A Study of the Technique of Western Blot for Diagnosis of Lyme Disease caused by Borrelia afzelii in China

Objective To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. Methods FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and irnmunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples （159 patients with Lyme disease and 292 controls）, all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. Results Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively.

Hepatitis B virus induces expression of cholesterol metabolism-related genes via TLR2 in HepG2 cells

AIM:To investigate whether hepatitis B virus(HBV) exacerbates hepatic cholesterol accumulation,and explore the underlying mechanisms.METHODS:HepG2 cells were infected with adenovirus(Ad) containing 1.3-fold overlength HBV genome.Realtime polymerase chain reaction and Western blotting were used to measure mRNA and protein expression of target genes.Cholesterol accumulation was measured by fluorescence microscopy.Cell toxicity due to Ad-HBV treatment was determined by the mitochondrial tetrazolium assay.The protein levels of toll-like receptors(TLRs) were determined by Western blotting.RESULTS:Ad-HBV increased hepatic cholesterol accumulation and enhanced the mRNA and protein levels oflow-density lipoprotein receptor(LDLR) and 3-hydroxy3-methylglutharyl-coenzyme A reductase(HMGCoAr) mRNA and protein expression in HepG2 cells.In addition,these inductive effects were partly offset by suppressing TLR2 expression levels by small interfering RNA in HepG2 cells.CONCLUSION:Ad-HBV increases LDLR and HMGCoAr expression,resulting in exacerbated cholesterol accumulation in HepG2 cells,which was mediated via the TLR2 pathway.

Effect of HCMV IE1 Protein on Cytokines Secretion and Apoptosis of Macrophages

Objective： To investigate the effect of human cytomegalovirus （HCMV） IE1 protein on the secretory activity and apoptosis of macrophages. Methods： The eukaryotic expression vector pEGFP-C1/IE1 was used to transfect THP-1-macrophages. 48 h after transfection, the expression and localization of GFP or GFP-IE1 was observed under fluorescent microscope. The levels of IL-1β and TNF-α in the culture media were examined by ELISA, and the mRNA expression of them was analyzed by RT-PCR. Cell undergoing apoptosis were determined by flow cytometry using the propidium iodide （PI） staining method. The data were analyzed by SPSSI3.0. Results： As observed under fluorescent microscope, the expressions of GFP-IEI and GFP by plasmid pEGFP-C1/IE1 or pEGFP-C1 in THP-1-macrophages could be found in nuclei or whole cells. Conclusion： As demonstrated by RT-PCR and ELISA, mRNA and protein expressions of IL-1β and TNF-α and promotes apoptosis in THP-1-macrophages.

Hepatitis B Virus X Protein Up-regulates TNF-α and IL-1β Secretion of Macrophages

Objective： To provide the experimental basis for further studying the molecular transformation mechanism of Hepatitis B virus （HBV） X protein （HBx） on hepatocellular carcinoma. Methods： Reconstructed plasmid pcDNA3.1（＋）-HBx was transfected into THP-1 macrophages. Expression of HBx was assayed in macrophages lysate by Western-blotting, and TNF-α and IL-1β contents were detected respectively by ELISA. All the data were analyzed by SPSS13.0. Results： In THP-lmacrophages, the pcDNA3.1（＋）-HBx plasmid expressed HBx with a molecular weight of about 17 KDa demonstrated by Western-blotting. The secreted TNF-α and IL-1β from macrophages were determined by ELISA, the results from analysis of all groups showed as following： control group was different from LPS group and pcDNA3.1（＋） group （P〈0.01）, and so was pcDNA3.1（＋）-HBx group; but there was no obvious difference between pcDNA3.1（＋） group and LPS group （P〉0.05）, all of which indicated that transient overexpression of HBx enhanced LPS-induced production of TNF-α and IL-1β by macrophages. Conclusion： Transient overexpression of HBx up-regulates LPS-induced TNF-t~ and IL-113 secretion of macrophages.

Transient over-expression of human papillomavirus type 16 E6 protein down-regulate the secretion of TNF-αor IL-1β LPS-induced from macrophages

In order to provide the experimental basis for the further studies on the oncogenic mechanism of the E6 protein from human papillomavirus type 16(HPV16),the eukaryotie expression vector pcDNA3.1(-)/E6 was used for the study on the effect of E6 protein to influence the secretory activity of LPS-indueed THP-1-macrophages,and the reconstructed plasmid pcDNA3.1(-)/E6 was transfected into THP-1-maerophages.The expression of E6 gene was assayed in macrophage lysates by using Western blot analysis and the level of TNF-αor IL-1βwas examined by ELISA.All of data were analyzed by SPSS12.0.As demonstrated by Western blot analysis,the expression of E6 protein with a molecular weight of about 18 kDa by plasmid pcDNA3.1(-)/E6 in THP-1-macrophages could be detected.Howev- er,as demonstrated by ELISA assay,the level of TNF-αor IL-1βin lysates of THP-1-macrophages showed an obvious difference between the pcDNA3.1(-)/E6 group and the LPS control group or the pcDNA3.1(-)control group(P<0.01),but no significant difference existed between pcDNA3.1(-) control group and LPS control group(P>0.05).All these results illustrate that the transient over-ex- pression of HPV6 E6 protein reduces the production of TNF-αand IL-1βinduced by LPS in THP-1-mac- rophages.

