Detection of hepatitis viruses in dendritic cells by in situ PCR.

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We have already reported the defective function of splenic dendritic cells in hepatitis B virus (HBV)-transgenic mouse (Tg), a murine model of human chronic HBV-carrier state. Moreover, a role of dysfunction of dendritic cells have been shown in human hepatitis C virus infection. However, the mechanism underlying the functional defects of dendritic cells could not be clarified in full detail and it is unknown whether dendritic cells are infected with hepatitic viruses or dendritic cells act as a vehicle for the replication of these viruses.In this context, existence of HBV-DNA in dendritic cells and peripheral blood mononuclear cells (PBMC) was examined in patients infected with HBV by PCR and in situ PCR hybridization. In order to optimize the experimental conditions, first we used mononuclear cells from spleen of HBV-Tg as positive controls and undertook a series of preliminary experiments. HBV-DNA was reproducibly detected in HBV-Tg spleen cells using this methods. HBV-DNA was visua … More lized in almost all of the nuclei of spleen cells from HBV-Tg.Next, dendritic cells and PBMC were obtained from patients with chronic hepatitis B. Dendritic cells were isolated from the peripheral blood following a 8 days enrichment procedure in GM-CSF and IL-4. DNA was extracted from dendritic cells and PBMC from patients with chronic hepatitis B, and PCR was done using PCR primers specific for HBV. HBV-DNA was detected in both dendritic cells and PBMC from patients with chronic hepatitis. However, HBV-DNA was not stably detectable in human dendritic cells and PBMC by this method because the structure of these cells were distorted during the manipulation of cells for in situ PCR methodology.Further study is needed to clarify the infectivity of dendritic cells by HBV in human HBV-carriers. The effect of existence of HBV in dendritic cells also need in situ PCR and immunological investigations. However, our data has provided a direct evidence that human peripheral blood dendritic cells is either infected with HEV or HEV is attached to dendritic cells in patients with chronic hepatitis B. Less