Objective To establish the ELISA method by using synthetic peptide for detection of IgM antibodies to human cytomegalovirus(HCMV).Methods According to HCMV PP150 sequence of amino acid and nucleotide,the peptide sequence was decided by computer with some program of software of protein.The ELISA method using synthetic peptide as antigen was developed and was applied to detected anti-HCMV IgM in sera of normal pregnant woman and habitual abortion woman.The HCMV DNA were detected by polymerase chain reaction(PCR).Results The positive rates of anti-HCMV IgM in different populations were as follows:9 17%(11/120) in normal pregnant woman,37 5%(24/64) in habitual abortion woman.The ELISA method have good speciality.The positive rate of HCMV DNA was significantly related to that of anti-HCMV IgM(P

BACKGROUND: Microglial cells are prominently involved in certain neurologic diseases such as Parkinson disease and Alzeheimer disease. In vitro primary culture is commonly used in studies on the functions of microglia.However, these classical culture methods have some defects including complex procedures and low out-put.OBJECTIVE: To establish a simplified high-output primary culture of microglia.DESIGN: An explorative experiment with microglial cells as the single sample.SETTING: Department of Neurology, Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The study was finished at the Central Laboratory of Union Hospital from April to October 2004. Microglial cells were obtained from 10 newborn(one day) male Kunming mice that were selected.METHODS: The author' s culture method was based on McCarthy method, we developed a new culture method and made some improvements,including the increased cell density for primary culture and nutritional deprivation. The microlglial cells were isolated with low-concentration trypsin-EDTA(ethylene diamine tetraacetic acid) digestion and immunochemically labeled with MAC-1 antibody, so as to measure the output and purity of microglia.MAIN OUTCOME MEASURES: ① Morphologic features of microglial cells, observed with inverted microscope; ② Purity and activity of microglia cultured with these two methods, were measured immunohistochemically.RESULTS: For microglia cultured with McCarthy method, the culture cycle was 20 days and the output was 2 × l05 cells per flask with a purity of 95% -97%. The new method shortened the culture cycle to 15 days and the output reached 1 × 106 cells per flask with a purity of 96-98%. Cell purity and activity had no significant difference between these two culture methods.CONCLUSION: The new method has a similar purity and activity with classical method; however, it may simplify procedures, shorten cycle, and increase output, and therefore can be a useful method for studies on microglia function and for nerve repair.

The expression of the cytokines IL-2, IL-10, TNF-alpha and their roles in mice with herpes simplex viral encephalitis (HSE) were studied. By using semiquantitative reverse transcription polymerase chain reaction (RT-PCR), the expressions of IL-2, IL-10 and TNF-alpha mRNA in control group, HSE group and acyclovir (ACV)-treated group were detected and the pathological changes of brain were observed. It was found that after HSV1 infection, the cerebral lesions of haemorrhage and necrosis in mice were observed under the microscopy, and the levels of IL-2, IL-10 and TNF-alpha were increased remarkably. After treatment with ACV after HSV1 infection, the cerebral lesions in mice were improved, the level of IL-2 maintained stable, IL-10 was increased consistently, and TNF-alpha was decreased significantly as compared with those in HSE group. In acute HSE, many cytokines are upregulated, including IL-2, IL-10 and TNF-alpha to eliminate virus and TH1 type response is dominant. In convalescence, there is a shift in the cytokine expression profile from TH1 profile to TH2 profile and the shift can inhibit the overexpression of immune response in animals. ACV has remarkable effects in the treatment of HSE.

Objective To explore the inhibitory effects of Tanshinone Ⅱ A on the radiationinduced microglia activation and the possible mechanism.Methods Microglia cells BV-2 were irradiated with 2,4,8,16,and 32 Gy doses or sham-irradiated in presence or absence of 1.0 μg/ml Tanshinone Ⅱ A for 12 h,respectively.The effects of Tanshinone Ⅱ A on radiation-induced pro-inflammatory cytokines were evaluated using real-time PCR.The expression level of NF-κB p65 in cytoplasm and nucleus was measured by using Western blot.Immunofluorescence staining and confocal microscopy analysis were applied to detect the expression of γ-H2AX and p65 post-irradiation.Results The microglia cells were activated at 16,32 Gy post-irradiation.Radiation-induced release of the pro-inflammatory cytokines in BV-2 cells was detectable after irradiation.Tanshinone Ⅱ A decreased radiation-induced release of proinflammatory cytokines(t=5.56,P ＜ 0.05).Furthermore,western blotting showed that Tanshinone Ⅱ A could attenuate the nuclear translocation of NF-κB p65 submit post-irradiation.Immunofluorescence staining showed that γ-H2AX foci formation while p65 translocation into nucleus post-irradiation.Conclusions Tanshinone Ⅱ A exerts anti-inflammatory properties by suppressing the transcription of proinflammatory cytokine genes that might be associated with NF-κB signaling pathway.It is postulated that irradiation causes immediate cellular reaction and DSB triggers the molecular response which leads to NFκB pathway activation.

