Geoffrey W. McCaughan

CD26: A Multifunctional Integral Membrane and Secreted Protein of Activated Lymphocytes

CD26 has proved interesting in the fields of immunology, endocrinology, cancer biology and nutrition owing to its ubiquitous and unusual enzyme activity. This dipeptidyl aminopeptidase (DPP IV) activity generally inactivates but sometimes alters or enhances the biological activities of its peptide substrates, which include several chemokines. CD26 costimulates both the CD3 and the CD2 dependent T‐cell activation and tyrosine phosphorylation of TCR/CD3 signal transduction pathway proteins. CD26 in vivo has integral membrane protein and soluble forms. Soluble CD26 is at significant levels in serum, these levels alter in many diseases and soluble CD26 can modulate in vitro T‐cell proliferation. CD26, being an adenosine deaminase binding protein (ADAbp), functions as a receptor for ADA on lymphocytes. The focus of this review is the structure and function of CD26 and the influence of its ligand binding activity on T‐cell proliferation and the T cell costimulatory activity of CD26.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I.

Real‐time quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) is the method of choice for rapid and reproducible measurements of cytokine or growth factor expression in small samples. Fluorescence detection methods for monitoring real‐time PCR include fluorogenic probes labelled with reporter and quencher dyes, such as Taqman probes or Molecular Beacons and the dsDNA‐binding dye SYBR Green I. Fluorogenic (Taqman) probes for a range of human and rat cytokines and growth factors were tested for sensitivity and compared with an assay for SYBR Green I quantification using real‐time fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detector). SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. Fluorogenic probes provided sensitive and reproducible detection of targets that ranged from low (<10 copies/reaction) to high (>107 copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (≥1000 copies/reaction), but did not give consistently reproducible quantification when the target gene was expressed at low levels. Although optimization of melting temperature improved the specificity of SYBR Green I detection, in our hands it did not equal the reproducible sensitivity and specificity of fluorogenic probes. The latter method is the first choice for measurement of low‐level gene expression, although SYBR Green I is a simple and reproducible means to quantify genes that are expressed at moderate to high levels.

The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity

Hepatic immunobiology is paradoxical: although the liver possesses unusual tolerogenic properties, it is also the site of effective immune responses against multiple pathogens and subject to immune-mediated pathology. The mechanisms underlying this dichotomy remain unclear. Following previous work demonstrating that the liver may act as a site of primary T cell activation, we demonstrate here that the balance between immunity and tolerance in this organ is established by competition for primary activation of CD8+ T cells between the liver and secondary lymphoid tissues, with the immune outcome determined by the initial site of activation. Using a transgenic mouse model in which antigen is expressed within both liver and lymph nodes, we show that while naive CD8+ T cells activated within the lymph nodes were capable of mediating hepatitis, cells undergoing primary activation within the liver exhibited defective cytotoxic function and shortened half-life and did not mediate hepatocellular injury. The implications of these novel findings may pertain not only to the normal maintenance of peripheral tolerance, but also to hepatic allograft tolerance and the immunopathogenesis of chronic viral hepatitis.

Does alcohol directly stimulate pancreatic fibrogenesis? Studies with rat pancreatic stellate cells

BACKGROUND & AIMS Activated pancreatic stellate cells have recently been implicated in pancreatic fibrogenesis. This study examined the role of pancreatic stellate cells in alcoholic pancreatic fibrosis by determining whether these cells are activated by ethanol itself and, if so, whether such activation is caused by the metabolism of ethanol to acetaldehyde and/or the generation of oxidant stress within the cells. METHODS Cultured rat pancreatic stellate cells were incubated with ethanol or acetaldehyde. Activation was assessed by cell proliferation, alpha-smooth muscle actin expression, and collagen synthesis. Alcohol dehydrogenase (ADH) activity in stellate cells and the influence of the ADH inhibitor 4-methylpyrazole (4MP) on the response of these cells to ethanol was assessed. Malondialdehyde levels were determined as an indicator of lipid peroxidation. The effect of the antioxidant vitamin E on the response of stellate cells to ethanol or acetaldehyde was also examined. RESULTS Exposure to ethanol or acetaldehyde led to cell activation and intracellular lipid peroxidation. These changes were prevented by the antioxidant vitamin E. Stellate cells exhibited ethanol-inducible ADH activity. Inhibition of ADH by 4MP prevented ethanol-induced cell activation. CONCLUSIONS Pancreatic stellate cells are activated on exposure to ethanol. This effect of ethanol is most likely mediated by its metabolism (via ADH) to acetaldehyde and the generation of oxidant stress within the cells.

