The implementation of a hepatitis C testing service in community pharmacies: I-COPTIC consensus statement.

OBJECTIVES: This aimed to develop a blueprint for an effective community pharmacy Hepatitis C virus (HCV) testing service by producing a consensus statement. STUDY DESIGN: This was a modified Delphi process. METHODS: We recruited a heterogenous panel of experts (who had been involved in the setup or delivery of a community pharmacy HCV testing service) by purposive and chain referral methods. We had three rounds of a modified Delphi process. The first was a series of questions with free text responses and was analysed using thematic analysis, and the second and third were statements for the respondents to rate using a 7-point Likert scale. Consensus was predefined in a published protocol, and the results were reviewed by a public and patient involvement panel before the statement was finalised. RESULTS: We had 24 participants, including community and hospital-based pharmacists, local pharmaceutical committee members, charity representatives (Hepatitis C Trust), local clinical service lead, nurse specialists and doctors. The response rate of the first, second and third rounds were 100%, 96% and 88%, respectively. After the third round, we had 60 statements that reached consensus. We discussed the accepted statements with a patient and public involvement group. We used these statements to produce the I-COPTIC statement and a graphical summary. CONCLUSIONS: We developed a blueprint for the design of a gold standard community pharmacy HCV testing service. We believe this will support the successful implementation of community pharmacy testing for HCV. Community pharmacy testing is an important service to help achieve and maintain HCV elimination.

Vasoactive intestinal peptide induces proliferation of human hepatocytes.

OBJECTIVES: Proliferation of hepatocytes in vitro can be stimulated by growth factors such as epidermal growth factor (EGF), but the role of vasoactive intestinal peptide (VIP) remains unclear. We have investigated the effect of VIP on maintenance and proliferation of human hepatocytes. MATERIALS AND METHODS: Human hepatocytes were isolated from liver specimens obtained from patients undergoing liver surgery. Treatment with VIP or EGF was started 24 h after plating and continued for 3 or 5 d. DNA replication was investigated by Bromodeoxyuridine (BrdU) incorporation and cell viability detected by MTT assay. Cell lysate was analysed by western blotting and RT-PCR. Urea and albumin secretion into the culture supernatants were measured. RESULTS: VIP increased DNA replication in hepatocytes in a dose-dependant manner, with a peak response at day 3 of treatment. VIP treatment was associated with an increase in mRNA expression of antigen identified by monoclonal antibody Ki-67 (MKI-67) and Histone Cluster 3 (H3) genes. Western blotting analysis showed that VIP can induce a PKA/B-Raf dependant phosphorylation of extracellular signal-regulated kinases (ERK). Although EGF can maintain hepatocyte functions up to day 5, no marked efffect was found with VIP. CONCLUSIONS: VIP induces proliferation of human hepatocytes with little or no effect on hepatocyte differentiation. Further investigation of the role of VIP is required to determine if it may ultimately support therapeutic approaches of liver disease.

Hepatocellular carcinoma: Prospects for natural killer cell immunotherapy.

Liver disease is a growing cause of death in the United Kingdom and the incidence of hepatocellular carcinoma (HCC) is rising (http://www.cancerresearchuk.org/). The combination of an immunosuppressive environment within the liver and suboptimal host anti-tumour immune responses may account for the poor survival outcome of HCC. Understanding how tumours evade immune recognition coupled with new insights into the unique immunological environment within the liver will be critical to developing liver-specific immunotherapies.

STAT4-associated natural killer cell tolerance following liver transplantation.

