Gene expression profiles of T cells from hepatitis E virus infected patients in acute and resolving phase.

BACKGROUND AND AIMS: Approximately 50% of acute viral hepatitis in young adults and in pregnant women is due to hepatitis E virus (HEV) infection in developing countries. T cell-mediated immune injury probably plays a key role in the pathogenesis of acute hepatitis illness. However, there is a paucity of data on the global gene expression programs activated on T cells, which are subsequently responsible for T cell recruitment to the liver and triggering of immune injury. PATIENTS AND METHODS: We performed a flow cytometric analysis of T cells in individuals with acute hepatitis E (AVH-E; n=10), resolving phase of HEV (n=9), and ten healthy controls (HC). Further transcriptional profiling analysis was performed using Affymetrix GeneChip DNA microarrays to identify the genes that were differentially expressed in AVH-E and HC. RESULTS: Patients with AVH-E showed higher frequencies of CD8+ (27 ± 4%; P=0.02) and activated CD38+ CD69+ T cells (25% ± 3%; P=0.04) than in resolving phase patients (20 ± 2% and 9.1 ± 4%, respectively), who in turn exhibited higher CCR9 expression than cells from patients in active phase. The naïve T cell population (CD3+ CD45RA+) was decreased upon HEV infection (29 ± 4% in AVH-E vs. 53.1 ± 3.2% in HC; P=0.05); however, the CD11a high subpopulation within CD4+ CD45RA+ cells was increased in both AVH-E (6.1%) and resolving phase (7.7%) patients. Gene ontology analysis suggested that during AVH-E infection, there is in CD4+ T cells an activation of genes involved in pro-inflammatory responses. Additional RT-PCR analysis confirmed that in cells from AVH-E patients, there is an increased expression of CCR5, CCR9, CXCR3, CXCR4, STAT1, IRF-9, IFN-α, and TNF-α, together with a down-regulation of IL-2, SOCS3, and IL-10, with respect to cells from resolving phase patients. CONCLUSIONS: Our findings suggest the involvement of a circulating CD45RA+ CD11a high population with CCR5 expression in the pathogenesis processes of AVH-E. The obtained results help to understand the underlying inflammatory process occurring in HEV infection, which can lead to either resolution or immunopathology.

Gene expression signatures of peripheral CD4+ T cells clearly discriminate between patients with acute and chronic hepatitis B infection.

UNLABELLED: CD4+ T and regulatory T cells (Tregs) seem to play a key role in persistence of hepatitis B virus (HBV) infection. However, the molecular events by which Tregs exert their modulatory activity are largely unknown. The transcriptional profiles of CD4+ T cells of healthy controls (HCs) and patients affected by acute hepatitis B (AVH-B) or chronic hepatitis B (CHB) infection were established using a custom expression array consisting of 350 genes relevant for CD4+ T cell and Treg function. These studies were complemented by real-time reverse-transcription polymerase chain reaction. Peripheral blood mononuclear cells (PBMCs) were also analyzed for the presence of Tregs, which were more abundant in the acute stage of the disease (7%) than in HCs and CHB infection (HCs versus AVH-B, P = 0.003; AVH-B versus CHB, P = 0.04). One hundred eighteen genes (34%) intrinsically differentiate HBV-infected patients from HCs. Using gene ontology, we identified T cell receptor signaling and clusterization, mitogen-activated protein kinase kinase signaling, cell adhesion, cytokines and inflammatory responses, cell cycle/cell proliferation, and apoptosis as the most prominent affected modules. A higher expression of CCR1, CCR3, CCR4, CCR5, and CCR8 was seen in AVH-B than in CHB-infected patients and HCs. Annotation of the interconnected functional network of genes provided a unique representation of global immune activation during acute infection. Almost all genes were down-regulated in patients with CHB infection. CONCLUSION: The fingerprints enable clear discrimination between patients suffering from AVH-B or CHB infection. The observed profiles suggest accumulation of effector T cells with a potential role in necro-inflammation during the acute stage. Subsequent down-regulated effector functions support the hypothesis of suppressed CD4+ effector T cells favoring viral persistence in the chronic infection stage.

Signatures of human regulatory T cells: an encounter with old friends and new players.

