T-cell subsets,interleukin-2 receptor expression and production of interleukin-2 in minimal change nephrotic syndrome

This study was designed to investigate T-lymphocyte subsets interleukin-2 receptor (IL-2R) expression and IL-2 production in minimal change nephrotic syndrome (MCNS). Peripheral blood T-lymphocytes and IL-2R expression were analysed using fluorescein isothiocyanatelabelled CD3, CD4, CD8 and CD25 monoclonal antibodies with flow cytometry. IL-2 production was determined by enzyme immunoassay. Ten children with MCNS in relapse and in remission were evaluated. Thirteen healthy children served as controls. The patients in relapse demonstrated a moderate decrease in the total absolute lymphocyte counts and CD8(+) T-lymphocytes compared with controls (P<0.05) and had a greatly increased IL-2R expression in frashly isolated, unstimulated peripheral lymphocytes compared with patients in remission and controls. While this was not statistically significant, IL-2R expression on cultured lymphocytes stimulated with phytohaemagglutinin was significantly elevated in relapse compared with those in remission and controls (P<0.05). IL-2 production did not correlate well with IL-2R expression and there was no significant difference between the groups. Our results suggest that T-cell subset changes and high IL-2R expression on peripheral lymphocytes may indicate the presence of stimulated T-cell populations in MCNS which could contribute to the immunopathogenesis.     

The urinary podocyte as a marker for the differential diagnosis of idiopathic focal glomerulosclerosis and minimal-change nephrotic syndrome

BACKGROUND/AIMS: Detection of podocytes in the urine sediment of children indicates that severe podocyte injury occurred in the glomerulus. Focal glomerulosclerosis (FGS) and minimal-change nephrotic syndrome (MCNS) are kidney diseases characterized by massive proteinuria. The aim of the present study was to determine whether urinary podocytes can be detected in patients with idiopathic FGS or MCNS and whether immunosuppression therapy alters these cells. METHODS: Twenty patients with MCNS (nephrotic stage, n = 12; remission stage, n = 8), 15 patients with FGS and 20 healthy controls were included in the present study. Urinary podocytes were stained by immunofluorescence. All patients with MCNS at the nephrotic stage received prednisolone for 6 months, and all patients with FGS received some form of immunosuppression therapy including prednisolone, cyclophosphamide or mizoribine for 12 months. RESULTS: The 12 nephrotic-stage MCNS patients achieved remission after treatment. Seven of the 15 FGS patients also achieved remission, but the other 8 remained in the nephrotic stage. Urinary podocytes were not detected in any patient with MCNS nor were they detected in healthy controls. Urinary podocytes were detected in all FGS patients (mean, 4.2 cells/ml) before treatment and the number of cells decreased in the 7 patients who achieved remission. The number of podocytes was unchanged in the other 8 patients even after treatment. CONCLUSION: Urinary podocytes may be a useful diagnostic indicator for differentiation between FGS and MCNS. These cells may also mark disease progression in cases of FGS.     

Studies of glomerular permeability factor (GPF) in focal segmental glomerular sclerosis and the relationship between GPF and vascular permeability factor (VPF)

BACKGROUND: We previously demonstrated that the supernatants of cultured concanavalin-A (con-A) stimulated peripheral blood mononuclear cells (PBMC) from patients with minimal change nephrotic syndrome (MCNS) increased the urinary protein excretion in injected rats and suggested that PBMC released a factor, which we called glomerular permeability factor (GPF), changes in the glomerular permeability and thus resulted in proteinuria in MCNS. MATERIAL AND METHODS: In this study we investigated the GPF activity in focal segmental glomerular sclerosis (FGS) and other conditions of chronic glomerulonephritis (CGN), and also the relationship between GPF and vascular permeability factor (VPF). In experiment 1 the supernatants of the cultured con-A stimulated PBMC from patients with 10 FGS, 5 other CGN and 10 controls were tested regarding their ability to produce GPE The GPF activity was defined as positive when the 8-hour urinary protein excretion after the injection of the supernatant in Sprague-Dawley rats exceeded the mean value plus 2 standard deviations (M + 2 SD) of that before injection. RESULTS: Three out of 10 FGS patients and 1 membranous nephropathy patient out of the 5 other CGN patients were positive for GPF activity. In experiment 2 the relationship between GPF and VPF was analyzed using culture supernatants of PBMC from 10 nephrotic MCNS patients and 15 controls. The VPF activity was measured following the method developed by Ovary [1975]. All 7 cases that were positive for GPF activity were simultaneously positive for VPF activity. On the other hand, 16 cases that were positive for VPF activity were not always positive for GPF activity (7 cases were positive and 9 were negative for VPF activity). CONCLUSION: Experiments 1 and 2 thus suggested that GPF was not active in MCNS alone, but also in other CGN conditions and it was therefore not considered to be the same factor/substance(s) as VPF.     

