A micro cell culture method utilizing modified microscope slides for use in fluorescent antibody and enzyme immunoassays

Summary A technique for culturing small quantities of mammalian cells on modified microscope slides is described. The modified microscope slides were Bellco Glass, Inc., toxoplasmosis slides and the cell cultures used were early passage bovine embryonic lung cells and continuous cell lines of porcine and canine origins. The slide cell cultures were either uninfected or infected with selected viruses or the obligate intracellular protozoanEncephalitozoon caniculi for utilization in direct and indirect fluorescent antibody testing or in peroxidase antiperoxidase immunosorbant assays.     

Generation of the eosinophil chemotactic factor (ECF) from various cell types by melittin

The peptide melittin, the main constituent of bee venom is a potent stimulus for the generation of an eosinophil chemotactic factor (ECF) from human polymorphonuclear neutrophils, rat mast cells and rat peritoneal cells depleted in mast cells. Optimal EFC induction required a sublytic activation of the cells. With each cell type the kinetics of ECF generation were similar in that after an early rise in activity a steep fall off occurred at later times of incubation suggesting a mechanism of inactivation. The induction of ECF by melittin is increased in the presence of calcium. The polar portion of the melittin molecule (aminoacids 20–26) is responsible for the generation of the chemotactic activity. Other peptides of honey bee venom such as the mast cell degranulating peptide (MCD) or apamine do not initiate ECF release. It appears that melittin leads to ECF induction via the phospholipase A₂-arachidonic acid dependent pathway of cell activation. Our data suggests that the lipid mediator ECF can be obtained from phagocytes and mast cells thus indicating the interdependence of inflammatory reactions.     

Different patterns of the L-histidine decarboxylase (HDC) gene expression in mice resistant and susceptible to experimental cutaneous leishmaniasis

Objective and design: In the present study the experimental murine Leishmania major (L. major) infection model was used to investigate the role of histamine biosynthesis in cutaneous leishmaniasis.Subjects, treatment and methods: A novel RNase Protection Assay (RPA) was developed and applied for the assessment of L-histidine decarboxylase (HDC) gene expression in organs of resistant C57BL/6 and susceptible BALB/c mice after infection with L. major.Results: In the acute phase of infection a rapid but transient induction of HDC expression was observed in the infected lymph nodes of both strains correlating both temporally and spatially with parasite spread. The signal was present in the draining popliteal lymph nodes of both hosts, however, only susceptible mice known to be unable to control parasite dissemination showed induction of HDC in their distant periaortic lymph nodes as well. During the chronic phase of infection only the heavily parasitized organs of BALB/c mice showed high HDC gene expression.Conclusions: These data suggest that expression of the histamine-producing enzyme HDC in the decisive acute phase of leishmaniasis is not coupled with development of either appropriate Th1 or inadequate Th2 responses to L. major. We hypothesize however, that during the chronic phase of infection elevated HDC levels, possibly of mast cell origin, are associated with Th2-dominated responses and serious disease development.Received 12 May 2003; returned for revision 3 July 2003; returned for final revision 11 September 2003; accepted by M. Parnham 24 September 2003     

新生大鼠海马神经元原代培养方法的研究

目的 :研究和比较 3种不同培养神经元的方法 ,提供一种纯化效率和存活率均较高的培养技术。方法 :取新生SD大鼠的海马区 ,应用一般培养法、加阿糖胞苷法、加阿糖胞苷和神经生长因子法培养神经元 ,观察神经元的形态。用倒置显微镜观察和MTT比色分析检测神经元的存活率。用ABC免疫组化染色法 ,比较 3种不同培养方法在不同培养时间所获得神经元的纯度。结果 :加阿糖胞苷和神经生长因子培养组神经元生长良好 ,纯度和存活率均较高。结论 :加阿糖胞苷和神经生长因子培养神经元的方法具有简便可行 ,易于生长的优点 ,可作为神经元体外培养的良好模型     

树突状细胞负载肝癌抗原肽疫苗体外诱导特异性免疫学反应的研究

目的：研究树突状细胞(dendritic cell，DC)负载肝癌抗原肽EPVTKAEML体外诱导特异性CTL的能力及其抑癌效果。方法：用顺序特异引物聚合酶链反应技术(PCR—SSP)选择HLA—B7表型供者，从脾组织中分离、培养DC-EPVTKAEML特异性CTL。用^51Cr释放法检测CTL的杀伤活性，并用抗HLA-1分子单抗(mAb)进行杀伤抑制实验。结果：找到4例HLA-B7杂合子供者，用DC负载HLA-B7限制的抗原肽EPVTKAEML可诱导特异性CTL反应，对肝癌细胞HHCC有较强的杀伤作用。结论：DC负载抗原肽EPVTKAEML在体外可诱发较强的特异性免疫反应。     

CD4+CD56+ hematodermic neoplasms bear a plasmacytoid dendritic cell phenotype

CD4+CD56+ hematodermic neoplasms (HNs) with initial presentation in the skin are characterized by highly aggressive behavior and poor prognosis. Recent studies indicate that malignant cells, which are devoid of common T-, B-, NK-, and myeloid lineage markers, may be of plasmacytoid dendritic cell (pDC) origin. We undertook a study to assess the expression of several pDC-associated molecules on a series of 5 CD4+CD56+ HN cases. CD123 was expressed in all 5 cases, with some heterogeneity in individual cases. All but one case revealed fine membranous BDCA-2 staining of the dermal infiltrate. pDC-like phenotype of the malignant infiltrating cells was confirmed by costaining of BDCA-2+ cells with CD123 and CD4. MxA protein, representing the surrogate marker for lesional type I interferon activity, was expressed in 4 of 5 evaluated cases. Our findings further substantiate the putative pDC origin of CD4+CD56+ HNs.     

