Simon M. Rushbrook

Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection.

ABSTRACT The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8+ T cells. The role of CD4+CD25+ T regulatory (Treg) cells in priming and expanding virus-specific CD8+ T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8+ T-cell proliferation and gamma interferon (IFN-γ) frequency were analyzed with/without depletion of Treg cells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4+CD25+ Treg cells inhibited anti-CD3/CD28 CD8+ T-cell proliferation and perforin expression. Depletion of CD4+CD25+ Treg cells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P = 0.0005 and P = 0.002, respectively) and also the number of HCV- and EBV-specific IFN-γ-expressing CD8+ T cells. Although stimulated CD8+ T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4+CD25+ regulatory T cells on CD8+ T-cell proliferation. In conclusion, marked CD4+CD25+ regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8+ T-cell responses and viral persistence.

Compromised lymphocytes infiltrate hepatocellular carcinoma: the role of T-regulatory cells.

Hepatocellular carcinoma (HCC) has a poor prognosis with limited therapeutic options. We propose that local immune responses in patients with HCC are held in check by tumor‐infiltrating CD4+CD25+ T‐regulatory lymphocytes (Treg cells), which suppress the activity and proliferation of effector CD4+ and CD8+ T cells. The phenotype and cell cycle status of tumor‐infiltrating lymphocytes (TILs) in HCC were analyzed via immunohistochemistry of sections from patients undergoing surgery for HCC and via flow cytometry of peripheral blood mononuclear cells and TILs isolated from patients with HCC. Circulating and tumor‐infiltrating T‐cell function and activation status were assessed via proliferation and flow cytometry. More than 96% of TILs were quiescent as measured via Mcm‐2 or Ki‐67 expression, while less than 10% of CD8+ T cells expressed perforin or granzyme B. CD4+CD25+ Treg cells comprised 8.7% (1.4–13.8) of TILs and always exceeded the proportion in distant nontumor tissue (2.4% [1.5–5.6]; P = .014). Treg cells isolated from HCC suppressed proliferation of autologous circulating CD4+CD25− cells and perforin expression and proliferation of autologous CD8+ T cells. The proportion of circulating Treg cells in patients with HCC was similar in healthy controls (7.2% [1.2–23.3] and 9.2% [1.6–30.2], respectively), but the proportion of circulating Treg cells that were also transforming growth factor β1+ was elevated in HCC compared with controls (55.5% [8.2–73.9] and 2.0% [0–4.9], respectively; P = .003). In conclusion, TILs are compromised and contain a subpopulation of suppressive CD4+CD25+Foxp3+ Treg cells. Functional deletion of tumor‐infiltrating Treg cells could enhance tumor‐specific immunotherapy. (HEPATOLOGY 2005;41:722–730.)

A novel immunohistochemical method to estimate cell-cycle phase distribution in archival tissue: implications for the prediction of outcome in colorectal cancer

An immunohistochemical method for assessing cell‐cycle phase distribution in colorectal resection specimens would enable phase data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in colorectal cancer. In contrast to flow cytometry, an immunohistochemical method would also allow the phase distribution to be examined within morphologically heterogeneous regions of neoplasms. Paraffin sections of normal colon (n = 25), colonic adenoma (n = 15), and colonic adenocarcinoma (n = 30) were analysed by immunohistochemistry using antibodies against markers of cell‐cycle entry, Mcm‐2 and Ki67, and putative markers of the cell‐cycle phase, cyclins D1 and E (putative markers of G1 phase), cyclin A (S phase), cytoplasmic cyclin B1 (G2 phase), and phosphohistone H3 (M phase). The phase specificity of each marker was assessed by examining the degree of co‐expression of adjacent phase markers using double‐antibody fluorescence confocal microscopy and by comparison with flow cytometric analysis performed on adjacent tissue sections. The S‐phase specificity of detectable cyclin A was also assessed in combination with in situ DNA replication using fluorescence confocal microscopy. All cells expressing phase markers co‐expressed Mcm‐2. Adjacent phase markers were not significantly co‐expressed, confirming the relative specificity of these markers in tissue sections of colon. Cell‐cycle phase distribution, calculated by immunohistochemistry, compared well with phase analyses obtained by flow cytometry. No cells expressed cyclin A in the absence of active DNA replication. The S‐phase labelling index, as defined by detectable cyclin A expression, showed a positive correlation with the Mcm‐2 labelling index and increased in the progression from normal colon to adenocarcinoma. In conclusion, a combination of these cell‐cycle phase markers can be used to calculate the distribution of cells throughout each phase of the cell cycle in colorectal tissue sections. Detectable cyclin A can be used as a surrogate marker of S phase and may be of value in predicting prognosis and response to adjuvant therapy. Copyright

Histological changes in HCV antibody–positive, HCV RNA–negative subjects suggest persistent virus infection