Serum IgE in primary glomerular diseases and its clinical significance

Total serum IgE was measured in 119 cases of primary glomerular diseases and 33 normal healthy persons. Statistically significant higher levels were noted in minimal change disease (MCD; median: 630 U/ml), IgM nephropathy (IgMN; 618 U/ml), focal glomerulosclerosis (FGS; 373 U/ml) and membranous glomerulonephritis (MGN; 144 U/ml). A higher level of serum IgE was noted in association with more frequent relapse or steroid resistance in MCD and IgMN and in FGS with nephrotic syndrome. A small group of IgA nephropathy with nephrotic range proteinuria was also noted to have extraordinarily high serum IgE. These findings suggest that IgE may play an important role in the pathogenesis of MCD, IgMN, and FGS and may serve as a prognostic indicator in terms of steroid responsiveness in MCD and IgMN.     

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.     

Implication of serum IgE in childhood nephrotic syndrome

Elevated serum IgE levels have been related to glomerular diseases. We investigated the relationship between serum total and specific IgE levels and their modulating factors [interleukin-4 (IL-4) and sCD23] and the outcome of childhood nephrotic syndrome (NS) after steroid treatment. We found that children with NS had significantly higher serum IgE levels than age-matched allergic patients and normal controls. Patients with steroid-resistant nephrotic syndrome (SRNS) had higher serum IgE levels than patients with steroid-sensitive nephrotic syndrome (SSNS) both pre and post treatment. Elevated initial serum IgE levels appeared to be associated with poor outcome. Although one-half of nephrotic children had detectable specific IgE to common allergens (dust mites or milk), the presence of specific IgE was not correlated with disease outcome. Serum IL-4 levels were not different among normal controls and patients with SRNS or SSNS. However, serum sCD23 levels in NS patients were significantly higher than in normal controls both pre and post treatment. Serum sCD23, but not IL-4, levels were correlated with serum total IgE levels. Our results suggest that regulation of total IgE production correlates with the disease activity and outcome of NS, although the presence of common specific IgE may not be linked to the pathogenesis.     

A CD200FC immunoadhesin prolongs rat islet xenograft survival in mice

BACKGROUND: A solubilized form of the CD200 molecule, CD200Fc, has been shown to suppress allograft rejection and development of collagen-induced arthritis in mice. We investigated whether the same molecule could prolong survival of rat islet xenografts. METHODS: Streptozocin-treated mice, receiving injections with anti-asialo-GM1 antibody, received rat islets ( approximately 400/mouse) under the kidney capsule or injected into the portal vein, along with rapamycin treatment. Thereafter mice received injections of CD200Fc (10 microg/mouse/injection) or control mouse IgG2. Blood glucose was monitored daily. Some mice received additional injections of anti-CD200/-CD200R monoclonal antibodies. RESULTS: Portal vein delivery of islets led to more extended resolution of diabetes than did transplantation under the kidney capsule. CD200Fc further prolonged survival in either case, an effect abolished by anti-CD200 or F(ab')2 anti-CD200R mAbs, but not by whole anti-CD200R (anti-CD200R Ig). Spleen cells taken from CD200Fc-treated mice showed polarization to type-2 cytokine production (interleukin-4, interleukin-10) on restimulation with rat splenocytes in culture, in comparison to cells from control mice (type-1 cytokines, interlulin-2, interferon-gamma). CONCLUSION: CD200:CD200R interactions are important in regulating rat islet xenograft survival.     

Nephrotic syndrome due to focal glomerulosclerosis and undifferentiated carcinoma

In neoplastic disorder-related nephrotic syndrome, focal glomerulosclerosis (FGS) has been reported mainly in hematological disorders like minimal change nephrotic syndrome (MCNS) in association with presumed T lymphocyte dysfunction. The association of FGS with cancer or solid tumor is rare. We report a case of nephrotic syndrome due to FGS in a patient with undifferentiated adenocarcinoma of the cystic duct. Although the underlying mechanism is unclear, the development of FGS seemed to be related to the poor histological differentiation of the cancer in the possibility of production of an active peptide.     