OBJECTIVE: Natural killer (NK) cells are important mediators of liver inflammation in chronic liver disease. The aim of this study was to investigate why liver transplants (LTs) are not rejected by NK cells in the absence of human leukocyte antigen (HLA) matching, and to identify a tolerogenic NK cell phenotype. DESIGN: Phenotypic and functional analyses on NK cells from 54 LT recipients were performed, and comparisons made with healthy controls. Further investigation was performed using gene expression analysis and donor:recipient HLA typing. RESULTS: NK cells from non-HCV LT recipients were hypofunctional, with reduced expression of NKp46 (p<0.05) and NKp30 (p<0.001), reduced cytotoxicity (p<0.001) and interferon (IFN)-γ secretion (p<0.025). There was no segregation of this effect with HLA-C, and these functional changes were not observed in individuals with HCV. Microarray and RT-qPCR analysis demonstrated downregulation of STAT4 in NK cells from LT recipients (p<0.0001). Changes in the expression levels of the transcription factors Helios (p=0.06) and Hobit (p=0.07), which control NKp46 and IFNγ expression, respectively, were also detected. Hypofunctionality of NK cells was associated with impaired STAT4 phosphorylation and downregulation of the STAT4 target microRNA-155. Conversely in HCV-LT NK cell tolerance was reversed, consistent with the more aggressive outcome of LT for HCV. CONCLUSIONS: LT is associated with transcriptional and functional changes in NK cells, resulting in reduced activation. NK cell tolerance occurs upstream of major histocompatibility complex (MHC) class I mediated education, and is associated with deficient STAT4 phosphorylation. STAT4 therefore represents a potential therapeutic target to induce NK cell tolerance in liver disease.

A national survey of the provision of ultrasound surveillance for the detection of hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC), the sixth most common cancer worldwide and third most common cause of cancer related death, is closely associated with the presence of cirrhosis. Survival is determined by the stage of the cancer, with asymptomatic small tumours being more amenable to treatment. Early diagnosis is dependent on regular surveillance and the primary objective of this survey was to gain a better understanding of the baseline attitudes towards and provision of ultrasound surveillance (USS) HCC surveillance in the UK. In addition, information was obtained on the stages of cancer of the patients being referred to and discussed at regional multidisciplinary team meetings. DESIGN: UK hepatologists, gastroenterologists and nurse specialists were sent a questionnaire survey regarding the provision of USS for detection of HCC in their respective hospitals. RESULTS: Provision of surveillance was poor overall, with many hospitals lacking the necessary mechanisms to make abnormal results, if detected, known to referring clinicians. There was also a lack of standard data collection and in many hospitals basic information on the number of patients with cirrhosis and how many were developing HCC was not known. For the majority of new HCC cases was currently being made only at an incurable late stage (60%). CONCLUSIONS: In the UK, the current provision of USS based HCC surveillance is poor and needs to be upgraded urgently.

The interaction of genetic determinants in the outcome of HCV infection: evidence for discrete immunological pathways.

Diversity within the innate and adaptive immune response to hepatitis C is important in determining spontaneous resolution (SR) and treatment response. The aim of this study was to analyze how these variables interact in combination; furthering our understanding of the mechanisms that drive successful immunological clearance. Multivariate analysis was performed on retrospectively collected data for 357 patients previously genotyped for interferon (IFN)-λ3/4, killer cell immunoglobulin (KIR), human leukocyte antigen (HLA) class I and II and tapasin. High resolution KIR genotyping was performed for individuals with chronic infection and haplotypes determined. Outcomes for SR, IFN response and cirrhosis were examined. Statistical analysis included univariate methods, χ(2) test for trend, multivariate logistic regression, synergy and principal component analysis (PCA). Although KIR2DL3:HLA-C1C1 (P = 0.027), IFN-λ3/4 rs12979860 CC (P = 0.027), tapasin G in individuals with aspartate at residue 114 of HLA-B (TapG:HLA-B(114D) ) (P = 0.007) and HLA-DRB1*04:01 (P = 0.014) were associated with SR with a strong additive influence (χ(2) test for trend P < 0.0001); favorable polymorphisms did not interact synergistically, nor did patients cluster by outcome. In the treatment cohort, IFN-λ3/4 rs12979860 CC was protective in hepatitis C virus (HCV) G1 infection and KIR2DL3:HLA-C1 in HCV G2/3. In common with SR, variables did not interact synergistically. Polymorphisms predictive of viral clearance did not predict disease progression. In summary, different individuals resolve HCV infection using discrete and non-interacting immunological pathways. These pathways are influenced by viral genotype. This work provides novel insights into the complexity of the interaction between host and viral factors in determining the outcome of HCV infection.

