The association between production of under-O-glycosylated IgA1 in the patient with IgA nephropathy and the staphylococcal cell membrane antigen

（1）Staphylococcer细胞膜抗原，后MRSA感染glomerulonepritis和IgA nephropathy中的可能抗原，直接与中间细胞的相互作用：Staphylococcer细胞膜抗原（GenBank登录号： BAB41819.1）是一种可能的抗原，被纳入后MRSA感染GN和IgA nephropathy 2。自从Toll-like受体（TLRs）激活宿主的innate immune响应以识别组件常见于细菌的范围，临床发现建议使用staphylococcal细胞膜抗原相互作用直接与肾脏维亚TLRs的体内细胞有关。方法：小鼠甲基细胞（ATCC ：CRL-1927-用纯净的旗帜混合标记的淀粉样球菌细胞膜抗原在各种条件下，并被保留在荧光素异硫氰酸酯（FITC）-结合抗FLAG抗体。FLAG标记的抗原的细胞附着是由流式细胞分析的。这些细胞也具有或没有抗小鼠TLR 2或TLR 4 IgG抗体，以及 ... More 该抗原对这些细胞的生长刺激效应已被确定为基于ELISA的溴代氧尤利定（BrdU） incorporation。基因组DNA在孵化或没有孵化的淀粉样细胞膜抗原被电泳分析。结果：淀粉样细胞膜抗原的附着到培养阶段的培养中，当超氟细胞膜抗原时，当超氟细胞没有与抗原发生交互时。在37 ° C时与抗原孵化15分钟，结果在流动细胞中高荧光强度，当与孵化比较时，在4 ° C时。在混合细胞中使用低剂量的重组淀粉样细胞膜抗原的缺失-TLR抗体在BrdU上没有加速增加BrdU的微囊细胞，在与抗TLR抗体附属的BrdU增加的地方进行治疗。一种中等到没有抗TLR抗体的高剂量也是一种依赖剂量的BrdU增加。从中间细胞中孵化的基因组DNA.总结：这些结果建议与中间细胞直接和传输的中间细胞中的抗原相互作用通过TLRs和抑制信号通过适应性途径的弱增长加速信号。（2）对IgA肾上腺病中的低O糖基化IgA 1的阿伯兰糖基化进行的一项新分析在IgA肾上腺病的病理学中发挥了重要作用（IgA-N）。这一不正常性是由IgA 1的铰链区O-link glycans中半乳糖的缺陷描述的。当与健康对照患者比较时，β 1，3-半乳糖基转移酶活动中的降解和N-乙酰半乳糖胺特定α 2，6-半乳糖基转移酶活动被观察到IgA肾病患者中的降解。这种非常正常的O-糖基化的IgA 1很可能会在IgA神经病理学的病理学中发生。However、临床参数之间的相关性（蛋白质、血细胞、病理学发现和类似）、临床治疗效果（类固醇治疗、tonsillectomy等）而这个不含糖的IgA 1仍然是不含糖的。这个研究的目的是确定这种不含糖的转化是如何受到IgA肾上腺治疗的影响的。Fcα/μ受体（Fcα/μR）成本地表达了B淋巴细胞和宏观及其人类同源物的多数，其中结合的IgA和IgM都具有高亲和力。我们建立了一种方法来从小鼠Fcα/μR转染细胞中捕获血清IgA。免疫球蛋白域的Fcα/μR which can fix IgA and IgM has marked homology for the domain of polymeric immunoglobulin receptor （poly-IgA）。我们也怀疑这个系统可以评估的underglycosylation of serum polymeric IgA主要观察到的患者' glomeruli.BW5147 （parent）或moFc？/？R·BW 5147 transfectant was treated with normal serum。在流式细胞中检测到了FITC附着的反人类IgA。我们也通过这个流程细胞分析测量了Lectin绑定（Helix Aspersa-Phycoerythrin ; HA-PE）。转染者没有绑定到单体IgA，但绑定到聚合物IgA。通过一个一致的激光显微镜揭示的IgA结合在细胞表面上的Fcα/μR组。在患者与IgA肾病（n=33）和健康控制（n=22）之间的HAA-PE绑定分析中，患者显示出明显高于HA绑定的显著性，并指示IgA的不良糖基化，当与控制进行比较时。Less（低）

(1)Staphylococcal cell membrane antigen, a possible antigen in post-MRSA infection glomerulonephritis and IgA nephropathy, interacts directly with mesangial cellsBackground : The staphylococcal cell membrane antigen (GenBank accession number : BAB41819.1) is a possible antigen involved in post-MRSA infection GN and IgA nephropathy 2. Since Toll-like receptors (TLRs) activate the host innate immune response to recognize components common to a range of bacteria, the clinical findings suggest that the staphylococcal cell membrane antigen interacts directly with intrinsic cells of the kidney viaTLRs.Methods : Mouse mesangial cells (ATCC : CRL-1927) were mixed with purified FLAG-tagged staphylococcal cell membrane antigen under various conditions and stained with fluorescein isothiocyanate (FITC)-conjugated anti-FLAG antibody. Cellular attachment of the FLAG-tagged antigen was analyzed by flow cytometry. The cells were also cultured with or without anti-mouse TLR2 or TLR4 IgG antibody, and … More the growth stimulation effect of the antigen on these cells was determined using ELISA based on bromodeoxyuridine (BrdU) incorporation. Genomic DNA obtained from mesangial cells after incubation with or without staphylococcal cell membrane antigen was analyzed by electrophoresis.Results : Attachment of staphylococcal