It is unclear whether hepatitis C virus (HCV) has been eradicated or persists at a low level in HCV antibody–positive HCV RNA–negative individuals. The natural history and liver histology are not well characterized. One hundred seventy‐two HCV antibody–positive, serum HCV RNA–negative patients underwent diagnostic liver biopsy between 1992 and 2000 and were followed a median 7 years (range, 5–12). Patients with any possible cause of liver injury other than HCV were excluded. A single histopathologist scored sections using Ishak criteria. Characterization of the inflammatory infiltrate in selected cases used a novel semiquantitative technique and compared with HCV RNA–positive patients and healthy controls. One hundred two patients were excluded because of a risk factor for liver injury other than HCV. Seventy patients met the study criteria; four (5.7%) became HCV RNA–positive during follow‐up. Sixty‐six cases remained HCV RNA–negative; five (7.5%) had a normal liver biopsy; 54 (82%) had fibrosis (stage 2 or 3 in 16 (24%)). Nonviremic cases revealed expanded portal tracts (P < 0.05), with fewer CD4+ (P < 0.05) and more CD8+ cells (P < 0.05) than healthy controls, but were indistinguishable from HCV RNA–positive cases for these parameters. Lobular CD4 staining, absent in healthy controls, was noted in both HCV RNA–negative and –positive cases and was more marked in the latter (P < 0.05) with a sinusoidal lining cell distribution. Conclusion: Nonviremic HCV antibody–positive patients have a liver biopsy that is usually abnormal. Fibrosis was present in most with similar inflammatory infiltrate to viremic cases. The presence of a CD8+ rich inflammatory infiltrate suggests an ongoing immune response in the liver, supporting the view that HCV may persist in the liver in the majority of HCV RNA–negative cases. (HEPATOLOGY 2008;48;1737‐1745.)

Genome-wide association study of primary sclerosing cholangitis identifies new risk loci and quantifies the genetic relationship with inflammatory bowel disease

Primary sclerosing cholangitis (PSC) is a rare progressive disorder leading to bile duct destruction; ∼75% of patients have comorbid inflammatory bowel disease (IBD). We undertook the largest genome-wide association study of PSC (4,796 cases and 19,955 population controls) and identified four new genome-wide significant loci. The most associated SNP at one locus affects splicing and expression of UBASH3A, with the protective allele (C) predicted to cause nonstop-mediated mRNA decay and lower expression of UBASH3A. Further analyses based on common variants suggested that the genome-wide genetic correlation (rG) between PSC and ulcerative colitis (UC) (rG = 0.29) was significantly greater than that between PSC and Crohns disease (CD) (rG = 0.04) (P = 2.55 × 10−15). UC and CD were genetically more similar to each other (rG = 0.56) than either was to PSC (P < 1.0 × 10−15). Our study represents a substantial advance in understanding of the genetics of PSC.

Improved detection of hepatocyte proliferation using antibody to the pre-replication complex: an association with hepatic fibrosis and viral replication in chronic hepatitis C virus infection

Summary. To test the hypothesis that hepatitis C virus (HCV) might induce hepatocyte proliferation directly, thereby predisposing HCV carriers to cirrhosis and hepatocellular carcinoma, we have used a new method to identify proliferating hepatocytes, employing a novel monoclonal antibody to minichromosome maintenance (Mcm) proteins, essential components of the pre‐replication complex. Antibody to Ki‐67, a conventional marker of cell division, was also studied. Eighty‐seven patients with chronic HCV infection and a broad spectrum of histological change were studied. Proliferation was observed rarely in hepatocytes from normal liver from healthy controls (always less than 0.01%). However, proliferating hepatocytes were detected in all HCV‐infected patients and the proportion of hepatocytes expressing Mcm‐2 (3–40%) always exceeded that expressing Ki‐67 (1–14%) and correlated positively with increasing stage of fibrosis (P = 0.0001) and viral replication (P = 0.0004). There were weaker but significant associations between the proportion of hepatocytes expressing Mcm‐2 and inflammatory indices including interface hepatitis, portal tract inflammation, lobular inflammation and steatosis. There was no association between the proportion of hepatocytes expressing Mcm‐2 and age, gender or past alcohol consumption, but there was a weak association with current consumption of alcohol (P = 0.0067). The proportion of Ki‐67 hepatocytes did not correlate with any clinical, laboratory or histological parameter. Mcm‐2 was also detected in bile duct cells, sinusoidal lining cells and infiltrating lymphocytes, but at low frequency. These data indicate first, that Mcm‐2 is a more sensitive marker of hepatocyte proliferation than Ki‐67, second that many hepatocytes in chronic HCV infection have entered the cell cycle and third, suggest that interference with the hepatocyte cell cycle might be an alternative approach to therapy.

Immunohistochemical estimation of cell cycle entry and phase distribution in astrocytomas: applications in diagnostic neuropathology.