Innate and adaptive genetic pathways in HCV infection.

Infection with hepatitis C virus (HCV) leads to a wide spectrum of clinical manifestations. This heterogeneity is underpinned by the host immune response and the genetic factors that govern it. Polymorphisms affecting both the innate and adaptive immunity determine the outcome of exposure. However the innate immune system appears to play a greater role in determining treatment-associated responses. Overall the effects of IFNL3/4 appear dominant over other polymorphic genes. Understanding how host genetics determines the disease phenotype has not been as intensively studied. This review summarizes our current understanding of innate and adaptive immunogenetic factors in the outcome of HCV infection. It focuses on how they relate to resolution and the progression of HCV-related liver disease, in the context of current and future treatment regimes.

Genome-wide association study of hepatitis C virus- and cryoglobulin-related vasculitis.

The host genetic basis of mixed cryoglobulin vasculitis is not well understood and has not been studied in large cohorts. A genome-wide association study was conducted among 356 hepatitis C virus (HCV) RNA-positive individuals with cryoglobulin-related vasculitis and 447 ethnically matched, HCV RNA-positive controls. All cases had both serum cryoglobulins and a vasculitis syndrome. A total of 899 641 markers from the Illumina HumanOmni1-Quad chip were analyzed using logistic regression adjusted for sex, as well as genetically determined ancestry. Replication of select single-nucleotide polymorphisms (SNPs) was conducted using 91 cases and 180 controls, adjusting for sex and country of origin. The most significant associations were identified on chromosome 6 near the NOTCH4 and MHC class II genes. A genome-wide significant association was detected on chromosome 6 at SNP rs9461776 (odds ratio=2.16, P=1.16E-07) between HLA-DRB1 and DQA1: this association was further replicated in additional independent samples (meta-analysis P=7.1 × 10(-9)). A genome-wide significant association with cryoglobulin-related vasculitis was identified with SNPs near NOTCH4 and MHC Class II genes. The two regions are correlated and it is difficult to disentangle which gene is responsible for the association with mixed cryoglobulinemia vasculitis in this extended major histocompatibility complex region.

The allele 4 of neck region liver-lymph node-specific ICAM-3-grabbing integrin variant is associated with spontaneous clearance of hepatitis C virus and decrease of viral loads.

L-SIGN is a C-type lectin expressed on liver sinusoidal endothelial cells involved in the capture of hepatitis C virus and trans-infection of adjacent hepatocyte cells. The neck region of L-SIGN is highly polymorphic, with three to nine tandem repeats of 23 residues. This polymorphism is associated with a number of infectious diseases, but has not been explored in HCV. We therefore investigated the impact of L-SIGN neck region length variation on the outcome of HCV infection. We studied 322 subjects, 150 patients with persistent HCV infection, 63 individuals with spontaneous clearance and 109 healthy controls. In healthy subjects, we found a total of nine genotypes, with the 7/7 genotype being the most frequent (33%) followed by the 7/6 (22.9%) and the 7/5 (18.3%). The frequencies of the alleles were as follows: 7-LSIGN (56.4%), 6-LSIGN (20.2%), 5-L-SIGN (18.3%) and 4-L-SIGN (5%). The frequency of the 7/4 genotype was higher in spontaneous resolvers (14.3%) as compared with the persistent group (4%) (OR = 0.25, 95% CI = 0.07-0.82, p 0.022). In addition, we found that 4-L-SIGN was associated with spontaneous resolution of HCV infection (OR = 0.30, 95%CI, 0.12-0.74, p 0.005). Interestingly, patients with 4-L-SIGN had lower viral loads when compared with carriers of the 5 (p 0.001), 6 (p 0.021) and 7-alleles (p 0.048). The results indicate that neck region polymorphism of L-SIGN can influence the outcome of HCV infection and the four-tandem repeat is associated with clearance of HCV infection.

Association of NKG2A with treatment for chronic hepatitis C virus infection.

