RNA in liver without detectable anti-HCV and serum HCV-RNA) may have viral RNA in peripheral blood mononuclear cells (PBMCs) and in serum after ultracentrifugation, and may present HCV-specific T-cell responses, but it is unknown whether these markers persist to be detectable over time. AIM: To perform a prospective virological long-term follow up of patients with occult HCV. METHODS: Viral markers were tested every 3-4 months during 55.7 ± 20.3 months in 37 patients with occult HCV who were under ursodeoxycholic acid treatment. RESULTS: Viral RNA was detectable in PBMCs of 31 patients during the follow up. In 23 of them, viral RNA in PBMCs was detected intermittently and in the other eight patients HCV-RNA was positive in a single sample. After ultracentrifugation, serum HCV-RNA was detected in 33 patients, being the viraemia intermittently detectable in 28, whereas in the remaining five patients, serum HCV-RNA was positive only once. Only one patient tested always HCV-RNA negative in PBMCs and in ultracentrifuged serum during follow up. Specific Core, NS3, and/or NS4 T-cell responses were found in 31 of the patients. The patient who was always HCV-RNA negative in PBMCs and in ultracentrifuged serum had specific HCV-T-cell responses. CONCLUSIONS: Occult HCV infection persists over time with fluctuating viraemia levels that induce and maintain specific T-cell responses against viral proteins.

Diagnosis of occult hepatitis C without the need for a liver biopsy.

The diagnosis of occult hepatitis C virus (HCV) infection is based on the presence of HCV-RNA in the liver. This study aimed to evaluate the use of combining non-invasive assays to diagnose occult HCV. A total of 122 patients with occult HCV (HCV-RNA in the liver without detectable anti-HCV and serum HCV-RNA) and 45 patients with cryptogenic chronic hepatitis (without HCV-RNA in the liver and negative for anti-HCV and serum HCV-RNA) were included. HCV-RNA was tested in peripheral blood mononuclear cells (PBMCs) and in 2 ml of ultracentrifuged serum. Anti-core HCV was examined by a non-commercial enzyme-linked immunosorbent assay. All controls were negative for the three HCV markers studied. Among patients with occult HCV, 36% were anti-core HCV positive, 57% had serum HCV-RNA after ultracentrifugation, and 61% had HCV-RNA in PBMCs. Combining the results of the assays, 91% of the patients were positive for at least one marker. Intrahepatic HCV-RNA load was significantly higher in patients who were positive simultaneously for the three HCV markers than in patients who were negative for all markers (P = 0.006) and than in those with one or two HCV markers (P = 0.039). Replication of HCV in liver was detected more frequently in patients with three (93%, P = 0.002), two (82%, P = 0.001), and one HCV marker (73%, P = 0.011) than in those without markers (27%). In conclusion, testing for all these markers allows diagnosis of occult HCV without the need for a liver biopsy and these assays may help to elucidate the clinical significance of occult HCV infection.

Effect of hemodialysis schedules and membranes on hepatocyte growth factor and hepatitis C virus RNA levels.

Hemodialysis induces production of the hepatocyte growth factor (HGF) and decrease of serum hepatitis C virus (HCV) RNA in patients with HCV infection, but it is not known if the hemodialysis schedule or type of membrane affect both the HGF production and HCV viremia. The effects on both parameters of alternate-day intermittent hemodialysis and short-daily hemodialysis and high and low flux membranes were investigated in 41 patients treated by hemodialysis. Sixteen (39%) patients were anti-HCV positive and 11 (69%) had HCV RNA. Twenty-six patients were on alternate-day intermittent and 15 on short-daily hemodialysis. High flux membranes were used for 29 patients and low flux membranes for 12 patients. A decrease in HCV RNA was observed at the end of hemodialysis (8.6 x 10(5) +/- 1.1 x 10(6) IU/ml vs. 4.4 x 10(5) +/- 7.3 x 10(5) IU/ml, P = 0.003). The proportion of HCV RNA decrease was similar in patients dialyzed with both schedules and with both types of membranes. The HGF levels increased from 2,605.9 +/- 1,428.7 to >8,000 pg/ml at 15 min. At the end of the session, the HGF levels decreased to 5,106.7 +/- 2,533.9 pg/ml. The HGF levels at the start of the next session were similar to those at baseline (2,680.0 +/- 1,209.3 pg/ml). The increase and dynamics of the HGF levels were similar in patient's hemodialyzed with both schedules and with both types of membranes. These results suggest that changes in HCV RNA and HGF levels during hemodialysis are not influenced by the schedule or type of membrane used.

Hepatitis C virus infection in the family setting of patients with occult hepatitis C.

