Proto-oncogene expression in peripheral blood mononuclear cells in IgA nephropathy.

We have investigated the expression of proto-oncogenes in mononuclear cells obtained from patients with IgA nephropathy using a RNA hybridization technique. Patients with IgA nephropathy expressed more c-myc, c-raf, c-fos, and c-jun proto-oncogene RNA than did normal controls. However, no significant expression of c-N-ras, c-mos or c-myb genes was found in the mononuclear cells of these patients. When the amount of urinary protein excretion was used as an indicator of disease activity (greater than 1 g/day), a positive correlation was found between c-myc, c-raf, c-fos, and c-jun expression and urinary protein excretion (P less than 0.01). The expression of these genes correlated also with the serum IgA concentration (P less than 0.01), IgA immune complex (P less than 0.01), and histopathological changes in renal tissues obtained from patients with IgA nephropathy (P less than 0.01). The results of this survey suggest that abnormally regulated proto-oncogene expression in mononuclear cells may play an important role in the progression of IgA nephropathy and may be useful as an indicator of disease activity and/or prognosis.     

Adrenomedullin gene transcription is decreased in peripheral blood mononuclear cells of patients with IgA nephropathy

We measured mRNA levels of adrenomedullin (AM), C-type natriuretic peptide (CNP), vascular endothelial growth factor (VEGF), interleukin 1beta (IL-1beta) and interleukin 6 (IL-6) in peripheral blood mononuclear cells (PBMC) of patients with IgA nephropathy. To evaluate these mRNA levels, we employed a real-time quantitative PCR method which was performed using a hybridization probe labeled with two fluorescence dyes. This strategy was found to afford the standard curves with a high correlation, suggesting that this method is useful for evaluations of mRNA levels. By this method, levels of AM, CNP, VEGF, IL-1beta and IL-6 mRNA in PBMC of 49 IgA nephropathy patients and 35 healthy volunteers were evaluated. Among the mRNAs examined, AM mRNA levels were significantly lower in severe-grade than in mild-grade IgA nephropathy patients. Furthermore, AM mRNA levels correlated with CNP mRNA levels in PBMC of patients with IgA nephropathy, and each peptide generated from these mRNAs has antiproliferative effects on mesangial cells. These data indicate that gene expression of AM in PBMC is regulated according to the pathophysiological states of IgA nephropathy and that decreased AM production may contribute to the progression of IgA nephropathy.     

In vitro immunoglobulin production by peripheral blood mononuclear cells as a prognostic factor in IgA nephropathy

To determine whether immune system disorders are involved in the exacerbation of IgA nephropathy, the immunoglobulin production of peripheral blood mononuclear cells obtained from 45 IgA nephropathy patients was measured and then compared with that of healthy individuals. The level of IgA production was classified into an elevated group and a non-elevated group and comparisons were made with various clinical factors considered to be related to exacerbation of this disease. The results indicated that although there was no significant difference in immunoglobulin production of the peripheral mononuclear cells between IgA nephropathy cases and healthy individuals in the group not stimulated with pokeweed mitogen (PWM), the group stimulated with PWM revealed a production of IgA, IgG, and IgM which was significantly elevated (P less than 0.01). Also, within the group stimulated with PWM, hypertension, severe proteinuria and microscopic hematuria, elevated BUN and serum creatinine values, decreased 15-min PSP and creatinine clearance values, severe histological damage, and severe IgA deposition were observed more in the elevated IgA production group than in the non-elevated group. These findings suggest that an elevated IgA production plays an important role in the excerbation of this disease.     

