[Expression, purification and polyclonal antibody preparation for a novel gene BC097361].

OBJECTIVE: To express and purify of the BC097361 recombinant protein, and to prepare the BC097361 specific rabbit polyclonal antibody. METHODS: BC097361 cDNA was ligated into the prokaryotic expressive vector pET-32a (+), and the resulting plasmid was transformed into E.coli BL21 (DE3). The protein expression was induced with IPTG and the protein was analyzed with SDS-PAGE and western blotting. The expressed product was purified using Ni+ affinity column chromatography.Then the purified pET-32a (+) -BC097361 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by Western blot and ELISA. RESULTS: The BC097361 fusion protein was highly expressed.The protein was mainly in the inclusion body. ELISA indicated the titer of polyclonal antibody more than 1:320000. The high specificity was confirmed with Western blot. CONCLUSIONS: The recombinant BC097361 fusion protein and the BC097361 specific polyclonal antibody will be valuable tools for the investigation on the biological function of BC097361.

[Expression of intrahepatic CD4+ CD25+ regulatory T cells in patients with chronic hepatitis B].

OBJECTIVE: To investigate the expression and distribution of intrahepatic CD4+ CD25+ regulatory T cells in immuno-tolerant and immuno-clearance phase of patients with chronic hepatitis B. METHODS: The expression of FoxP3 was detected in 19 cases of immuno-tolerant phase and 12 cases of immuno-clearance phase by immunohistochemistry. The relation between the intrahepatic expression of FoxP3 and the clinicopathological features were analyzed. RESULTS: The positive signal of FoxP3 is located in nuclear of lymphocyte and mainly aggregated in portal areas as well as occasionally scattered in hepatic sinusoids. The expression of intrahepatic FoxP3 in the group of immuno-tolerant phase was significantly increased than those in normal control (P < 0.01), and greatly decreased than those in immuno-clearance phase (P < 0.01). No correlation was observed among the expression of intrahepatic FoxP3, ALT, levels of HBV DNA, HBeAg positive, in patients of immuno-clearance phase, respectively. There were significant differences between immuno-tolerant phase and immuno-clearance phase age, ALT, TBIL, PTA, HBV-DNA and detection of HBeAg but not in sex and family history of HBV infection. CONCLUSION: CD4+ CD25+ regulatory T cells may play important roles in the clearance of HBV as well as in liver inflammation and injury during chronic HBV infection.

[Detection of CD4+CD25+ regulatory T cells in liver tissues of fibrosing cholestatic hepatitis after liver and kidney transplantation].

OBJECTIVES: To study the expression and distribution of CD4+CD25+ regulatory T cells (Treg) in liver tissues of patients with fibrosing cholestatic hepatitis (FCH) after liver and kidney transplantation and to investigate their roles in the pathogenesis of FCH. METHODS: Liver biopsy specimens from five patients with FCH were studied histopathologically. A specific marker for CD4+CD25+ regulatory T cells in those specimens was detected with anti-FOXP3 monoclonal antibody by immunohistochemistry. Apoptoses of hepatocytes were detected with in situ apoptosis detection TUNEL kit. RESULTS: Fibrosis in portal and around portal areas, cholestasis in some of the hepatocytes and canaliculi, widespread ballooning and ground-glass appearance of liver cells, and positivity of HBsAg and HBcAg and Pre-S1 protein were seen in the livers of all cases. The positive signal of FOXP3 was located in the cytoplasm of lymphocytes and the positive cells were mainly aggregated in the portal areas as well as occasionally appearing in the hepatic sinusoids. There were many more apoptotic hepatocytes near the portal areas. CONCLUSION: Fibrosing cholestatic hepatitis has specific pathological characteristics which might be caused by high expressions of FOXP3 in liver tissues.

[Pathology of AIDS-related lymphadenopathy and its relationship to expression of CD4 + CD25 + regulatory T cells in lymphoid node tissue].

OBJECTIVE: To observe the pathology of AIDS-related lymphadenopathy and its relationship to the expression and distribution of CD4 + CD25 + regulatory T cells in lymphoid node tissue. METHODS: Totally 22 biopsy and 13 autopsy lymphoid node tissues from HIV-positive patients were examined under microscopy and pathological staging was performed. Specific marker for CD4 + CD25 + regulatory T cells in lymphoid node tissue was detected with anti-Foxp3 monoclonal antibody by immunohistochemistry. RESULTS: Among all the 35 specimens, 5, 4, 14, and 12 specimens were histopathologically staged from 1 to 4, respectively. FoxP3 were detected in all lymphoid node tissues. The distribution of FoxP3-positive lymphocytes were mainly in intermediate zone of follicle and cortical area in stages 1 and 2. The counts of FoxP3-positive lymphocytes remarkably decreased in stages 3 and 4, following depletion of lymphocytes. CONCLUSIONS: CD4 + CD25 + regulatory T cells exist in lymphoid node tissue of patients with HIV infection. Their amounts decrease or deplete along with the progression of AIDS-related lymphadenopathy.

[Effects of traditional Chinese medicine on CD4 + T cell counts and HIV viral loads during structured treatment interruption in highly active antiretroviral therapy].

OBJECTIVE: To explore the impacts of traditional Chinese medicine (TCM) on CD4 + T cell counts and human immunodeficiency virus (HIV) viral loads during the course of structured treatment interruption (STI) in highly active antiretroviral therapy (HAART). METHODS: Nineteen HIV/ADIS patients were treated for 14 months as follows: initiated with zidovudine/lamivudine + efavirdine for 6 months, then discontinued the therapy and treated with TCM instead for 2 months. HAART was then reinitiated for another 3 months, and then discontinued and replaced with TCM for another 3 months. The changes of CD4 + T cell counts and HIV viral loads were measured. RESULTS: During the first STI of HAART, 43.8% of patients had no viral rebounds one month later, and 62.6% had stable or increased immune functions; 18.8% had no viral rebounds two months later, and 43.8% had stable or increased immune functions. Changes of viral loads were not significantly different between these two months (P = 0.097), while CD4 + T cell counts significantly decreased two months later compared with one month later (P = 0.043). During the second STI of HAART, 33.3% of patients had no viral rebounds one month later, and 64.3% had stable or increased immune functions; 13.3% had no viral rebounds 3 months later and 46.6% had stable or increased immune functions. Changes of viral loads had significant difference (P = 0. 017), while CD4 + T cell counts at month 12 elevated significantly compared with the baseline (P = 0.014). CONCLUSIONS: TCM can suppress the viral rebounds during STI-HAART, maintain immune functions. However, this effect may decrease along with the prolongation of STI-HAART.