HPV16 E6蛋白与hDaxx的相互作用及其对HeLa细胞凋亡的影响(英文)

Objective: To study the interaction of human papillomavirus type 16 (HPV16 E6) protein and human death domain associated protein (hDaxx) and its effect on apoptosis of HeLa cells to provide the experimental basis for exploring the oncogenic mechanism of HPV16 E6 protein. Methods: Recombinant vector of pGADT7/E6 or pGBKT7/hDaxx was con- structed. The interaction of E6 protein and hDaxx was detected by yeast two-hybrid system. Their expression in yeast was detected by Western blotting. The eukaryotic plasmids of E6 and hDaxx were co-transfected into HeLa cells. Apoptosis was induced by 5-FU. The apoptotic rate was measured by flow cytometry (FCM). Results: E6 protein had intracellular interaction with hDaxx. The apoptotic rate was rising with the increase in the transfection quantity of pcDNA3.1 (-) / hDaxx in pcDNA3.1 (-) /E6 and pcDNA3.1 (-) / hDaxx co-transfected cells. The difference was significant ( P < 0. 01). Conclusion: There is intracellular interaction between HPV16 E6 protein and hDaxx. The over-expression of hDaxx can increase the sensitivity of E6 protein positive HeLa cells to 5-FU. The effect was in a dose dependent manner. HPV16 E6 protein inhibited the apoptosis of HeLa cells by interacting with hDaxx.

Apoptosis of macrophages induced by human papollimavirus tgpes 18 E2 protein

Objective To investigate the effect of over-expression of E2 protein GFP-E2,GFP-TAD(N-extremity domain of HPV18 E2)and GFP-DBD(C-extremity domain of HPV18 E2)fusion proteins on the apoptosis of macrophages in uterine cervix cancer was studied.Methods TAD or DBD gene was amplified by PCR from pEGFP-C1/HPV18 E2,and cloned into pEGFP-C1 vector.Then the subclone of pEGFP-C1/TAD or pEGFP-C1/DBD was screened respectively.Forty-eight hours after transfection of pEGFP-C1/HPV18 E2,pEGFP-C1/TAD,pEGFP-C1/DBD or pEGFP-C1 into macrophages,the localization or expression of them was analyzed by fluorescent microscope or western blot,respectively.The apoptotic rate of macrophages was detected by flow cytometry.Results The plasmid of pEGFP-C1/TAD or pEGFP-C1/ DBD was constructed respectively.Macrophages transfected with different plasmid,over-expression of GFP-E2 or GFP-TAD fusion protein up-regulated apoptotic rate of macrophages.Furthermore,the effect of GFP -TAD was more powerful than GFP-E2.But the GFP-DBD over-expressed had not the same effects.Conclusion Over-expression of GFP-E2 or GFP-TAD fusion protein could induce apoptosis of macrophages.

mTOR regulates TLR-induced c-fos and Th1 responses to HBV and HCV vaccines

Although IL-12 plays a critical role in priming Th1 and cytotoxic T lymphocyte(CTL) responses, Toll-like receptor(TLR) signaling only induces low amounts of IL-12 in dendritic cells and macrophages, implying the existence of stringent regulatory mechanisms. In this study, we sought to uncover the mechanisms underlying TLR-induced IL-12 expression and the Th1 response. By systemic screening, we identified a number of protein kinases involved in the regulation of TLRinduced IL-12 expression. In particular, PI3 K, ERK, and m TOR play critical roles in the TLR-induced Th1 response by regulating IL-12 and IL-10 production in innate immune cells. Moreover, we identified c-fos as a key molecule that mediates m TOR-regulated IL-12 and IL-10 expression in TLR signaling. Mechanistically, m TOR plays a crucial role in c-fos expression, thereby modulating NFκB binding to promoters of IL-12 and IL-10. By controlling the expression of a special innate gene program, m TOR can specifically regulate the TLR-induced T cell response in vivo. Furthermore, blockade of m TOR by rapamycin efficiently boosted TLR-induced antigen-specific T and B cell responses to HBV and HCV vaccines. Taken together, these results reveal a novel mechanism through which m TOR regulates TLR-induced IL-12 and IL-10 production, contributing new insights for strategies to improve vaccine efficacy.