IgA肾病患者腭扁桃体单个核细胞IgA类别转换基因表达变化

目的 研究脂多糖(LPS)或溶血性链球菌(HS)刺激IgA肾病和非肾脏疾病慢性扁桃体炎患者腭扁桃体单个核细胞Iα-Cα胚系转录本、激活诱导的胞嘧啶脱氨酶(AID)mRNA和蛋白的表达,以探讨IgA肾病腭扁桃体单个核细胞IgA及IgA1产生异常的分子机制.方法 入组2009年1月到2010年2月在我院住院的IgA肾病患者27例,非肾脏疾病慢性扁桃体炎患者27例作为对照.通过单个核细胞分离液和密度梯度离心法分离出腭扁桃体单个核细胞.IgA肾病组及非肾脏疾病慢性扁桃体炎组腭扁桃体单个核细胞分别分为3组:LPS刺激组,HS刺激组和未刺激组.ELISA法检测培养上清中IgA和IgA1的浓度.实时PCR检测Iα-Cα胚系转录本和AID mRNA的表达;Western印迹检测AID蛋白的表达.结果 IgA肾病组腭扁桃体单个核细胞IgA和IgA1的分泌,特别是IgA1/IgA较慢性扁桃体炎组显著增加(P＜0.05),Iα-Cα和AID mRNA和AID蛋白的表达较慢性扁桃体炎组显著增加(均P＜0.05).IgA肾病组腭扁桃体单个核细胞IgA和IgA1的水平在刺激后明显增加(P＜0.05);Iα-Cα和AID mRNA的表达明显上调(均P＜0.05);AID蛋白表达明显增加(LPS刺激组P＜0.05,HS刺激组P＜0.01).结论 LPS和HS均能够诱导IgA肾病患者腭扁桃体单个核细胞IgA和IgA1的分泌、AID和Iα-Cα的表达增加,提示IgA肾病患者腭扁桃体IgA和IgA1的分泌增加可能与IgA类别转换相关基因AID和Iα-Cα高表达有关.     

Interleukin-6 production induced in peripheral blood mononuclear cells by a serum factor from IgA nephropathy patients is inhibited in vitro by specific sugars

The basal production of IL-6 by cultured peripheral blood mononuclearcells (PBMC) from patients with IgAN, is markedly higher (A,109 pg/ml) as compared to that of PBMC of either patients withoutclinical signs (I, 39 pg/ml) or appropriate controls (C, 44pg/ml). When PBMC from healthy subjects were incubated in thepresence of sera from patients A, the IL-6 production was stronglyenhanced. No such an effect was observed by stimulating PBMCwith sera from the other two groups of subjects (I, C). In anotherexperiment we observed that the IL-6 production stimulated byserum from patients A could be inhibited by addition of specificmonosaccharides. The inhibitory effect was rapidly abolishedwhen the sugar-containing medium was substituted with the originalone. Finally molecular components from serum of A were grosslyseparated by gel column chromatography. Individual fractionswere incubated with PBMC of C: fractions with Mr > 30 000highly stimulated the release of IL-6 (up to 1320 pg/ml); fractionswith lower molecular weight were inactive. The data suggest the presence of an IL-6 releasing factor inthe serum of IgAN patients. Although the chemical nature ofsuch a factor is not yet established, the observations reportedfocus our attention to the lectins family. Since this factorseems potentially important in the understanding of the pathogenesisof IgAN, both its isolation and structural/functional characterizationdeserve further efforts.     

Increased expression of HLA-DR molecule on peripheral blood Th lymphocytes from patients with IgA nephropathy

Background. IgA nephropathy is the commonest type of glomerulonephritis. Recent studies have shown a decrease in the expression of HLA class I antigens on peripheral blood mononuclear leukocytes (PBML) from patients with HLA class II-associated autoimmune diseases. In this study, the expression of HLA molecules on T cells from patients with IgA nephropathy was examined in order to investigate the immunological events contributing to the pathogenesis of this disorder. Methods. Thirty Japanese patients with IgA nephropathy were studied. Nine patients with membranous nephropathy and 21 sex- and age-matched healthy individuals were enrolled as controls. Heparinized PBML with or without stimulation by an anti-CD3 monoclonal antibody were analyzed in regard to the expression of HLA-class I, HLA-DR, CD4, CD8, CD11a, CD11b, and CD56, by two-color fluorescence flow cytometry. Results. The expressions of HLA-class I, HLA-DR, CD11a, CD11b, and CD56 on resting CD3-positive, CD4-positive, CD8-positive, or CD20-positive cells from patients with IgA nephropathy were found to be comparable with those from the controls. However, after stimulation by anti-CD3 antibody, the expression of HLA-DR on CD4-positive cells from these patients was significantly higher than that from the controls. Further, the expression of HLA-DR on CD4-positive cells from patients with proteinuria of more than 1 g/day was much higher than that in patients with proteinuria of less than 1 g/day. Conclusions. In this study, the expression of HLA-DR on stimulated Th cells from IgA nephropathy patients was shown to be significantly higher than the expression in the stimulated T cells from the controls. This finding suggests that Th cells may acquire antigen-presenting activity by HLA-DR expression, present antigens to other Th cells, promote B cells to produce antibodies, and, presumably, lead to the development of IgA nephropathy. Received: October 26, 1998 / Accepted: February 16, 1999     

Increase of CD23-positive cells in peripheral blood from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis.