Objective To explore the effect of SFI in radiation-induced mice brain injury after 20 Gy cranial radiation.Methods The mice were divided into three groups:(1) control group,(2) RT-only group:the whole brain was irradiated with a dose of 20 Gy,(3) RT and SFI group:SFI at 20 ml/kg/d from 4 weeks after 20 Gy cranial radiation theraty(CRT).Results Morris water maze test showed that the latency of the irradiated group was longer than control group and SFI improved the cognitive function of mice (t =6.34,6.70,P ＜0.05).The expression of TNF-α reached to the highest level at 3 h after irradiation,and then it decreased but got the second higher level again at 4 weeks after irradiation.The expression of IL-1 β reached to the highest level at 72 h after irradiation and decreased until 4 weeks after irradiation.SFI decreased both expressions of TNF-α (t =11.34,9.70,6.07,P ＜ 0.05) and IL-1 β (t =12.27,5.70,7.52,P ＜ 0.05).Immune florescence staining showed that SFI reduced the number of activated microglia (t =12.35,8.64,7.82,P ＜ 0.05)and inhibited the translocation of p65 of microglia after irradiation.Conclusions Findings suggest that SFI may decrease microglial activation and suppress the expression of TNF-α and IL-1β by inhibiting the translocation of NF-κB p65 and then attenuate irradiation-induced brain injury.

Objective To explore the inhibitory effects of Corilagin on the production of proinflammatory cytokines in microglia induced by radiation. Methods The cytotoxicity of Corilagin was measured by MTT assay. Microglia BV-2 cells were irradiated 0 or 32 Gy after pretreated with Corilagin for 12 hours. Realtime-PCR was used to detect the mRNA levels of inflammatory cytokines, such as IL-1β,TNF-α on several time-points. The content of nitric oxide (NO) was determined with nitrate reductase method. The translocation of NF-κB was measured by Western blot and immunocytochemical stain.Confocal microscopy was used to observe the expression of Iba-1 and Nemo. Results No cytotoxicity was detected on BV-2 cells with 1-10 μg/ml Corilagin. Iba-1 expression in microglia cells was activated by irradiation, the expression levels of inflammatory cytokines, such as IL-1β, TNF-α and NO were also elevated. Whereas, the production of IL-1 β, TNF-α in activated microglia cells was significantly inhibited with 5 μg/mL corilagin ( tIL-1β = 6. 341, tTNF-α = 3.41 1, tNO = 3. 134, P ＜ 0. 05 ). Corilagin significantly inhibited the expression of Nemo and the translocation of NF-κB p65. Conclusion Corilagin could inhibit the activation of irradiated microglia cells and down-regulate the expression of inflammatory cytokines, via inhibition of the NF-κB signaling pathway.

AIM:We examined the efficacy ofanti-L3T4 McAb in the T cell signaling pathway in treating ex-Derimental antoimmune cardiomyopathy in BALB/c mice,as a model of the autoimmune mechanism involved in human di-latedardiomyopathy(DCM).METHODS:ADP/ATP carrier peptides were used to induce autoimmune cardiomyopathy in BALB/c mice.Afier 3 months.anti-L3T4 McAb WaS administered to deplete CD4+ T ceHs in the mice.Real-time PCR were used to detect the expression of intracellular signaling molecules(p561ck,p59fyn and Zap-70)and cytokine production(IFN-r,IL-2 and IL-4)in T cells.The expression of CIM5 was determined by immunohistochemistry a-nalysis.RESULTS:Reduced expression of p561ck,p59fyn and Zap-70 and the reduced cytokine production of IFN-r, IL-2 and IL-4 in T cells of anti-IJ3T4-treated DCM mice were found.Also,the expression of CD45 in spleen T cells was significantiv decreased in the anti-L3T4-treated group.In contrast,immunization with irrelevant Ab did not protect the mice.the expression of T cell signaling molecules,CD45,and cytokine were not inhibited.CONCLUSION:Thesestudies provide direct evidenee that anti-L3T4 McAb can be all effective immunomodulator to T cell signal molecules and 8ubsequent cytokine production events in ADP/ATP carrier-induced DCM in BALB/c mice.

