Detection of IgA protease from Haemophilus influenzae by immunoblotting

IgA protease produced by various strains of Haemophilus infuenzae can digest serum IgA and yield its fragments which can react with anti-IgA serum. We assayed IgA protease activity by detecting the digests of IgA by SDS-PAGE and immunoblotting. The digests were separated with SDS-PAGE, transferrend to nitrocellulose membranes and detected with anti- ( chain of human IgA, its Fab and its Fc) immunoglobulin conjugated peroxidases.Using this method, we can determine which type of IgA protease is produced by various of H. infuenzae strains. All the 20 strains isolated from respiratory tracts produced IgA protease.     

Alpha-SM actin expression as prognostic indicator in IgA nephropathy (Berger's disease)

Recent studies have demonstrated that α-Smooth Muscle actin expression in glomerular and tubulointerstitial compartments of renal tissue could represent a prognostic marker in several renal diseases. Our objective was to identify the prognostic value of α-SM actin actin expression on the evolution of renal damage in Primary IgA nephropathy (Berger’s Disease). 43 patients followed up from 1988 to 1999 at the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil, was studied. Clinical-laboratory data were obtained from the medical records of the patients using a protocol containing name, race, gender, origin, profession, age at clinical presentation of the disease and personal and family history. The parameters assessed in the approach to IgA nephropathy were serum creatinine, creatinine clearance, serum albumin, total serum protein, 24 hours proteinuria, glycaemia, serum sodium, potassium, calcium and phosphorus ions, analysis of urinary sediment, serum complement profile, blood count, and renal biopsy. Morphological evaluation was performed by renal biopsy using common light and immunofluorescence microscopy. Immunohistochemical studies were performed using a murine monoclonal antibody to α-SM actin. Our data showed that α-SM actin expression in the glomerular and tubulointerstitial compartments are not correlated with unfavorable clinical course of primary IgA nephropathy.     

IL-10-driven immunoglobulin production by B lymphocytes from IgA-deficient individuals correlates to infection proneness

In search for a possible explanation of the phenotypic heterogeneity in IgA deficiency, we studied the function of B cells from IgA-deficient (IgAd) individuals. Two groups of IgAd individuals, one frequently infected and one clinically apparently healthy, as well as normal controls, were studied. Peripheral blood mononuclear cells (PBMC) and B cells from IgAd individuals and controls were cultured with Staphylococcus aureus Cowan I strain and with anti-CD40 MoAb presented on the CD32-transfected fibroblast cell line in the presence of IL-10. In this experimental system PBMC and B cells from the infection-prone IgAd individuals produced only minute amounts of IgA. In contrast, PBMC and B cells from healthy IgAd subjects secreted significantly more IgA1 and IgA2 in comparison with infection-prone IgAd patients (P < 0.05). These data suggest that the abnormalities of B cell differentiation in IgAd could be of heterogeneous origin. Thus, whereas in healthy IgAd subjects IgA production may be efficiently up-regulated in vitro by addition of IL-10 to CD40-activated B cell culture, the corresponding B cell differentiation does not occur in infection-prone IgAd patients. These observations provide a conceptual framework for phenotypic heterogeneity in IgAd subjects.     

Etiology of IgA nephropathy syndrome

Since Berger's original paper on mesangial IgA-IgG deposition with hematuria, there have been a number of clinical and pathological studies regarding IgA immune complexes, the mechanisms of glomerular IgA deposition leading to glomerular injury and animal models of IgA nephropathy. During the last quarter of this century, glomerular changes such as IgA nephropathy have also been observed in cases associated with other diseases, such as systemic lupus erythematosus, Schoenlein-Henoch purpura, liver cirrhosis and chronic inflammatory diseases of the lung. This evidence supports the idea of an IgA nephropathy syndrome. On the other hand, IgA is thought to be an important humoral factor at the mucosal immune system and appears to have an antibody function against various etiologic candidates of extrinsic or intrinsic substances at the mucosal and systemic immune system. Glomerular IgA deposition in IgA nephropathy syndrome is thought to result from elevated levels of circulating immune complexes or aggregated IgA due to an overproduction of polymeric IgA as antibodies in the serum and due to the clearance impairment of IgA immune complexes in the hepatic and splenic phagocytic system. The glomerular IgA subclass is not one-sided, but should be evaluated in comparison with the age of patients at renal biopsy; this indicates the approximate age of onset. Cirrhotic IgA glomerulonephritis is not related to Hepatitis B or C virus infection, but to the pathophysiologic condition of liver cirrhosis. Various etiologic candidates such as viral, microbial, dietary antigens or auto-antigens have been listed and experimental models of IgA nephropathy syndrome have provided some clues in understanding the etiology of primary IgA nephropathy. However much still remains to be clarified and some specific epitopes common among these etiologic candidates will have to be identified.     