CD23 is a surface marker of activated B cells as well as a low-affinity Fc receptor for IgE. In this study, we enumerated CD23-positive peripheral blood lymphocytes and evaluated their clinical significance in patients with IgA nephropathy (IgAN). Twenty-five patients with IgAN and 16 patients with non-IgA proliferative glomerulonephritis (PGN) were studied. Twenty-seven healthy adults served as controls. CD23-bearing cells were enumerated by flow cytometry, and serum IgE levels were measured by latex photometric immunoassay. Significant increases in the number of CD23-positive cells were observed in patients with IgAN (p less than 0.01) and PGN (p less than 0.05) compared with controls. A significant elevation of serum IgE levels was also observed in the patients with IgAN and PGN (p less than 0.05). No positive correlation between the number of CD23-positive cells and serum IgE levels was observed. We also examined the induction of surface CD23 expression on peripheral lymphocytes by interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, interferon (IFN)-gamma, IFN-alpha, phytohemagglutinin, concanavalin A, pokeweed mitogen, lipopolysaccharide and phorbol myristate acetate. IL-4 was revealed to have a significantly potent effect on the induction of cell surface CD23 compared with other stimulants. It was concluded that many patients with IgAN or PGN show high serum IgE levels and/or high CD23-positive cell counts in their peripheral blood, suggesting that hyperactivation of B cells might be involved in the development of IgAN and non-IgA PGN. It appeared that IL-4 may play a significant role in the etiology of these types of glomerulonephritis.     

二期梅毒患者外周血单个核细胞基因表达谱研究

目的:分析二期梅毒患者外周血单个核细胞基因表达谱特征,探索机体抗梅毒免疫的分子机制。方法:采用全基因组高通量的Illumina测序技术,对4例二期梅毒患者和4例健康对照者外周血单个核细胞行数字基因转录组分析。后行实时PCR对其结果进行验证。结果:与健康对照者相比较,二期梅毒患者外周血单个核细胞共发现差异表达基因78个,其中有16个免疫应答相关基因。肿瘤坏死因子超家族成员17(TNFRSF17)、IL-17C、IL-21、IL-31受体A(IL-31RA)、C-X-C配体10(CXCL10)、趋化因子配体1(CCL1)等炎症因子和相关受体、CD4+T细胞激活标志CD38、Fc类吞噬性受体(FcγR1A、FcγR3B)以及补体(C2、SERPING1)等均显著上调。结论:二期梅毒患者多种天然免疫和适应性免疫分子参与机体系统性抗梅毒应答。     

Expression and function of fibronectin receptors on peripheral mononuclear cells in IgA nephropathy

The ß1 integrin family, major adhesive receptors forthe extracellular matrix (ECM), have been reported to be presentin normal and diseased kidneys. Attachment of glomerular cellsto ECM is mediated by ß1 integrins. Several membersof the ß1 integrins are referred to as VLA (very lateactivation) antigens. Peripheral mononuclear cells also expressVLA antigens in both resting and activated states. We examinedthe expression and function of VLA antigens on peripheral lymphocytesand monocytes in patients with IgA nephropathy using monoclonalantibodies (mAbs) specific for VLA -chains. Peripheral lymphocytesfrom patients with IgA nephropathy expressed VLA-4 and 5, butnot VLA-1 2 or 3. Peripheral monocytes from patients with IgAnephropathy expressed VLA-2 4 and 5, but not VLA-1 or 3. Theexpression of VLA adhesive receptors was observed in healthyindividuals. Adhesion assay to fibronectin revealed augmentedadhesion of mononuclear cells in IgA nephropathy (P<0.05),and this increased adhesion was inhibited by mAbs to VLA-4 and5. The expression of ß1 integrins in IgA nephropathywas similar to that of healthy individuals, but the functionof these molecules in terms of adhesion to fibronectin thoughVLA-4 and VLA-5 is increased in these patients. These findingssuggest that the activation of fibronectin receptors on peripheralmononuclear cells plays an important role in the pathogenicprocess of IgA nephropathy.     