An immunohistochemical method for assessing cell cycle phase distribution in neurosurgical biopsies would enable such data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in glial neoplasms, without the requirement for flow cytometric analysis. Paraffin‐embedded sections of intracerebral gliomas (n = 48), consisting of diffuse astrocytoma (n = 9), anaplastic astrocytoma (n = 8) and glioblastoma (n = 31), were analysed by immunohistochemistry using markers of cell cycle entry, Mcm‐2 and Ki67, and putative markers of cell cycle phase, cyclins D1 (G1‐phase), cyclin A (S‐phase), cyclin B1 (G2‐phase) and phosphohistone H3 (Mitosis). Double labelling confocal microscopy confirmed that the phase markers were infrequently coexpressed. Cell cycle estimations by immunohistochemistry were corroborated by flow cytometric analysis. There was a significant increase in Mcm‐2 (P < 0.0001), Ki67 (P < 0.0001), cyclin A (P < 0.0001) and cyclin B1 (P = 0.002) expression with increasing grade from diffuse astrocytoma through anaplastic astrocytoma to glioblastoma, suggesting that any of these four markers has potential as a marker of tumour grade. In a subset of glioblastomas (n = 16) for which accurate clinical follow‐up data were available, there was a suggestion that the cyclin A:Mcm‐2 labelling fraction might predict a relatively favourable response to radical radiotherapy. These provisional findings, however, require confirmation by a larger study. We conclude that it is feasible to obtain detailed cell cycle data by immunohistochemical analysis of tissue biopsies. Such information may facilitate tumour grading and may enable information of prognostic value to be obtained in the routine diagnostic laboratory.

Fine mapping and replication of genetic risk loci in primary sclerosing cholangitis

Abstract Background and aims. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts eventually leading to biliary cirrhosis. Recent genetic studies in PSC have identified associations at 2q13, 2q35, 3p21, 4q27, 13q31 and suggestive association at 10p15. The aim of this study was to further characterize and refine the genetic architecture of PSC. Methods. We analyzed previously reported associated SNPs at four of these non-HLA loci and 59 SNPs tagging the IL-2/IL-21 (4q27) and IL2RA (10p15) loci in 992 UK PSC cases and 5162 healthy UK controls. Results. The most associated SNPs identified were rs3197999 (3p21 (MST1), p = 1.9 × 10-6, ORA vs G = 1.28, 95% CI (1.16–1.42)); rs4147359 (10p15 (IL2RA), p = 2.6 × 10-4, ORA vs G = 1.20, 95% CI (1.09–1.33)) and rs12511287 (4q27 (IL-2/IL-21), p = 3.0 × 10-4, ORA vs T = 1.21, 95% CI (1.09–1.35)). In addition, we performed a meta-analysis for selected SNPs using published summary statistics from recent studies. We observed genome-wide significance for rs3197999 (3p21 (MST1), P combined = 3.8 × 10-12) and rs4147359 (10p15 (IL2RA), P combined = 1.5 × 10-8). Conclusion. We have for the first time confirmed the association of PSC with genetic variants at 10p15 (IL2RA) locus at genome-wide significance and replicated the associations at MST1 and IL-2/IL-21 loci in a large homogeneous UK population. These results strongly implicate the role of IL-2/IL2RA pathway in PSC and provide further confirmation of MST1 association.

Endoscopic ultrasound: a review of current diagnostic and therapeutic applications

Endoscopic ultrasound (EUS) has become important in a variety of clinical settings. Echoendoscopes may be categorised into radial and linear configurations. Radial devices are used for diagnostic imaging, whereas linear echoendoscopes also facilitate image guided tissue sampling and intervention. EUS is an established primary diagnostic tool for a number of conditions including choledocholithiasis and biliary microlithiasis. It is therefore well suited to the investigation of the aetiology of pancreatitis where simpler measures fail to identify the aetiology. It can also be used to identify chronic non-calcific pancreatitis. EUS is important in the secondary evaluation of abnormalities detected by other imaging modalities—for example, cystic pancreatic lesions. The high resolution of EUS allows more detailed image based analysis than other imaging modalities. The ability to sample cyst fluid significantly increases the accuracy of lesion characterisation. Most importantly, EUS has become indispensable in the staging of a variety of upper gastrointestinal tract tumours. If resection is being considered, the high resolution images obtained via EUS are invaluable for local tumour staging. EUS guided tissue sampling permits accurate nodal staging without relying on lymph node size as proxy for malignant infiltration. In patients with contraindications to magnetic resonance imaging, EUS is an alternative for the staging of rectal carcinoma. It is used in the staging of lung cancer, often in combination with endobronchial ultrasound. Finally, EUS is used therapeutically in image guided drainage (such as gastrocystostomy in pancreatic pseudocyst) and coeliac plexus neurolysis in patients with abdominal pain caused by pancreatic cancer or pancreatitis.

T-regulatory lymphocytes and chronic viral hepatitis

Both hepatitis B virus (HBV) and hepatitis C virus (HCV) can cause persistent viral infection in humans. Chronic infection is associated with a risk of cirrhosis and hepatocellular carcinoma. The cause of chronic infection is unknown. A large body of evidence suggests that a failure of the adaptive immune response is critical in the establishment of chronic infection. Recently a new group of T cells (T-regulatory cells), that express CD4+CD25+ and Foxp3, which can inhibit the cellular (CD4+/CD8+) immune response have been described. In this review the authors explore the thoughts regarding immune responses to HBV and HCV infections and the role of these T-regulatory cells in relation to the pathogenesis of chronic HBV and HCV infection and the potential for therapeutic intervention.