Induction and persistence of local rotavirus antibodies in relation to serum antibodies

The induction and persistence of local rotavirus antibodies, including stool IgA, jejunal IgA, and jejunal neutralizing antibody, were evaluated in 14 adult volunteers infected with the CJN strain of human rotavirus. In addition, the relationships between local rotavirus IgA and serum rotavirus IgA, IgG, and neutralizing antibody were determined. Both stool and serum rotavirus IgA appeared to have similar kinetics. Both antibodies peaked by days 14-17 after inoculation in all subjects, then decreased rapidly. By days 26-28, titers had fallen to 13% and 30% of their respective peaks. Serum rotavirus IgG peaked somewhat later, occurring in five subjects on days 26-28. Serum neutralizing antibody peaked on days 26-28 in all but three subjects. Both serum IgG and neutralizing antibodies also declined more slowly than rotavirus IgA. Although all antibody concentrations had decreased to only a fraction of their peak responses by days 270-365 after rotavirus inoculation they remained higher than baseline levels. For example, stool rotavirus IgA concentrations were 13.5-fold higher than baseline, while jejunal rotavirus IgA and neutralizing antibody were 8.9- and 4.3-fold above baseline, respectively. Similarly, serum antibodies remained 3.7- to 11.2-fold higher than baseline at 270-365 days after rotavirus inoculation. These studies imply that serum rotavirus IgA is a good indicator of local antibody responses. Furthermore, although both serum and local antibody titers peaked within 2-4 weeks after infection, these antibodies persisted at above baseline concentrations for at least 9-12 months after infection.     

旋毛虫Ts87重组蛋白诱导的小鼠黏膜免疫保护性研究

本实验探讨旋毛虫Ts87重组蛋白灌胃免疫小鼠诱导的黏膜免疫保护性作用。实验分对照组、佐剂组(霍乱毒素B亚单位组,CTB)和免疫组(CTB Ts87重组蛋白),灌胃免疫,间隔1周共免疫3次。末次免疫后第7天用400条旋毛虫感染期幼虫攻击,比较3组小鼠肠道成虫数、雌虫生殖力和肌幼虫数。且于末次免疫后第7天刮取肠黏液、取血检测特异性sIgA、IgG抗体水平。结果显示免疫组小鼠成虫减虫率、新生蚴减虫率、肌幼虫减虫率分别是81·34%、67·02%、84·49%;小鼠肠黏液sIgA水平及血清IgG水平显著高于对照组和佐剂组。结果表明Ts87重组蛋白黏膜免疫能够诱导小鼠产生抗旋毛虫的保护性免疫。灌胃免疫能显著提高肠黏液特异性sIgA水平,对促进肠道成虫的排出有明显作用。     

上海人群中TAP与IgA肾炎相关性的研究

目的探讨上海人群中抗原处理相关转运蛋白（ＴＡＰ）与ＩｇＡ肾炎的相关性。方法用ＰＣＲ－ＳＳＯ方法对上海地区８８名正常人及４０名ＩｇＡ肾炎患者ＴＡＰ进行分型。结果在病人组及对照组中共发现３种ＴＡＰ１（ＴＡＰ１Ａ、１Ｂ、１Ｃ）及４种ＴＡＰ２（ＴＡＰ２＊０１０１、＊０１０２、＊０２０１、＊０２０２）等位基因。未发现病人组与对照组之间ＴＡＰ等位基因分布的差异。结论本文结果未能证明ＴＡＰ基因与ＩｇＡ肾炎相关，但不排除未检测的多态位点与ＩｇＡ肾炎关联的可能性。     