Efficacy of triptolide on the apoptosis of tonsillar mononuclear cells from patients with IgA nephropathy

Aims: This study aimed to evaluate the extent of apoptosis of tonsillar mononuclear cells (TMCs) derived from patients with IgA nephropathy (IgAN) and the effects of triptolide (TP) on the apoptosis of these TMCs. Methods: TMCs were isolated from tonsillar tissues of patients with IgAN or chronic tonsillitis (control group). Rates of TMCs apoptosis were measured by annexin V-fluorescein isocyanate (FITC)/propidium iodide (PI)-labeled flow cytometry (FCM). Expression levels of Bcl-2 family proteins were quantified by immunohistochemistry of fixed tonsillar sections and Western blot analyzes of TMCs lysates. TMCs from IgAN patients were treated 10, 20, or 30?ng/mL TP for 24?h and then evaluated for viability by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, for the percentage of apoptotic cells by FCM, and for the relative expression levels of Bcl-2 family proteins by Western blot analysis. Results: Compared to TMCs from the control group, TMCs from the IgAN group demonstrated lower rates of apoptosis, higher expression levels of the anti-apoptosis proteins Bcl-2 and Bcl-xL, and lower expression levels of the pro-apoptosis protein Bax. Treatment of IgAN patient-derived TMCs with 10, 20, or 30?ng/mL TP for 24?h suppressed the viability and promoted the apoptosis of TMCs in a dose-dependent manner. Western blot analysis revealed a TP dose-dependent decrease in Bcl-2 and Bcl-xL expression levels, in parallel with increased Bax protein levels. Conclusion: TMCs from IgAN patients may be in a state of inhibited apoptosis mediated by Bcl-2 family proteins, which may be reversed by TP treatment.     

Interleukin-6 gene expression in peripheral blood mononuclear cells from patients undergoing hemodialysis or continuous ambulatory peritoneal dialysis

To compare the interleukin-6 (IL-6) gene expression in the peripheral blood mononuclear cells (PBMCs) and plasma IL-6 levels in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) with those in patients undergoing hemodialysis. Eleven hemodialysis patients, 10 CAPD patients, 15 non-dialyzed patients with end-stage kidney disease (ESKD), and 7 healthy controls were included in this study. PBMCs were collected by differential centrifugation. Plasma IL-6 concentration was measured by enzyme immunoassay. Plasma IL-6 levels were significantly increased in the hemodialysis and CAPD patients as compared with non-dialyzed ESKD patients and normal subjects (p < 0.01). Following hemodialysis, plasma IL-6 levels exceeded those before hemodialysis. No significant difference was found in plasma IL-6 levels in CAPD patients and in hemodialysis patients when blood was drawn before hemodialysis. Low but steady-state levels of IL-6 mRNA expression were observed in the non-dialyzed ESKD patients. The expression of IL-6 mRNA in PBMCs was significantly increased in the patients undergoing hemodialysis or CAPD as compared with the non-dialyzed ESKD patients. The PBMC IL-6 mRNA was significantly lower in CAPD patients than in hemodialysis patients (p < 0.01). A significant correlation was found between the plasma concentration of IL-6 and the expression of IL-6 mRNA in PBMCs from patients undergoing hemodialysis or CAPD (p < 0.01). The hemodialysis or CAPD procedure contributed to the increase in PBMC IL-6 mRNA expression and plasma IL-6 concentration. CAPD treatment stimulated the production of IL-6 to a lesser extent than hemodialysis treatment.     