蛋白酶激活受体2激动剂对肥大细胞释放组胺的影响

目的 研究PAR-2激动剂(tc-LIGRLO-NH2与SLIGKV-NH2)和胰蛋白酶对结肠肥大细胞释放组胺的影响。方法 结肠组织经酶消化后,细胞成份用全HBSS重新悬浮。激发过程在LP4试管中、37℃条件下完成。组胺水平用以玻璃纤维为基础的荧光方法测定。结果PAR-2激动剂tc-LIGRLO-NH2和SLIGKV-NH2均可诱导人结肠肥大细胞剂量依赖性组胺释放。浓度为100μmol／mL时,tc-LIGRLO-NH2和SLIGKV-NH2可分别引起比基础分泌量多出2．5倍和2倍的组胺释放,而反PAR-2激动剂tc-OLRGIL-NH2和VKGILS-NH2在实验浓度高至300μmol／mL时仍对组胺释放无影响。胰蛋白酶在1．0～100μg/mL间可引起剂量相关性组胺释放,胰蛋白酶抑制剂可抑制之。PAR-2激动剂tc-LIGRLO-NH2的作用从加样后1min开始,3min后完成。细胞经过百日咳毒素和代谢抑制剂预先处理后,几乎失去了对tc-LIGRLO-NH2、SLIGKV-NH2和胰蛋白酶刺激的反应性。结论 PAR-2激动剂和胰蛋白酶是高效的组胺释放刺激剂,其在人结肠炎症性疾病中起一定的作用。     

Primary cutaneous B-cell lymphoma in Japanese patients

Primary cutaneous B-cell lymphoma (PCBCL) is a rare group of lymphoproliferative disorders. There have been few reports of Japanese patients with PCBCL, so the present study investigated the clinicopathological and immunological features and Bcl-2 gene rearrangement and protein expression in 28 Japanese patients with PCBCL. According to the Revised European-American Lymphoma (REAL) classification, there were 25 diffuse large B-cell lymphomas (DLBCL), one Burkitt type lymphoma, one lymphoblastic lymphoma and one marginal zone cell lymphoma. Of the 25 DLBCL, 17 were in males and eight in females, with an average age of 69.4 years. Follow-up data were available in 19 cases of DLBCL of which seven died and 12 were alive. The overall 5-year survival rate was 61%. Cases of DLBCL involving the legs were found to have poorer clinical outcomes; two of four cases with leg lesions died, with a mean survival of 13 months. Of 14 cases with non-leg lesions, four died, and the mean survival was 38.9 months. Only one case of Burkitt type lymphoma was CD10 positive. Bcl-2 rearrangement was not observed in 13 cases studied by polymerase chain reaction. Bcl-2 expression was observed in nine of 13 cases studied. All five cases with leg lesions exhibited Bcl-2 expression, but four of six cases with non-leg lesions also expressed the protein. These results show that DLBCL is the most frequent subtype of PCBCL in Japanese patients and that the prognosis of Japanese patients with DLBCL is worse than that of reported European cases. The study also found that PCBCL was frequently associated with Bcl-2 expression, which was not site-confined, and that there was no evidence for a follicular center origin of PCBCL.     

Increased c-Fos/activator protein-1 confers resistance against anergy induction on antigen-specific T cell

We have studied the contribution of c-Fos/activator protein-1 (AP-1) to antigen-specific T cell response with reference to T cell anergy by increasing c-Fos/AP-1 in vivo and in vitro. First, after injection of a high dose of staphylococcus enterotoxin B (SEB), clonal deletion of SEB-reactive V(beta)8(+) CD4 T cells occurred both in control B6 and H2-c-fos transgenic (fos) mice, whereas proliferation of T cells against SEB was profoundly depressed in B6 but not in fos mice. Second, the keyhole limpet hemocyanin-specific CD4 T(h)1 cell clone produced decreasing amounts of IL-2 in response to increasing amounts of concanavalin A (Con A) in vitro, whereas the decrease was less significant in the T(h)1 clones stably transfected with c-fos gene. Electrophoretic mobility shift assay with nuclear protein from the transformants showed that overexpression of the c-fos gene compensated the amounts of AP-1 in the nuclei of Con A-treated T(h)1 clones. Thus, increased c-Fos/AP-1 confers resistance against anergy induction on antigen-specific T cells.