Anti-P30 IgA antibodies as prenatal markers of congenital toxoplasma infection

This study extends a previous study and confirms that the detection of anti-P30 IgA antibodies is very helpful in the diagnosis of acute acquired or congenital toxoplasmosis. Moreover, we demonstrate that an anti-P30 IgA response can be mounted in the fetuses infected by Toxoplasma gondii during their intra-uterine life as early as week 23 of gestation. A double-sandwich ELISA described in our previous work was used to detect anti-P30 IgA antibodies in 1378 human serum samples collected from 551 patients, including 162 fetuses whose mothers had been infected by T. gondii during pregnancy, 46 congenitally infected and 90 uninfected newborns and 253 women suspected of having been infected during pregnancy, including the mothers of fetuses and newborns previously described. Anti-P30 IgA antibodies were detected in all cases of acute toxoplasmosis but in no case of chronic toxoplasmosis: in the majority of cases, the IgA antibody titre fell below cut-off in 3-9 months. Among the 46 congenitally infected newborns, anti-P30 IgA antibodies were detected in sera of 41 infected newborns (38 at birth, two in the first months of life, one in the seventh month of life), while anti-P30 IgM antibodies were detected in only 30 cases at birth and in one case during the first month of life. Among 162 fetuses, anti-P30 IgA response was observed in five infected fetuses, but was not detected in either 152 uninfected fetuses or in five fetuses considered as infected. The absence or presence of anti-P30 IgA antibodies in the fetus is discussed in relation to the date of maternal infection and collection of the fetal blood. It clearly appears from our study that the combined testing of both IgM and IgA in the fetus and the newborn is essential for a more efficient diagnosis of infection.     

Increase in tonsillar germinal centre B-1 cell numbers in IgA nephropathy (IgAN) patients and reduced susceptibility to Fas-mediated apoptosis

IgAN is a common form of primary glomerulonephritis and also a disease of tonsillar focal infection. The comprehensive mechanism underlying this disease remains to be defined. To better understand its pathogenesis, we investigated tonsillar CD5+ B cells (B-1 cells) with respect to IgA synthesis. Germinal centre (GC) B cells were isolated from the tonsils of IgAN patients and the number of B-1 cells in the GC determined by flow cytometry. GC B-1 and B-2 (CD5- B) cells were purified by cell sorter, the cells were incubated with agonist anti-CD40 MoAb and the ability for antibody production by B-1 and B-2 cells determined by ELISPOT assay. GC B-1 cells and B-2 cells were incubated with agonist anti-Fas MoAb, and apoptosis in GC B-1 cells and B-2 cells was analysed by flow cytometry. Although B-1 cells do not usually take part in the GC reaction, an increase in B-1 cell numbers was observed in the GC of tonsils from IgAN patients. These B-1 cells were likely IgA1 antibody-producing cells, since the prominent IgA subclass in IgAN is generally considered to be IgA1. Although Fas-dependent apoptosis is essential for the elimination of activated B cells, these B-1 cells showed a reduced susceptibility to Fas-mediated apoptosis. It is conceivable that activated B-1 cells may survive in the GC due to impaired apoptosis and thus produce abnormal antibodies. These findings suggest that the immune responses of B-1 cells in the tonsillar GC could thus have an impact on the pathogenesis of IgAN.     

IgA polyspecific autoantibodies in IgA nephropathy.

The specificity of circulating and kidney-bound IgA during IgA nephropathy is still a matter of discussion. In the present study, high levels of IgA antibodies directed against a panel of self and non-self antigens were found in the serum from patients with IgA nephropathy and were eluted from four out of the seven kidney biopsies studied. After immunoadsorption of pooled selected serum samples on TNP and actin-coated columns, polyspecific IgA antibodies were eluted. This supports the hypothesis that IgA-bearing B cells clones most probably producing polyspecific antibodies are a major feature of human IgA nephropathy. These findings also suggest that it may be hazardous to draw conclusions from the finding of apparently monospecific IgA antibodies in this condition.