T lymphocytes interact with hepatocytes through fenestrations in murine liver sinusoidal endothelial cells

The liver has an established ability to induce tolerance. Recent evidence indicates that this unique property might be related to its distinctive architecture allowing T cells to be activated in situ independently of lymphoid tissues. Unlike lymph node–activated T cells, liver‐activated T cells are short‐lived, a mechanism that might contribute to the “liver tolerance effect.” Although the potential role of hepatocytes as tolerogenic antigen‐presenting cells has been demonstrated, the question as to whether these cells are able to interact with CD8+ T cells in physiological settings remains controversial. Contradicting the immunological dogma stating that naïve T lymphocytes are prevented from interacting with parenchymal cells within non‐lymphoid organs by an impenetrable endothelial barrier, we show here that the unique morphology of the liver sinusoidal endothelial cell (LSEC) permits interactions between lymphocytes and hepatocytes. Using electron microscopy, we demonstrate that liver resident lymphocytes as well as circulating naïve CD8+ T cells make direct contact with hepatocytes through cytoplasmic extensions penetrating the endothelial fenestrations that perforate the LSECs. Furthermore, the expression of molecules required for primary T cell activation, MHC class I and ICAM‐1, is polarized on hepatocytes to the perisinusoidal cell membrane, thus maximizing the opportunity for interactions with circulating lymphocytes. In conclusion, this study has identified, at the ultrastructural level, a unique type of interaction between naïve T lymphocytes and liver parenchymal cells in vivo. These results hold implications for the pathogenesis of viral hepatitis in which hepatocytes may represent the main antigen‐presenting cell, and for the development of immune tolerance as lymphocytes pass through the liver. (HEPATOLOGY 2006;44:1182–1190.)

Improved prediction of fibrosis in chronic hepatitis C using measures of insulin resistance in a probability index.

We sought to develop a clinically useful index comprising standard and physiologically relevant variables to predict the probability of significant hepatic fibrosis in subjects with chronic hepatitis C virus (HCV) infection. Fibrosis was graded as mild (stages F0 or F1) or significant (stages F2–F4). Thirty‐five clinical and laboratory parameters were analyzed initially in 176 patients with detectable HCV RNA to derive a fibrosis probability index (FPI) to predict significant fibrosis. This index then was validated in a second group of 126 subjects. Among 18 variables associated with severe fibrosis on univariate analysis, multiple logistic regression analysis identified age, aspartate aminotransferase (AST), total cholesterol level, insulin resistance (by homeostasis model), and past alcohol intake as independent predictors of significant fibrosis. The area under the receiver operating characteristic (ROC) curves was 0.84 for the initial cohort and 0.77 for the validation cohort. In the initial cohort, the sensitivity of the FPI based on these five predictors was 96%, and the negative predictive value was 93% at a score of ≥0.2. At scores ≥0.8, the FPI was 94% specific and had a positive predictive value of 87%. In conclusion, an FPI using routinely assessed markers and incorporating a measure of insulin resistance can reliably predict the probability of significant hepatic fibrosis in most patients with chronic HCV infection. Such an index should prove useful to guide decision making regarding the need for liver biopsy, and potentially for avoiding or deferring biopsy in a large proportion of patients with mild liver disease. (HEPATOLOGY 2004;39:1239–1247.)

Antigen-Specific Primary Activation of CD8+ T Cells Within the Liver

It is generally accepted that naive T cells recirculate via the blood and lymph, but do not enter nonlymphoid tissues without prior activation and differentiation. In this study, we demonstrate that the liver is an exception to this rule. Naive Des-TCR transgenic CD8+ T cells specific for H-2Kb were selectively retained in the liver within a few minutes of adoptive transfer into transgenic Met-Kb mice expressing H-2Kb in the liver. Activated CD8+ cells were found in the liver, but not the blood, as soon as 2 h after transfer and underwent cell division and started to recirculate within 24 h of transfer. In contrast, CD8+ cells activated in the lymph nodes remained sequestered at that site for 2 days before entering the blood. Our results therefore suggest that, in addition to its previously described role as a non Ag-specific activated T cell graveyard, the liver is involved in Ag-specific activation of naive recirculating CD8+ T cells. This particular property of the liver, combined with the previously demonstrated ability of hepatocytes to induce tolerance by means of premature CD8+ T cell death, may be a major mechanism contributing to the acceptance of liver allografts and the chronicity of viral hepatitis.

