Effects of interleukin 4 on CD25+CD4+ regulatory T cell function

CD25+CD4+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance against self and non-self. The modulatory effects of cytokines, such as interleukin 4 (IL-4) on the function of Tregs have not been explored in detail. We here report that IL-4 prevents spontaneous apoptosis and the decline of foxp3 mRNA which were found to occur during culture of isolated Tregs. Tregs exposed to IL-4 were more potent in suppressing the proliferation of na?ve CD4+ T cells and they better inhibited IFN-gamma production by CD4+ T cells as compared to Tregs cultured in medium. IL-4 also enhanced membrane IL-2Ralpha (CD25) expression on Tregs above the levels observed on freshly isolated cells. IL-4-mediated effects on Treg function persisted in Tregs from Stat6-/- mice, pointing to a Stat6-independent intracellular transduction pathway. In conclusion, our data suggest that the anti-inflammatory function of IL-4 could partly be mediated by effects on Tregs function.     

No association between schizophrenia and homozygosity at the D3 dopamine receptor gene

The D₃ dopamine receptor gene is an important candidate gene for schizophrenia, since (because of its almost exclusive expression in the limbic system) it combines the dopamine receptor hypothesis with the limbic system hypothesis of schizophrenia. A BalI restriction fragment length polymorphism of the D₃ dopamine receptor gene has been typed in 107 schizophrenic patients and 98 normal controls from Sichuan (China). With regard to alleles or genotypes, no significant differences were obtained between controls from Europe and China, between patients and controls, and between patient subgroups and controls. These results indicate a lack of association between schizophrenia and the D₃ dopamine receptor gene in our sample. Our findings are at variance with reports of a significant excess of homozygosity at the D₃ dopamine receptor gene in schizophrenic patients from Wales (United Kingdom) and Alsace (France). In conclusion, further studies will be needed with larger samples of patients from Wales and Alsace as well as with samples of different racial groups to prove or disprove the initial positive association between schizophrenia and genotypes of the D₃ dopamine receptor gene. © 1993 Wiley-Liss, Inc.     

Development of a Western Blot Assay for Detection of Antibodies against Coronavirus Causing Severe Acute Respiratory Syndrome

To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.     

小鼠趋化因子COL19Ig融合蛋白表达载体的构建、表达及鉴定

通过特异引物扩增出去掉终止密码的mCCL19编码序列，经酶切、亚克隆、拼接构建了真核表达载体pcDNA3．1-CCL19Ig，酶切和测序鉴定插入序列；将重组质粒转染CHO细胞进行体外表达，通过RT—PCR、Western blot鉴定目的基因的表达，利用趋化小室法测趋化活性。结果测序证实重组表达载体含mCCL19编码序列和人IgGI Fc段序列，其序列分别与GenBank中公布序列比对一致，IgG1-Fc段读码框未发生改变；Western blot结果证实转染了pcDNA3．1-mCEL19Ig的CHO细胞培养上清中有相对分子质量为38000的融合蛋白mCCL19Ig表达；体外趋化实验表明融合蛋白mCCL19Ig对小鼠脾细胞有趋化活性，且呈剂量依耐关系。     

Multiple B- and T-cell epitopes on a major allergen of Kentucky Bluegrass pollen.

The B- and T-cell epitopes of a recombinant grass allergen, rKBG60, were delineated using a set of overlapping synthetic peptides. Direct binding by enzyme-linked immunosorbent assay (ELISA) utilizing serum pools led to the identification of 13 murine immunoglobulin-, and nine to 13 human IgG- and five to seven human IgE-reactive overlapping peptides. Of the peptides which bound to human IgE antibodies, all but three peptides bound to human and/or murine IgG antibodies. Furthermore, eight out of 12 synthetic peptides induced antigen-specific antibodies in mice, suggesting that these peptides contained epitopes that recognized and/or induced T cells. These results, in conjunction with cross-recognition of different peptides at the C-terminus of rKBG60 by antibodies to neighbouring or non-overlapping peptides suggest that the C-terminus of this antigen represents a dominant antigenic and allergenic site. Peripheral blood mononuclear cell (PBMC) proliferation studies using these synthetic peptides for 13 grass allergic individuals indicated that seven potential human T-cell epitopes exist on this allergen. Taken together, the results demonstrate that multiple B- and T-cell epitopes exist on this major group of grass allergens, the majority of which are localized at the C-terminus of this antigen.     

Production of monoclonal antibody to a phenolic glycolipid of Mycobacterium tuberculosis and its use in detection of the antigen in clinical isolates.

A monoclonal antibody (MAbIII604) specific to phenolic glycolipid Tb (PGL-Tb), a Mycobacterium tuberculosis-specific antigen, was produced and used in the detection of the antigen. MAbIII604 reacted with the PGL-Tb antigen but not with other phenolic glycolipids from Mycobacterium leprae, M. bovis, and M. kansasii, thus indicating the specificity of the monoclonal antibody to PGL-Tb. A dot enzyme-linked immunosorbent assay with MAbIII604 was employed to detect the PGL-Tb antigen in lipids purified from M. tuberculosis clinical isolates. Of 50 isolates, 32 (64.0%) showed clear evidence of the PGL-Tb antigen by the dot enzyme-linked immunosorbent assay, but there were marked variations in the intensities and sizes of spots. This suggests differences in PGL-Tb antigen production among M. tuberculosis strains even when they are grown in the same culture media and conditions. This was most evident from the fact that in only eight (16.0%) of the isolates examined was the PGL-Tb antigen detectable by thin-layer chromatography, which is much less sensitive for the detection of glycolipid antigens. This study shows that monoclonal antibodies specific to PGL-Tb are useful in detecting the antigen in lipid extracts and that there is a marked variation in the PGL-Tb production among M. tuberculosis clinical isolates.     

Development of a microsphere-based fluorescent immunoassay and its comparison to an enzyme immunoassay for the detection of antibodies to three antigen preparations from Candida albicans

A sensitive assay for the simultaneous detection of multiple serum antibodies by flow cytometry was developed. Polystyrene microspheres of 5, 7 and 9.3 micron in diameter were used as solid supports for the attachment of three different antigen preparations from Candida albicans. These antigens were a whole cell extract; a cytoplasmic protein extract and a cell wall polysaccharide. Microsphere-associated fluorescence was quantitated by flow cytometry, with the different sized microspheres analyzed separately using electronic volume gating. This procedure allowed for different antigen-coated microspheres with discrete sizes to be analyzed independently for immunofluorescence. The assay detected antibody levels in human serum at dilutions up to 10(-6) and provided complete discrimination, using all three antigen preparations, between antibody levels seen in healthy subjects and those seen in patients suspected of having a systemic Candida infection. A standard enzyme immunoassay (EIA) failed to provide complete discrimination between healthy subjects and patient samples: at least 17% of patient values fell within the healthy subject range using all three antigen preparations. The microsphere assay which allowed for the simultaneous detection of multiple antibodies, has increased dynamic range over EIA and provides for better discrimination of patients from healthy subjects in comparison to EIA. Precise quantitation of antibodies is possible and the rapid analysis of thousands of microspheres markedly enhances the statistical accuracy of the assay. We suggest this assay is likely to have many other important applications in immunologic testing.