cell membrane antigen to cultured mesangial cells occurred in the exponential growth phase, whereas supraconfluent cells did not interact with the antigen. Incubation of the mesangial cells with the antigen at 37℃ for 15 minutes resulted in higher fluorescence intensity in flow cytometry, when compared with incubation at 4℃. Treatment with a low dose of recombinant staphylococcal cell membrane antigen in the absence of anti-TLR antibodies did not accelerate incorporation of BrdU by the mesangial cells, whereas treatment with anti-TLR antibodies attenuated BrdU incorporation. A moderate to high dose of antigen without anti-TLR antibodies also attenuated BrdU incorporation in a dose-dependent manner. Genomic DNA from mesangial cells incubated with the antigen showed fragmentation.Conclusion : These results suggest that staphylococcal cell membrane antigen interacts with mesangial cells directly and transmits weak growth acceleration signals via TLRs and inhibitory signals via an apoptotic pathway.(2) A novel analysis of under-O-glycosylated IgA1 in IgA nephropathyAberrant glycosylation of IgA1 plays an essential role in the pathogenesis of IgA nephropathy(IgA-N). This abnormality is manifested by a deficiency of galactose in the hinge-region O-linked glycans of IgA1. When compared with the healthy control patients, a decrease in beta1,3-galactosyltransferase activity and an increase in N-acetylgalactosamine-specific alpha2,6-sialyltransferase activity were observed in IgA nephropathy patients. This abnormally O-glycosylated IgA1 is likely to be involved in the pathogenesis of IgA nephropathy. However, correlation between the clinilcal parameters, (proteinuria, hematuria, pathological findings and so on), clinical therapeutic efficacy (steroid therapy, tonsillectomy etc.) and this aberrant glycosylated IgA1 are still unclear.The purpose of this study is to determine how this aberrant glycosylation is effected by the treatment of IgA nephropathy.Fcα/μ receptor(Fcα/μR) is costitutively expressed on the majority of B lymphocytes and macrophages and its human homolog, which bind both IgA and IgM with high affinity. We established a method to capture serum IgA from whole serum by mouse Fcα/μR transfected cells. The immunoglobulin domain of Fcα/μR which can fix IgA and IgM has marked homology for the domain of polymeric immunoglobulin receptor (poly-IgR). We also hypothesized that this system can evaluate the underglycosylation of serum polymeric IgA mainly observed in the patients' glomeruli.BW5147 (parent) or moFcα/μR・BW5147 transfectant was treated with normal serum. Captured IgA was detected by FITC conjugated anti-human IgA in flow cytometry. We also measured lectin binding (Helix aspersa-phycoerythrin ; HA-PE) by this flow cytometry analysis. The transfectant did not bind to the monomeric IgA but bound to polymeric IgA. Observation by a confocal laser microscope revealed IgA binding of Fcα/μR grouped on the cell surface. In the HAA-PE binding assay between patients with IgA nephropathy (n=33) and healthy controls (n=22), the patients showed significantly higher HAA-binding, indicating underglycosylation of IgA, when compared with controls. Less