CAR regulates epithelial cell junction stability through control of E-cadherin trafficking.

CAR (Coxsackie and Adenovirus Receptor) is the primary docking receptor for typeB coxsackie viruses and subgroup C adenoviruses. CAR is a member of the JAM family of adhesion receptors and is located to both tight and adherens junctions between epithelial cells where it can assemble adhesive contacts through homodimerisation in trans. However, the role of CAR in controlling epithelial junction dynamics remains poorly understood. Here we demonstrate that levels of CAR in human epithelial cells play a key role in determining epithelial cell adhesion through control of E-cadherin stability at cell-cell junctions. Mechanistically, we show that CAR is phosphorylated within the C-terminus by PKCδ and that this in turn controls Src-dependent endocytosis of E-cadherin at cell junctions. This data demonstrates a novel role for CAR in regulating epithelial homeostasis.

Hepatitis B surface antigen: relation to hepatitis B replication parameters in HBeAg-negative chronic hepatitis B.

BACKGROUND & AIMS: Translation of HBsAg depends on transcription of the appropriate mRNAs from cccDNA, but its relation to other hepatitis B virus (HBV) replication parameters is not known, inasmuch as integrated sequences of HBV-DNA may also contribute to its serum levels, especially in HBeAg-negative chronic hepatitis B (CHB) patients. METHODS: We investigated HBsAg serum levels, its hepatocellular expression, and their relationship to HBV replicative- and host-response parameters before treatment in 54 HBeAg-negative CHB patients and in 15 of them after 40.1±33.3months of virological response on oral antiviral (NUC) therapy also. Liver cccDNA and HBV-DNA quantitation, HBsAg- and HBcAg-immunostaining were performed in the same needle biopsy material, while serum HBsAg and HBV-DNA levels were measured in samples drawn on the day of liver biopsy. RESULTS: In untreated patients, serum HBsAg correlated positively with HBsAg-positive hepatocytes/mm(2) (p=0.003) and weakly with serum HBV-DNA, but not with cccDNA, liver HBV-DNA, HBcAg-positive hepatocytes/mm(2), or ALT. cccDNA correlated significantly with liver HBV-DNA (p<0.00001), ALT (p=0.001), and serum HBV-DNA levels (p=0.012) but not with liver HBsAg or HBcAg. Antiviral therapy decreased serum HBsAg levels by 79.6% (p=0.012) and liver HBV-DNA by 84.4% (p=0.026) in paired comparisons and, as expected, significantly decreased serum HBV-DNA and ALT levels, but not cccDNA. CONCLUSIONS: In untreated HBeAg-negative CHB, serum HBsAg levels reflect liver HBsAg, but not cccDNA or liver HBV-DNA, suggesting that they are not solely dependent on the replicative cycle of HBV. Effective NUC therapy for 3.34 years significantly lowers serum HBsAg and liver HBV-DNA, but not cccDNA.

Core mutations in patients with acute episodes of chronic HBV infection are associated with the emergence of new immune recognition sites and the development of high IgM anti-HBc index values.

Acute exacerbations in HBeAg negative patients with chronic hepatitis B virus (HBV) infection are invariably associated with concurrent increases in the index of IgM class antibodies against the core protein (anti-HBc) of the virus. This study aimed to investigate whether this was related to the clearance of variants from the quasispecies pool and the appearance of new ones, with aminoacid substitutions in well recognized B-cell epitopes. In this study, 5 HBeAg negative patients (A to E) with 13 sequential serum samples (A1-A2, B1-B2-B3, C1-C2, D1-D2-D3, E1-E2-E3) were investigated after amplification of the entire core encoding region followed by cloning/sequencing studies. The sequences at different time points were compared with those from a single HBeAg positive patient with no apparent acute exacerbations. The results from sequence comparison showed that virus variants emerged in all (A2, B3, C2, D3, E2, and E3) but two (B2 and D2) subsequent sera with amino-acid substitutions affecting B-cell epitopes. It is concluded that the rise in the values of IgM anti-HBc may be attributed to the alteration of the antigenic epitopes leading to new antibody production in the majority of the cases. However, it appears that increases in IgM anti-HBc indexes in a few cases may relate to other possible mechanisms which are discussed.

Restricted quasispecies variation following infection with the GB virus B.

The extent of genetic variability following acute infection of tamarins with GB virus B (GBV-B) is not known. In this study we attempted to define the quasispecies variation of GBV-B 17 days post-infection, by PCR amplification of GBV-B RNA extracted from serum and liver. Cloning followed by sequencing revealed a small number of changes in the three regions studied, namely the 5' untranslated region, E2 and NS3. Moreover, there was no region of high amino acid variability in E2, akin to hypervariable region 1 of hepatitis C virus. This was further confirmed by analysing sequences from two additional animals obtained at a similar time point post-infection. Nevertheless, it was apparent that different variants with one or two amino acid substitutions in the region studied had been selected when comparing the sequences from the three animals. This restricted sequence variation of GBV-B during acute hepatitis may explain the infrequent progression of the infection to a chronic stage.

Citrate enhances in vitro metastatic behaviours of PC-3M human prostate cancer cells: status of endogenous citrate and dependence on aconitase and fatty acid synthase.

Prostate is a unique organ that produces and releases large amounts of citrate. This is reduced significantly in cancer and it is possible that citrate is (re)taken up and used as a metabolite to enhance cellular activity. The main purpose of this study was to determine how cytosolic citrate might affect in vitro metastatic cell behaviours (lateral motility, endocytosis and adhesion). Normal (PNT2-C2) and metastatic (PC-3M) human prostate cancer cells were used in a comparative approach. As regards intermediary metabolic enzymes, aconitase and fatty acid synthase, already implicated in prostate cancer, were evaluated. The level of intracellular citrate was significantly higher in PNT2-C2 cells under both control conditions and following preincubation in extracellular citrate. Supply of exogenous citrate enhanced endocytosis, lateral motility, decreased cell adhesion of PC-3M cells but failed to produce any effect on normal cells. Real-time PCR measurements showed that the mRNA levels of mitochondrial and cytosolic aconitases and fatty acid synthase were significantly higher in PC-3M cells. Correspondingly, aconitase activity was also higher in PC-3M cells. Using cerulenin (an inhibitor of fatty acid synthase), oxalomalate and fluorocitrate (inhibiting aconitases), we investigated the dependence of citrate-induced down-regulation of cellular adhesion on aconitase and fatty acid synthase activities. It was concluded: (1) that strongly metastatic PC-3M cells stored less/utilised more cytosolic citrate than the normal PNT2-C2 cells and (2) that cancer cells could metabolise cytoplasmic citrate via aconitase and fatty acid synthase to enhance their metastatic behaviour.