Zeta chain expression in T and NK cells in peripheral blood of children with nephrotic syndrome

The idiopathic nephrotic syndrome (INS) has been related to cellular immune disturbances. The zeta (ζ) chain, a component of the T-cell receptor/CD3 (TCR) complex and CD16 heterodimer in NK cells, plays a crucial role in T and NK cell activation and proliferation. The aim of our study was to examine zeta chain expression in CD4+, CD8+ T lymphocytes and NK cells in the peripheral blood of children with INS and to evaluate the effect of anti-CD3+rIL-2 stimulation on the level of zeta chain expression in the INS pediatric population. The study group consisted of 15 children with INS in relapse, 16 patients with INS in clinical remission, and 17 controls. The percentage of zeta-positive cells and the values of mean fluorescence intensity (MFI) were determined by flow cytometry. Compared with that in the controls, the percentage of zeta+ freshly isolated NK cells in children with INS in relapse was significantly lower, whereas, in CD3+/CD4+ and CD3+/CD8+ populations, no alteration was observed. There were no differences in the MFI values between the populations of freshly isolated cells either. Stimulation with anti-CD3+rIL-2 decreased the percentage of zeta+/CD4+ T cells and NKzeta+ cells in a significant way in all the groups analysed, whereas the percentage of zeta+/CD8+ T cells decreased significantly only in patients with INS in relapse. The altered pattern of zeta expression in fresh NK cells from children with INS in relapse, and the disturbed response of zeta+/CD8+ T cells to anti-CD3+rIL-2 stimulation in relapse, suggests the possible role of this chain in immune dysregulation in INS, particularly with regard to cytotoxic cells.     

Two-color analysis of lymphocyte subpopulations in membranous nephropathy and minimal change nephrotic syndrome

Two-color flow cytometry was carried out to determine the correlation between cell mediated immunity and the development of the nephrotic stage in patients with membranous nephropathy (MN) and minimal change nephrotic syndrome (MCNS). In this study, lymphocyte subpopulations were measured by two-color flow cytometry using various monoclonal antibodies of the Leu series. Thirty patients with MN and 25 patients with MCNS were examined. Clinically, those patients were divided into four stages as follows: (1) untreated nephrotic stage, (2) prednisolone (PSL) treated nephrotic stage, (3) persistent proteinuria stage (incomplete remission, ICR), and (4) complete remission (CR). Pathologically, the patients with MN also divided into four stages I-IV, according to Churg's classification. The values of the Leu 3a/Leu 2a ratio in patients in the untreated nephrotic stage of MN and MCNS were significantly higher than those in the remission stage in both diseases (P less than 0.01, P less than 0.05, respectively). Two-color flow cytometry showed that the reduction of Leu2a positive cells was mainly due to a decrease of Leu 2a+15+ subsets (suppressor T cells) in the untreated nephrotic stage and relative increase of Leu 3a+8+ subsets (suppressor inducer T cells). There was no significant difference in these findings among the histopathological stages in patients with MN. Patients with MN and MCNS showed a significant elevation of Leu 2a+DR+ cells after the treatment of PSL. The abnormalities of suppressor T cells and suppressor inducer T cells in the peripheral blood appear to be correlated with clinical activities of the nephrotic syndrome due to MN or MCNS, but not to be related to the pathogenesis of either disease. It is postulated that PSL might stimulate Leu 2a positive cells and Leu 3a positive cells, and then increase the number of Leu 2a+15+ cells in the peripheral blood of patients with MN and MCNS.     

Polymorphism of the interleukin-4, interleukin-13, and signal transducer and activator of transcription 6 genes in Indonesian children with minimal change nephrotic syndrome

BACKGROUND/AIMS: Minimal change nephrotic syndrome (MCNS) in children is frequently associated with allergy and immunoglobulin E (IgE) production. T-helper subtype 2 cytokines, such as interleukin (IL)-4 and IL-13, have been implicated in the regulation of IgE production. We investigated the associations of gene polymorphisms of IL-4, IL-13, and signal transducer and activator 6 (STAT6) in Indonesian children with MCNS (n = 84) and controls with neither allergic nor renal disease (n = 61). METHODS: Polymerase chain reaction-restriction fragment length polymorphism was used to determine the IL-4 promoter gene polymorphism (-590C/T) and IL-13 gene polymorphism (4257G/A), and direct sequencing was used for the STAT6 3S untranslated region (2964G/A) polymorphism. RESULTS: There was a significant difference between the MCNS group and the controls in the genotypic distribution of IL-4 and IL-13 gene polymorphism. In the case of the IL-4 promoter gene, the frequency of the CC homozygote was significantly lower in the MCNS group than in the controls, while, in the case of IL-13, the frequency of the GG homozygote was significantly lower in the MCNS group. However, there was no difference between the MCNS group and the controls in the STAT6 gene polymorphism. CONCLUSION: The genetic variations in the IL-4 and IL-13 genes may be associated with predisposition to MCNS.     