Family members of patients with chronic hepatitis C virus (HCV) infection are at increased risk of HCV infection but the prevalence of HCV among family members of patients with occult HCV infection is not known. Anti-HCV, serum HCV RNA and levels of liver enzymes were determined in 102 family members of 50 index patients with occult HCV infection and in 118 family members of 59 chronic hepatitis C index patients. HCV RNA and/or anti-HCV were detected in 10/102 (9.8%) relatives of patients with occult HCV infection and in 4/118 (3.4%) of patients with chronic hepatitis C. Fourteen additional family members (seven were relatives of index patients with occult HCV infection) had abnormal values of liver enzymes without serological markers of HCV infection. Two of these patients (who were relatives of two index patients with occult HCV infection) underwent a liver biopsy and were diagnosed with an occult HCV infection because HCV RNA was detected in the liver cells in the absence of serological HCV markers. In conclusion, the prevalence of HCV infection among family members of patients with occult HCV infection was similar to that found among family members of patients with chronic hepatitis C. This stresses the need to adopt strategies to prevent the transmission of HCV in the family setting of patients with occult HCV infection.

Occult hepatitis C virus infection among hemodialysis patients.

Occult hepatitis C virus (HCV) infection (i.e., detectable HCV-RNA in the liver or peripheral blood mononuclear cells) in the absence of both serum HCV-RNA and anti-HCV antibodies has not been investigated in hemodialysis patients. In this study, real-time PCR and in situ hybridization was used to test for the presence of genomic and antigenomic HCV-RNA in peripheral blood mononuclear cells of 109 hemodialysis patients with abnormal levels of liver enzymes. Occult HCV infection, determined by the presence of genomic HCV-RNA, was found in 45% of the patients; 53% of these patients had ongoing HCV replication, indicated by the presence of antigenomic HCV-RNA. Patients with occult HCV infection had spent a significantly longer time on hemodialysis and had significantly higher mean alanine aminotransferase levels during the 6 mo before study entry. Logistic regression analysis revealed that mortality was associated with age >60 yr (odds ratio 3.30; 95% confidence interval 1.05 to 10.33) and the presence of occult HCV infection (odds ratio 3.84; 95% confidence interval 1.29 to 11.43). In conclusion, the prevalence of occult HCV infection is high among hemodialysis patients with persistently abnormal values of liver enzymes of unknown cause. The clinical significance of occult HCV infection in these patients requires further study.

Detection of hepatitis C virus in thyroid tissue from patients with chronic HCV infection.

Thyroid dysfunctions are common in chronic hepatitis C virus (HCV) infection. HCV-RNA has been detected by reverse-transcription polymerase chain reaction (PCR) in thyroid from HCV infected patients with acquired immunodeficiency syndrome. However, morphological evidence of HCV replication in thyroid cells from immune competent patients has not been provided. In situ hybridization and real-time-PCR were used to analyze HCV-RNA replication in thyroid tissue from 11 patients (3 anti-HCV, serum HCV-RNA positive; 8 anti-HCV negative). Genomic and antigenomic HCV-RNA was detected in the thyroid of the 3 anti-HCV positive patients at concentrations of 2.6 x 10(4), 1.7 x 10(4), and 8.6 x 10(3) copies/microg of total RNA (genomic) and 3.2 x 10(2), 4.3 x 10(3) and 2.9 x 10(2) HCV-RNA copies/microg of total RNA (antigenomic). No HCV-RNA was detected in the thyroid tissue of the 8 anti-HCV negative patients. Presence of genomic/antigenomic HCV-RNA in the 3 anti-HCV positive cases was confirmed by in situ hybridization. Signals were observed in the cytoplasm of the thyroid cells. In conclusion, the data obtained indicate that HCV may infect cells of the thyroid in immune competent patients with chronic HCV infection. The pathogenic implications of this finding merit further research.

Hepatitis C virus replicates in the liver of patients who have a sustained response to antiviral treatment.

BACKGROUND: Positive-strand hepatitis C virus (HCV) RNA has been detected in the livers of patients who have achieved a sustained biochemical and virological response to antiviral therapy (hereafter, referred to as sustained responders), but negative-strand HCV RNA was undetectable in the hepatic tissue of these patients. We studied the presence of both positive- and negative-strand HCV RNA in the livers of 20 sustained responders with chronic hepatitis C whose response persisted for a mean (+/- standard deviation [SD]) of 47.4+/-32.8 months after treatment. METHODS: HCV RNA was tested by strand-specific, real-time reverse-transcriptase polymerase chain reaction and by in situ hybridization in posttreatment liver biopsy samples (obtained a mean [+/- SD] 35.4+/-35.0 months after therapy) and in patients' peripheral blood mononuclear cells. RESULTS: Positive-strand HCV RNA was found in 19 (95%) of 20 liver biopsy specimens, and negative-strand HCV RNA was found in 15 (79%) of the 19 samples that had positive-strand HCV RNA. These results were confirmed by in situ hybridization. Regarding peripheral blood mononuclear cells, 13 (65%) of 20 samples had positive-strand HCV RNA, and negative-strand HCV RNA was detected in 12 (92%) of the 13 samples with positive-strand HCV RNA. Liver necroinflammation was still present in the posttreatment liver biopsy specimens of 15 patients, and fibrosis was present in 7, although liver damage improved in all but 2 patients. CONCLUSIONS: HCV persisted and replicated in the livers and peripheral blood mononuclear cells of most sustained responders. Thus, these patients did not experience HCV infection clearance, despite apparent clinical disease resolution.