Natural killer (NK) cells are critical to the immune response to viral infections. Their functions are controlled by receptors for major histocompatibility complex (MHC) class I, including NKG2A and killer-cell immunoglobulin-like receptors (KIR). In order to evaluate the role of MHC class I receptors in the immune response to hepatitis C virus infection we have studied patients with chronic HCV infection by multi-parameter flow cytometry directly ex vivo. This has permitted evaluation of combinatorial expression of activating and inhibitory receptors on single NK cells. Individuals with chronic HCV infection had fewer CD56(dim) NK cells than healthy controls (4.9 +/- 3.4% versus 9.0 +/- 5.9%, P < 0.05). Expression levels of the inhibitory receptor NKG2A was up-regulated on NK cells from individuals with chronic hepatitis C virus (HCV) (NKG2A mean fluorescence intensity 5692 +/- 2032 versus 4525 +/- 1646, P < 0.05). Twelve individuals were treated with pegylated interferon and ribavirin. This resulted in a down-regulation of NKG2A expression on CD56(dim) NK cells. Individuals with a sustained virological response (SVR) had greater numbers of NKG2A-positive, KIR-negative NK cells than those without SVR (27.6 +/- 9.6% NK cells versus 17.6 +/- 5.7, P < 0.02). Our data show that NKG2A expression is dysregulated in chronic HCV infection and that NKG2A-positive NK cells are associated with a beneficial response to pegylated interferon and ribavirin therapy.

Speed and selection in the evolution of killer-cell immunoglobulin-like receptors.

The killer-cell immunoglobulin-like receptors (KIR) form a diverse family of receptors that control the functions of natural killer cells. Sequencing of KIR from primates has revealed the unexpected extent to which this gene family has diversified mostly likely in response to pathogens and to pathogen-mediated selection of their MHC class I ligands. Human KIR diversity is now a burgeoning area for disease association studies. This review examines the evolution of KIR from a primate-centric view in order to rationalize our current knowledge of the diversity of human KIR.

Comparison of chimpanzee and human leukocyte Ig-like receptor genes reveals framework and rapidly evolving genes.

The leukocyte receptor complex (LRC) on human chromosome 19 contains related Ig superfamily killer cell Ig-like receptor (KIR) and leukocyte Ig-like receptor (LIR) genes. Previously, we discovered much difference in the KIR genes between humans and chimpanzees, primate species estimated to have approximately 98.8% genomic sequence similarity. Here, the common chimpanzee LIR genes are identified, characterized, and compared with their human counterparts. From screening a chimpanzee splenocyte cDNA library, clones corresponding to nine different chimpanzee LIRs were isolated and sequenced. Analysis of genomic DNA from 48 unrelated chimpanzees showed 42 to have all nine LIR genes, and six animals to lack just one of the genes. In structural diversity and functional type, the chimpanzee LIRs cover the range of human LIRs. Although both species have the same number of inhibitory LIRs, humans have more activating receptors, a trend also seen for KIRs. Four chimpanzee LIRs are clearly orthologs of human LIRs. Five other chimpanzee LIRs have paralogous relationships with clusters of human LIRs and have undergone much recombination. Like the human genes, chimpanzee LIR genes appear to be organized into two duplicated blocks, each block containing two orthologous genes. This organization provides a conserved framework within which there are clusters of faster evolving genes. Human and chimpanzee KIR genes have an analogous arrangement. Whereas both KIR and LIR genes can exhibit greater interspecies differences than the genome average, within each species the LIR gene family is more conserved than the KIR gene family.

Rapid evolution of NK cell receptor systems demonstrated by comparison of chimpanzees and humans.

That NK cell receptors engage fast-evolving MHC class I ligands suggests that they, too, evolve rapidly. To test this hypothesis, the structure and class I specificity of chimpanzee KIR and CD94:NKG2 receptors were determined and compared to their human counterparts. The KIR families are divergent, with only three KIR conserved between chimpanzees and humans. By contrast, CD94:NKG2 receptors are conserved. Whereas receptors for polymorphic class I are divergent, those for nonpolymorphic class I are conserved. Although chimpanzee and human NK cells exhibit identical receptor specificities for MHC-C, they are mediated by nonorthologous KIR. These results demonstrate the rapid evolution of NK cell receptor systems and imply that "catching up" with class I is not the only force driving this evolution.

