Differential expression of VEGF ligands and receptors in prostate cancer.

BACKGROUND: Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer. METHODS: The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer. RESULTS: Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue. CONCLUSIONS: These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer.

Down-regulation of intra-hepatic T-cell signaling associated with GB virus C in a HCV/HIV co-infected group with reduced liver disease.

BACKGROUND & AIMS: Studies have shown that GB virus C (GBV-C) infection leads to reduced liver disease in hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infection. Considering that the underlying mechanism(s) are unknown, we aim to identify differential gene and protein expression associated with GBV-C in HCV/HIV co-infection that may be responsible for reduced liver disease. METHODS: Liver, peripheral blood mononuclear cells (PBMCs), and plasma samples were collected from 43 HCV/HIV patients. Plasma was tested for GBV-C RNA by RT-PCR with NS5B gene primers. A microarray was performed on the liver and RT-qPCRs on the liver/PBMC samples. Hepatic protein expression was measured by immunohistochemistry. RESULTS: Sixteen out of 43 patients had GBV-C RNA. GBV-C was associated with reduced hepatic fibrosis (p=0.005) and inflammation (p=0.007). The microarray analysis of the liver samples (n=10) showed down-regulation of genes critical to intra-hepatic T-cell signaling associated with GBV-C. Quantitative RT-PCR of the liver samples (n=13) confirmed the down-regulation of lymphocyte-specific protein tyrosine kinase (LCK) (p=0.02) and docking protein 2 (DOK2) (p=0.04). No differences in the expression levels of these genes were observed in PBMCs (n=22) according to the GBV-C status. The hepatic expression of the LCK protein, measured by immunohistochemistry (n=36), was decreased in CD3-positive T-cells within portal tracts associated with GBV-C (p=0.003). This remained significant in multivariate analysis controlling for hepatic fibrosis and inflammation (p=0.027). No differences were observed in plasma cytokine concentrations (n=25) or ex-vivo peripheral T-cell responses (n=13) versus GBV-C status. CONCLUSIONS: GBV-C infection is associated with down-regulation of critical genes involved in intra-hepatic T-cell signaling in HCV/HIV co-infection. This may be relevant to the pathogenesis of reduced HCV-related liver disease in HIV co-infection.

Virus-specific T-cell immunity correlates with control of GB virus B infection in marmosets.

GB virus B (GBV-B) is a hepatotropic virus that is closely related to hepatitis C virus (HCV). GBV-B causes acute hepatitis in infected marmosets and tamarins and is therefore a useful small-animal model for the study of HCV. We investigated virus-specific T-cell responses in marmosets infected with GBV-B. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay responses in the peripheral blood of two marmosets were assessed throughout the course of GBV-B infection. These T-cell responses were directed against the GBV-B nonstructural proteins 3 (NS3), 4A (NS4A), and 5B (NS5B), and their appearance was temporally associated with clearance of viremia. These marmosets were then rechallenged with GBV-B at least 3 months after clearance of the primary infection to determine if the animals were protected from reinfection. There was no detectable viremia following reinfection, although a sharp increase in T-cell responses against GBV-B proteins was observed. Epitope mapping of T-cell responses to GBV-B was performed with liver and blood samples from both marmosets after rechallenge with GBV-B. Three shared, immunodominant T-cell epitopes within NS3 were identified in animals with multiple common major histocompatibility complex class I alleles. IFN-gamma ELISPOT responses were also detected in the livers of two marmosets that had resolved a primary GBV-B infection. These responses were high in frequency and were directed against epitopes within GBV-B NS3, NS4A, and NS5B proteins. These results indicate that virus-specific T-cell responses are detectable in the liver and blood of GBV-B-infected marmosets and that the clearance of GBV-B is associated with the appearance of these responses.

Defining target antigens for CD25+ FOXP3 + IFN-gamma- regulatory T cells in chronic hepatitis C virus infection.

The mechanism behind the apparent lack of effective antiviral immune responses in chronic hepatitis C virus (HCV) patients is poorly understood. It remains unclear if natural regulatory T cells (Treg) contribute to the induction and maintenance of HCV persistence. We herein report for the first time that CD25(high)IFN-gamma(-)FOXP3(high) Tregs can be rapidly induced by culturing peripheral blood mononuclear cells (PBMCs) of HCV-positive patients with HCV protein-derived peptides. The HCV-specific Tregs, generally CD4(+)CD45RO(+), did not proliferate in response to HCV peptide and failed to produce interferon (IFN)-gamma, in distinct contrast to antiviral effector cells. Stimulation of healthy donor PBMCs with HCV peptides did not result in CD25 and FOXP3 upregulation above non-antigen background. To further investigate the antigen specificity of these potentially disease-associated natural Tregs, CD25(+) cells were isolated from PBMCs, labeled with carboxyfluorescein diacetate succinimidylester and added back to CD25-depleted PBMCs, and the co-cultures were then stimulated with individual peptides derived from the HCV core protein. We found that the actual peptide that can stimulate Treg varied between patients, but within any given subject only a small number of the peptides were able to stimulate Treg, suggesting the existence of dominant Treg epitopes. Although functional experiments for these peptides are ongoing in our laboratory, data presented here suggests that HCV-specific natural Tregs are abundant in infected individuals, in contrast to the extremely low frequency of anti-HCV effector T cells, supporting the view that natural Treg may be implicated in host immune tolerance during HCV infection.

Characterization of HCV-specific Patr class II restricted CD4+ T cell responses in an acutely infected chimpanzee.

Resolution of hepatitis C virus (HCV) infection is associated with strong and sustained virus-specific CD4+ T cell responses. In this study, we investigated the evolution of functional T cell responses during acute infection of a chimpanzee and the longevity of these lymphocytes in blood and liver after resolution of infection. Viremia increased through the first 3 weeks of infection and then remained stable until the onset of T cell responses at weeks 6 and 8 postinfection. CD4+ T cells targeting nonstructural HCV proteins were detected in proliferation assays by week 6 postinfection, but they failed to produce interferon gamma (IFN-gamma). HCV-specific CD4+ and CD8+ T cells with the ability to produce IFN-gamma appeared at week 8 when a rapid 10-fold reduction in plasma viremia was first observed. This cytokine response persisted through to week 24 when infection apparently resolved. T cell lines targeting 3 CD4+ T cell epitopes and 1 CD8+ T cell epitope were derived from liver and their Patr major histocompatibility complex (MHC) restriction elements were identified. In retrospective studies performed on cryopreserved peripheral blood mononuclear cells (PBMCs) collected at various timepoints after infection, the onset of an IFN-gamma response measured against the class II restricted epitopes correlated with viral clearance. In conclusion, the characterization of the HCV epitopes and MHC class II restriction elements described here will facilitate a detailed comparison of CD4+ T cell function in animals with resolved and persistent infections.

HCV persistence and immune evasion in the absence of memory T cell help.

Spontaneous resolution of hepatitis C virus (HCV) infection in humans usually affords long-term immunity to persistent viremia and associated liver diseases. Here, we report that memory CD4+ Tcells are essential for this protection. Antibody-mediated depletion of CD4+ Tcells before reinfection of two immune chimpanzees resulted in persistent, low-level viremia despite functional intra-hepatic memory CD8+ Tcell responses. Incomplete control of HCV replication by memory CD8+ Tcells in the absence of adequate CD4+ Tcell help was associated with emergence of viral escape mutations in class I major histocompatibility complex-restricted epitopes and failure to resolve HCV infection.