Structural characterization of the 5' untranslated RNA of hepatitis C virus by vibrational spectroscopy.

Raman and FTIR spectroscopy have been used to characterize the structure of 5'untranslated region (5'UTR, 342-mer RNA) of the HCV genome. The study of the 750-850 cm(-1) Raman spectral domain of the ribose-phosphate backbone reveals that the percentage of nucleobases involved in double helix-loop junctions is 19+/-1%, which is very close to that of a theoretical secondary structure model (18.7%) proposed on the basis of comparative sequence analysis and thermodynamic modelling. In addition, about 68+/-2% of the bases are helically ordered having C(3')-endo ribofuranose pucker. FTIR-monitored H/D exchange provides the following results: (a) base-paired guanine and cytosine nucleobases show the lowest rate of isotopic exchange, and some synchronous intensity changes of marker bands of A.U pair and single stranded adenine are consistent with the presence of A(*)A.U triplets; (b) the vibrational coupling between the ribose ether C-O stretching and 2'OH bending motions reveals that helical regions of 5'UTR RNA are characterized by hydrogen bonding between the 2'OH ribose groups and the ether oxygen atoms of neighbouring ribose residues.

Occult hepatitis C virus infection: a new form of hepatitis C.

Occult hepatitis C virus (HCV) infection is a new recently characterized entity. This occult infection can be present in two different clinical situations: in anti-HCV negative, serum HCV-RNA negative patients with abnormal liver function tests and in anti-HCV positive subjects with normal values of liver enzymes and without serum HCV-RNA. This review describes recent studies of occult HCV infection in both kinds of patients.

Percentage of hepatitis C virus-infected hepatocytes is a better predictor of response than serum viremia levels.

Pegylated alpha-interferon plus ribavirin is the current therapy for chronic hepatitis C virus (HCV) infection. Serum HCV-RNA concentration before treatment has been identified as an independent predictive factor of response. We have compared the percentage of HCV-infected hepatocytes with the concentration of serum HCV-RNA in baseline samples as predictors of response. We included 97 patients with chronic HCV infection (genotype 1), treated with pegylated-interferon-alpha2b plus ribavirin. Of these 97, 38 (39%) were sustained responders and 59 (61%) were not. Statistical differences between responders and nonresponders were found regarding the percentage of infected hepatocytes (6.83+/-4.50% versus 13.44+/-10.05%; P=0.00003) but not in serum HCV-RNA concentration [1.71+/-2.70 (x10(6) IU/L) versus 1.32+/-1.86 (x10(6) IU/L); P=0.40694]. Other factors associated with response were age, gamma-glutamyl transpeptidase level, and absence of previous therapy. Logistic regression demonstrated that percentage of infected hepatocytes (odds ratio, 1.160; 95% confidence interval, 1.065-1.264) and previous therapy (odds ratio, 0.294; 95% confidence interval, 0.109-0.795) were significant predictive factors for response. Therefore, the percentage of infected hepatocytes in liver biopsy before treatment is a better predictive factor of sustained response to 48 weeks of therapy with pegylated alpha-interferon plus ribavirin than serum HCV-RNA concentration in baseline serum sample.

Detection of hepatitis C virus RNA and core protein in keratinocytes from patients with cutaneous lichen planus and chronic hepatitis C.

Cutaneous lichen planus has been associated in patients with chronic hepatitis C virus infection. It is still unknown whether hepatitis C virus infects keratinocytes of lichen planus lesions. In this report we have analyzed the presence of genomic and anti-genomic hepatitis C virus RNA in skin biopsies from 26 patients with chronic hepatitis C and healthy skin and from 24 patients with cutaneous lichen planus (five with and 19 without hepatitis C virus infection) by in situ hybridization. Hepatitis C virus RNA was detected in the keratinocytes of 69% of the patients with healthy skin and chronic hepatitis C, in 100% of the patients with lichen planus and hepatitis C virus infection, and in none of lichen planus patients without hepatitis C virus infection. The percentage of keratinocytes showing genomic or anti-genomic hepatitis C virus RNA was statistically lower (p < 0.01 in all cases) in patients with healthy skin (mean +/- SD: 5.7 +/- 3.5% and 2.7 +/- 3.1% of keratinocytes with genomic or anti-genomic hepatitis C virus RNA, respectively) than in those with lichen planus lesions (31.7 +/- 7.9% and 18.8 +/- 7.4%, mean +/- SD) or the unaffected adjacent skin (24.8 +/- 6.9% and 14.3 +/- 3.8%, mean +/- SD). In conclusion, we have demonstrated that hepatitis C virus infects keratinocytes from patients with lichen planus and hepatitis C virus infection.