Cytotoxic T lymphocyte responses and CTL epitope escape mutation in HBsAg, anti-HBe positive individuals.

BACKGROUND/AIMS: Clearance of hepatitis B virus (HBV) is characterised by a strong cytotoxic T cell response. Persistence of HBV in chronic hepatitis B carriers may be related to failure of this response. The aim of this study was to determine whether HLA class I restricted cytotoxic T lymphocyte (CTL) responses persist in anti-hepatitis B e (HBe) positive / HBV DNA negative individuals, and to correlate the presence of viral CTL epitope mutation with clinical outcome. METHODS: An HLA/HBV dual transfectant model was used to demonstrate these CTL responses in individuals chronically infected with HBV. Subsequently, a known hepatitis B core (HBc) CTL epitope was sequenced in a family of five chronically infected individuals all sharing a HLA allele (HLA-A68.1). RESULTS: Low level HLA class I restricted cytotoxic T cell responses were detected in the peripheral blood of five of eight anti-HBe positive individuals. In the family of HLA-A68.1 positive chronically infected individuals, mutation of the HLA-A68.1 restricted hepatitis B core antigen (HBcAg) CTL epitope STLPETTVVRR was found in all four anti-HBe positive individuals but not in the sole hepatitis B e antigen (HBeAg) positive patient. CONCLUSION: These data are consistent with a continued immune selection pressure on HBV in anti-HBe positive chronically infected individuals with low replicating HBV infection and suggest that mutation of a CTL epitope may be a consequence of the immune response, as opposed to the cause of viral persistence.

Ribavirin and interferon alfa-2b in chronic hepatitis C: assessment of possible pharmacokinetic and pharmacodynamic interactions.

AIMS: The primary objective of this study was to determine whether pharmacokinetic interactions occurred between interferon alpha-2b (IFN) and ribavirin in patients with chronic hepatitis C infections. Additionally this study assessed the single and multiple-dose pharmacokinetics of ribavirin and IFN, and compared the safety, tolerability and antiviral pharmacodynamics of IFN plus ribavirin compared with either drug alone. METHODS: In this open label parallel group study, patients with chronic hepatitis C were randomized to receive IFN 3 million IU thrice weekly s.c. alone, ribavirin 600 mg twice daily p.o. alone or both drugs in combination over 6 weeks. Single and multiple dose pharmacokinetics and indices of antiviral pharmacodynamics were assessed during weeks 1 and 6, along with safety assessments during the study. RESULTS: The range of mean ribavirin terminal phase half-lives after single doses was 44-49 h. Comparison of week 1 and week 6 AUC(0,12h) values showed accumulation in plasma of approximately 6-fold. The range of mean washout half-lives after week 6 was 274-298 h, reflecting release of ribavirin from deep compartment stores. The range of single and multiple dose IFN terminal phase half-lives was 5-7 h. IFN demonstrated an increase in bioavailability (approximately 2-fold) upon multiple dose administration. Ribavirin and IFN pharmacokinetic parameters for combined ribavirin and IFN were similar to those during monotherapy with either compound, although the power of this study to detect differences was low. Serum HCV-RNA titers and ALT concentrations were reduced by IFN alone, ribavirin alone reduced ALT concentrations only, and combined IFN plus ribavirin produced numerically greater falls in both measurements than either treatment alone. Serum concentrations of neopterin and activity of 2',5'-oligoadenylate synthetase (2'5'-OAS) were increased by IFN alone and in combination with ribavirin, whereas serum 2'5'-OAS activity was decreased and neopterin concentrations unaltered by ribavirin monotherapy. IFN and ribavirin monotherapy produced characteristic changes in safety laboratory tests (IFN--reductions in white cells, neutrophils and platelets; ribavirin--reduced haemoglobin) and characteristic adverse event profiles (IFN--headache, flu-like symptoms, fatigue, anorexia, nausea, myalgia, and insomnia; ribavirin--headache, fatigue, myalgia, and pruritus). There was no additive effect of combination therapy on safety laboratory tests or reported adverse events. All changes were fully reversible upon treatment cessation. CONCLUSIONS: There was no evidence of pharmacokinetic interactions between IFN and ribavirin in this study. There were numerical trends indicating that the combination of IFN and ribavirin reduced titers of HCV-RNA to a greater extent than did either treatment alone, and the safety profile of combination therapy was similar to those of both monotherapy treatments.