BACKGROUND: Naturally occurring CD4+ CD25+ regulatory T cells (TReg) are involved in the control of autoimmune diseases, transplantation tolerance, and anti-tumor immunity. Thus far, genomic studies on TReg cells were restricted to murine systems, and requirements for their development, maintenance, and mode of action in humans are poorly defined. RESULTS: To improve characterization of human TReg cells, we compiled a unique microarray consisting of 350 TReg cell associated genes (Human TReg Chip) based on whole genome transcription data from human and mouse TReg cells. TReg cell specific gene signatures were created from 11 individual healthy donors. Statistical analysis identified 62 genes differentially expressed in TReg cells, emphasizing some cross-species differences between mice and humans. Among them, several 'old friends' (including FOXP3, CTLA4, and CCR7) that are known to be involved in TReg cell function were recovered. Strikingly, the vast majority of genes identified had not previously been associated with human TReg cells (including LGALS3, TIAF1, and TRAF1). Most of these 'new players' however, have been described in the pathogenesis of autoimmunity. Real-time RT-PCR of selected genes validated our microarray results. Pathway analysis was applied to extract signaling modules underlying human TReg cell function. CONCLUSION: The comprehensive set of genes reported here provides a defined starting point to unravel the unique characteristics of human TReg cells. The Human TReg Chip constructed and validated here is available to the scientific community and is a useful tool with which to study the molecular mechanisms that orchestrate TReg cells under physiologic and diseased conditions.

Expression and functional influence of cellular retinoic acid-binding protein II in renal cell carcinoma.

INTRODUCTION: Retinoic acid (RA) and its derivates possess antiproliferative and tumor-suppressive abilities and are successfully used in the treatment of various malignancies. However, in metastatic renal cell carcinoma (RCC), its application did not meet first expectations. As the exact mechanisms of RA action and especially the role of the cellular retinoic acid-binding proteins (CRABP) still remain unclear, we studied the expression of CRABP-II and its potential influence on RA response in RCC. MATERIALS AND METHODS: We used the real-time RT-PCR methodology to investigate CRABP-II expression in 12 RCC samples and corresponding normal kidney tissue. Moreover, CRABP-II was cloned and overexpressed in CAKI-2 RCC cells. CRABP-II (un)transfected CAKI-2 cells were stimulated with all-trans RA (ATRA) and 9-cis RA, and their antiproliferative effects were evaluated using 3H-thymidine-proliferation assays. RESULTS: Using RPS9 and RPLP0 to normalize its expression, the median tumor/kidney ratio for CRABP-II expression was 0.16 and 0.12, respectively. Using proliferation assays, CRABP-II overexpressing CAKI-2 cells did not exhibit a significant change in RA sensitivity, but appeared to be less sensitive toward RA-stimulation compared to CAKI-2 cells expressing naturally low levels of CRABP-II (maximum difference, 59% at 3 microM ATRA). CONCLUSIONS: We were able to demonstrate a downregulation of CRABP-II expression in primary RCC tumor samples compared to the corresponding normal kidney tissue. However, CRABP-II overexpression in CAKI-2 RCC cells did not significantly influence RA associated antiproliferative actions. Further experiments are necessary to define the exact role of CRABP-II and its downregulation in RCC including its influence and dependence on other molecules involved in RA signalling and metabolism.

Cellular retinoic acid binding protein I: expression and functional influence in renal cell carcinoma.

Despite the known anti-proliferative and tumor-suppressive effects seen with retinoic acid (RA), treatment of metastatic renal cell carcinoma (RCC) failed to meet the initial expectations. As the exact mechanisms of action of RA and especially the role of the cellular RA binding proteins (CRABP) have not been elucidated yet, we investigated the expression of CRABP-I and its potential influence on RA response in RCC. Real-time RT-PCR analysis disclosed a significant lack of CRABP-I expression in four RCC cell lines and 12 primary RCC samples; in contrast, high expression levels were found in the respective adjacent normal kidney tissue. To further investigate the impact of CRABP-I on RA response in RCC, A-498 RCC cells were employed as a cellular model system. CRABP-I was stably transfected into A-498 cells which consequently displayed substantial resistance to all-trans (ATRA) and 9-cis RA compared to vector controls lacking CRABP-I. Comparison of gene expression profiles of ATRA-treated CRABP-I-expressing A-498 cells and vector controls revealed specific regulation of 54 of approximately 20,000 genes tested on a selected human CodeLink UniSet Bioarray, with a prominent modulation of genes involved in transcriptional control, signaling, apoptosis, cell cycle regulation and metabolism. The genetic changes reported here contribute to a better understanding of the role of RA in RCC. They also provide new insights into CRABP-I-mediated signaling and gene expression.