Increases in intrahepatic CD68 positive cells, MAC387 positive cells, and proinflammatory cytokines (particularly interleukin 18) in chronic hepatitis C infection

BACKGROUND Upregulation of Th1 associated intrahepatic cytokines in chronic hepatitis C virus (HCV) infection should lead to a significant non-specific cellular immune response, a prerequisite for viral clearance. However, to date, the role of this non-specific response in HCV has been understudied. AIMS To analyse the intrahepatic macrophage activity in chronic HCV infection by immunostaining and by quantitation of cytokine mRNA. METHODS HCV positive liver tissues (chronic hepatitis, n=10; cirrhosis, n=5) were immunostained for CD68, MAC387, and semiquantitated by polymerase chain reaction for intrahepatic cytokine mRNAs (interferon γ (IFNγ), interleukin 1β (IL-1β), IL-6, IL-18, tumour necrosis factor α (TNFα), and macrophage inflammatory protein 1β (MIP1β)). HCV negative normal liver tissues (for cytokines, n=6; for immunostaining, n=5) were included as controls. RESULTS MAC387+ cells were focally increased in areas of erosion at the limiting plate while lobular staining was minimal. CD68+ staining was diffuse in both portal (increased in HCV) and lobular areas. The portal tract (mean) density of CD68+ and MAC387+ cells was significantly increased in patients with HCV compared with normal tissue. IFNγ and IL-18 mRNA levels were highly correlated and significantly upregulated in chronic hepatitis and cirrhotic tissue versus controls. TNFα mRNA was upregulated in chronic hepatitis without cirrhosis, while IL-6 mRNA was significantly downregulated. IL-1β, IL-6, and MIP1β mRNA levels were significantly correlated with portal tract MAC387+ cell density. CONCLUSIONS The significant upregulation of IFNγ and IL-18 mRNA and significant correlations between IFNγ and other proinflammatory cytokines, suggest a Th1/cell mediated intrahepatic immune response in chronic HCV infection. However, further clarification of the cellular sources of these cytokines is required.

Insights into the Pathobiology of Hepatitis C Virus-Associated Cirrhosis: Analysis of Intrahepatic Differential Gene Expression

The pathogenesis of hepatitis C virus (HCV)-associated liver injury involves many genes from multiple pathogenic pathways. cDNA array analysis, which examines the expression of many genes simultaneously, was used to achieve new insights into HCV liver injury. Membrane-based cDNA arrays of 874 genes compared HCV-associated cirrhosis with autoimmune hepatitis-associated cirrhosis as an inflammatory and cirrhotic control, and with nondiseased liver tissue. Array analysis identified many differentially expressed genes that are important in inflammation, fibrosis, proliferation, signaling, apoptosis, and oxidative stress. Genes up-regulated in HCV-associated cirrhosis were predominantly associated with a Th1 immune response, fibrosis, cellular proliferation, and apoptosis. Novel observations of differential gene expression included increased expression of secreted apoptosis-related protein 3, a Wnt pathway gene possibly involved in cellular apoptosis. EMMPRIN (CD147) and discoidin domain receptor 1 (CD167) were also shown to be increased and are likely to play a role in liver fibrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction confirmed the increased expression of 15 genes. The comparison of HCV cirrhosis with autoimmune hepatitis cirrhosis showed a marked difference in the apoptosis-associated gene profile with HCV cirrhosis characterized by increased proapoptotic gene expression whereas autoimmune hepatitis was characterized by increased expression of both antiapoptotic and proapoptotic genes. Furthermore, expression of beta-catenin and the fibrosis-associated protein EMMPRIN were localized by immunohistochemistry to the plasma membranes of hepatocytes and biliary epithelium. In conclusion, HCV-associated cirrhosis was characterized by a proinflammatory, profibrotic, and proapoptotic gene expression profile.

Asian Pacific Association for the Study of the Liver consensus statements on the diagnosis, management and treatment of hepatitis C virus infection.

Co-chairs: GW McCaughan, Royal Prince Alfred Hospital, University of Sydney, Sydney, Australia M Omata, Tokyo University Hospital, Tokyo, Japan Faculty Members: D Amarapurkar, Bombay Hospital, Mumbai, India S Bowden, Victorian Infectious Diseases Reference Laboratories, Melbourne, Australia WC Chow, Singapore General Hospital, Singapore A Chutaputti, Pramongkutklao Hospital, Bangkok, Thailand G Dore, National Center in HIV Epidemiology and Clinical Research, Sydney, Australia E Gane, NZ Liver Transplant Unit, Auckland, New Zealand R Guan, Mount Elizabeth Medical Center, Singapore SS Hamid, The Aga Khan University, Karachi, Pakistan W Hardikar, Royal Children’s Hospital, Melbourne, Australia CK Hui, Queen Mary Hospital, University of Hong Kong, Hong Kong, China W Jafri, The Aga Khan University, Karachi, Pakistan J-D Jia, Beijing Friendship Hospital, Capital Medical University, Beijing, China M-Y Lai, National Taiwan University Hospital, Taiwan L Wei, Peking University Peoples Hospital, Beijing, China N Leung, The Chinese University of Hong Kong, Hong Kong, China T Piratvisuth, Prince of Songkla University, Hat Yai, Thailand S Sarin, GB Pant Hospital, Delhi, India J Sollano, University Santo Tomas Hospital, Manilla, Philippines R Tateishi, University of Tokyo Hospital, Tokyo Japan