Lymphocyte and macrophage phenotypes in chronic hepatitis C infection. Correlation with disease activity.

The pathogenesis of chronic hepatitis C and the mechanisms underlying progressive liver disease in patients with chronic hepatitis C infection are poorly understood. To demonstrate which inflammatory cells might be responsible for the necroinflammatory damage in chronic hepatitis C infection, we have correlated the phenotype of the intrahepatic lymphocytes and macrophages with histological activity in liver biopsy and explant specimens from 19 patients with chronic hepatitis C infection. In all stages of disease, more CD8+ than CD4+ lymphocytes were found. However, histologically active versus histologically mild hepatitis was associated with a trend toward greater parenchymal concentrations of CD4+ lymphocytes (0.71 +/- 0.27 per 10(4) microns 2 versus 0.35 +/- 0.15; not significant), significantly less parenchymal CD8+ lymphocytes (0.90 +/- 0.1 versus 1.70 +/- 0.3; t = 2.32, P = 0.03) and a greater parenchymal CD4/CD8 ratio (4.1 +/- 2.8 versus 0.91 +/- 0.3; t = 1.65, P = 0.07). No difference was found in the number of cells containing cytotoxic granules between the two groups. Greater numbers of CD4+ lymphocytes were found in liver biopsy specimens with little or no staining for hepatitis C virus antigen (1.47 +/- 0.88 versus 0.27 +/- 0.27; t = 2.28, P < 0.05). No significant differences were found in the macrophage subsets between the three stages of disease. Our data suggest that active histological disease in chronic hepatitis C infection may be associated with an increase in CD4+ lymphocytes and suggest that CD4+ T cells may play an important role in the hepatic injury in these patients.

A clinical evaluation of a new method for HBV DNA quantitation in patients with chronic hepatitis B.

Selection of HBsAg-positive patients for antiviral therapy requires an estimation of disease activity and viral replication. Serum transaminases and histological analysis are commonly used to assess disease activity, and viral replication is assessed by serological testing of HBeAg and serum hepatitis B virus (HBV) DNA. Dot blot hybridisation may be insufficiently sensitive to corroborate low-grade replication in patients with active hepatitis, and polymerase chain reaction (PCR) may be testing too sensitive for this role. Theoretically an assay of intermediate sensitivity is therefore required. Our aim was to evaluate whether the branched chain DNA (bDNA) assay would fulfil this function. Seventy-one HBsAg-positive patients were tested for HBV DNA by the bDNA assay; 64 were also tested by dot blot hybridisation and, when appropriate, also by PCR. Thirty-seven (52%) patients were positive for HBV DNA by the bDNA assay. HBV DNA was detected in the majority (21/28; 75%) of HBeAg-positive patients but also in 14 of 36 (39%) anti-HBe-positive patients. HBV DNA was detected by the bDNA assay in 20 of 48 (42%) patients negative for HBV DNA by dot blot hybridisation assay. All patients positive for HBV DNA by dot blot hybridisation were also positive by the bDNA assay. Sixteen of twenty-five (64%) patients negative for HBV DNA by the bDNA assay were positive for HBV DNA by PCR. The bDNA assay is a sensitive and reliable method for the detection of HBV DNA. As nucleoside analogue therapy becomes more widely available, the assay should provide a useful tool for the selection for and monitoring of patients on antiviral therapy.