尿酸性肾病胰岛素样生长因子和成纤维细胞生长因子的表达

目的　探讨胰岛素样生长因子 1(IGF 1)和碱性成纤维细胞生长因子 (bFGF)与尿酸性肾病病变的关系。方法　采用原位杂交和免疫组织化学 (免疫组化 )方法 ,观察 30只腺嘌呤致尿酸性肾病模型大鼠肾病变发展过程中 ,IGF 1、bFGF、FGF受体 1(FGFR 1)、增殖细胞核抗原 (PCNA)及Ⅲ型胶原的表达。结果　受损伤及再生肾小管上皮细胞和肉芽肿内单个核细胞均有IGF 1mRNA和bFGFmRNA表达及蛋白合成 ,FGFR 1、PCNA和Ⅲ型胶原也广泛表达 ;肾小管间质bFGFmRNA和蛋白、FGFR 1、PCNA和Ⅲ型胶原阳性细胞随病程逐渐增多。结论　尿酸结晶对肾小管的损伤刺激IGF 1、bFGF在肾内表达增强且范围扩大 ,其表达程度与肾病变程度一致 ,提示IGF 1和bFGF在尿酸性肾病发展过程中发挥了重要作用。     

SOX13 gene downregulation in peripheral blood mononuclear cells of patients with Klinefelter syndrome

Klinefelter syndrome(KS)is the most common sex chromosome disorder in men.It is characterized by germ cell loss and other variable clinical features,including autoimmunity.The sex-determining region of Y(SRY)-box 13(Sox13)gene is expressed in mouse spermatogonia.In addition,it has been identified as islet cell autoantigen 12(ICA12),which is involved in the pathogenesis of autoimmune diseases,including type 1 diabetes mellitus(DM)and primary biliary cirrhosis.SOX13 expression has never been investigated in patients with KS.In this age-matched,case-control study performed on ten patients with KS and ten controls,we found that SOX13 is significantly downregulated in peripheral blood mononuclear cells of patients with KS compared to controls.This finding might be consistent with the germ cell loss typical of patients with KS.However,the role of SOX13 in the pathogenesis of germ cell loss and humoral autoimmunity in patients with KS deserves to be further explored.     

尿毒症患者外周血单个核细胞超保守区域转录表达水平的分析

目的 尿毒症是由于肾衰竭致使代谢产物和其他有毒物质在体内蓄积而引起的一种自身中毒综合征,超保守区域转录子(transcribed ultra-conserved regions,T-UCRs)是lncRNA重要组成部分,转录自人类基因组中481个极度保守区域,希望分析尿毒症外周血单个核细胞的超保守区域转录表达水平,从而发现尿毒症潜在的诊断和预后的生物标志物.方法 本研究采用T-UCRs芯片技术分析了15例尿毒症患者和15例健康对照组的外周血单个核细胞的超保守区域转录表达水平,并且用实时荧光定量PCR技术验证了尿毒症患者与健康对照组表达差异最显著的基因的表达水平.结果 对比尿毒症患者与健康对照组,通过芯片筛选出T UCRs发现21个T-UCRs表达水平上调,49个T-UCRs表达水平下调,在表达上调和下调的T-UCRs中,对应的超保守区域uc.458-表达水平最显著(P-2.47524E-24;FC=3118.5361),其相关基因为RBFOX2.结论 通过GO分析以及验证后发现uc.458-可能是尿毒症潜在的生物标志物,RBFOX2可能是尿毒症潜在的关键基因.     

Triiodothyronine stimulates growth of peripheral blood mononuclear cells in serum-free cultures in uremic patients.

The effect of triiodothyronine (T3) on the responses to mitogens and on the production of prostaglandin E2 and interleukin 2 were studied in serum-free cultures of peripheral blood mononuclear cells (PBMC) in 20 patients undergoing hemodialysis and in 30 control subjects. T3 increased the growth of PMBC induced by phytohemagglutinin and pokeweed mitogen in both groups. PBMC reached growth maximum at 0.5 nM T3 when stimulated by phytohemagglutinin in both groups. At higher concentrations of T3 the effect declined in the control group, but the response of uremic PBMC was constant. The response to T3 of pokeweed mitogen stimulated PBMC was lower in the uremic patients. The production of prostaglandin E2 by PBMC was higher in the uremic patients than in the controls. T3 had no effect on prostaglandin E2 production. Indomethacin alone and in combination with T3 had a stimulatory effect on cell growth in the patient group. T3 had no effect on the release of interleukin 2 by PBMC. An additive effect of interleukin 2 and T3 was observed in cultures stimulated by suboptimal concentrations of the mitogens. In conclusion, the impaired growth of PBMC in serum-free cultures from uremic patients was enhanced, however, not normalized, by external addition of T3, inhibition of prostaglandin E2 synthesis, and addition of interleukin 2.