Objective To investigate the effect of recombinant plasmid pshRNA-DNMT3b on expression of DNMT3b mRNA and protein and on the proliferation of bladder cancer T24 cells,and research the function of DNMT3b in the process of bladder tumor formation.Methods There were three groups in this study,which are blank controller,HK and pshRNA-DNMT3b(24h,48h,72h),respectively.T24 cells were cultured routinely and transfected by the recombinant plasmids with lipfectamine 2000.The cells were detected by methods of RT-PCR,western blot and MTT.The varying level of DNMT3b mRNA and expression protein,and the conditions of cellular survival rate were observed.Results The recombinant plasmids were successfully transfected into T24 cell lines.The grey valHe of RT-PCR elctrophoretogram was analyzed by the software of Gel-pro analyzer,the rate of blank controller,HK and pshRNA-DNMT3b(24h,48h,72h),was (99.56±1.24)%,(99.12±1.35)%,(75.77±1.42)%,(44.69±1.05)%and(20.52±0.89)%,respectively.The analytical resuit of western blot image was(99.43±1.28)%,(98.90±1.31)%,(67.83±1.02)%,(43.43±1.05)%and(21.92±0.89)%.There was no statistically difference in survival between blank control and HK(P＞0.05).The group of pshRNA-DNMT3b and other two groups had statistical difference only at the 72th hour and the cell inhibitory growth rate only increase 0.45%.Conclusions The recombinant ptasmid pshRNA-DNMT3b can inhibit the expression of mRNA and protein of DNMT3b effectively.However,it has slight function on inhibiting cell proliferation.

The cytokine repertoire of ADP/ATP carrier-specific humoral immune responses and the cytokine-dependent anti-ADP/ATP carrier antibody IgG subclasses were examined in a cohort of ADP/ATP carrier-immunized BALB/c mice treated with anti-CD4 monoclonal antibody. Eighteen male BALB/c mice (6-8 weeks old) were randomized into 3 groups: dilated cardiomyopathy (DCM) group, DCM-tolerance (Tol) group and control group. The mice in DCM group were immunized with the peptides derived from human ADP/ATP carrier protein for 6 months and mice in the control group were sham-immunized, while the mice in DCM-Tol group were immunized with ADP/ATP carrier protein and anti-CD4 McAb simultaneously. Serum autoantibody against ADP/ATP carrier and IgG subclasses were measured by ELISA, intracellular cytokines IFN-gamma and IL-4 of Th cells were monitored with flow cytometry, and splenic T cell cytokines IFN-gamma, IL-2, IL-4 and IL-6 were detected by using real-time fluorescent quantitative PCR. The results showed that the autoantibody against ADP/ATP carrier was found in all mice in DCM group, and the antibody level, serum IgG1 and IgG2a subclasses, cytokines in T cells and Th cells were all elevated in DCM group, as compared with those in control group (P<0.01). On the other hand, in DCM-Tol group, the autoantibody level and contents of all the cytokines were significantly different from those in DCM group (P<0.01), and were close to those in control group. And the levels of IgG1, IgG2a, IgG2b and IgG3 were influenced, to varying degrees, by anti-CD4 McAb as compared with those in DCM group. All these four types of IgG subclasses were substantially decreased in DCM-Tol group as compared with DCM group. It is concluded that the treatment with anti-CD4 McAb could prevent the activation of T cells, reverse the abnormal secretion of cytokines and the imbalance between Th1/Th2 cell subsets and abnormal production of autoantibody against ADP/ATP carrier, and eventually avoid myocardial injuries.

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-alpha, IL-1beta and TGF-beta1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-kappaB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA1, 2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-alpha, IL-1beta, IL-6 and COX-2, and inhibit NF-kappaB nuclear translocation. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-beta1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflammatory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.