Rituximab and minimal change nephrotic syndrome: a therapeutic option

Minimal change nephrotic syndrome (MCNS) usually responds to steroids but frequently relapses, requiring additional treatment with immunosuppressive agents. Rituximab is a chimeric murine/human monoclonal immunoglobulin G1 antibody that targets CD20, a B-cell differentiation marker. B-cell recovery begins at approximately 6 months following the completion of treatment. Rituximab has a beneficial effect, with the sustained remission or reduction of proteinuria in patients with steroid-dependent MCNS. Relapses are thought to be associated with an increase in CD19 cells. The mean serum half-life of rituximab was reported to be 10–15 days in patients with steroid-dependent nephrotic syndrome. Only infusion reactions, such as rash and chills, occurred after single-dose rituximab infusion and can be managed by pre-medication or infusion rate adjustments. Even though severe adverse effects of rituximab are not expected, physicians must be aware of potentially life-threatening adverse effects. Controlled randomized trials that include adult patients with steroid-dependent or steroid-resistant MCNS are required to prove the efficacy and safety of rituximab and to evaluate the cost-effectiveness of rituximab treatment.     

CD2 and CD3 receptor-mediated tolerance: constraints on T cell activation

BACKGROUND: Antigen specific allograft tolerance is induced in mice by anti-CD2 plus anti-CD3epsilon monoclonal antibody (mAb) treatment. Because anti-CD2 mAb inhibits several aspects of anti-CD3epsilon driven T cell activation, we investigated what components of T cell activation are required or may be dispensed with for tolerance induction. Anti-CD3epsilon-mediated T cell activation depends on FcgammaR interactions. METHODS: To assess the role of FcgammaR-mediated T cell activation in tolerance induction, FcgammaR binding IgG or non-binding IgG3 anti-CD3epsilon mAbs were examined. RESULTS: These mAbs, administered in conjunction with anti-CD2, were equally effective in inducing tolerance. Moreover, in vivo administration of a blocking mAb directed against the FcgammaR, or the use of allograft recipients deficient in FcgammaR, had no effect on tolerance induction. Blocking IL-2 using mAb directed against IL-2 or IL-2R also did not prevent the induction of tolerance. These results suggest that complete T cell activation was not required for tolerance induction. However, substitution of a partially activating mAb, directed against the T cell receptor (TCR) beta subunit for anti-CD3epsilon, failed to synergize with anti-CD2 mAb to induce tolerance. The anti-TCRbeta mAb and anti-CD3epsilon mAb were found to differentially down modulate expression of TCR/CD3 complex subunits. In particular, anti-CD3epsilon caused transient down modulation of the TCRbeta receptor subunit and the TCRzeta signaling module, and this pattern was enhanced and prolonged by anti-CD2. Anti-TCRbeta caused persistent TCRzeta modulation but no TCRbeta modulation, and anti-CD2 did not influence this pattern. CONCLUSIONS: These results suggest that, although full T cell activation is not required for the induction of tolerance by anti-CD2 plus anti-CD3epsilon mAb, a signal transduction pathway that is associated with TCRbeta and TCRzeta expression, and, specifically, is perturbed by mAb binding of the CD3epsilon epitope, is critical.     

Effects of anti-IgE in asthmatic subjects

A humanized murine monoclonal antibody directed to the Fc epsilon R1-binding domain of human IgE (rhuMAb-E25) has been shown to inhibit the binding of IgE to mast cells without provoking mast cell activation. To examine the effects of neutralizing IgE on allergic airway responses, we assessed the effects of 9 wk of treatment with rhuMAb-E25 in a parallel group, randomized, double-blind, placebo-controlled study of 19 allergic asthmatic subjects. We found that treatment with rhuMAb-E25 reduced the serum IgE, increased the dose of allergen needed to provoke an early asthmatic response, reduced the mean maximal fall in FEV1 during the early response (30 +/- 10% at baseline to 18.8 +/- 8%, versus 33 +/- 8% at baseline to 34 +/- 4% after placebo; p = 0.01), and reduced the mean maximal fall in FEV1 during the late response (24 +/- 20% at baseline to 9 +/- 10% versus 20 +/- 17% at baseline to 18 +/- 17% after placebo; p = 0.047). We conclude that an anti-IgE monoclonal antibody, which inhibits binding of IgE to its receptor, suppresses the early- and late-phase responses to inhaled allergen in allergic asthmatic subjects. Targeting IgE with rhuMAb-E25 might be a useful treatment for allergic asthma.     

Atopy,serum IgE,and interleukin-13 in steroid-responsive nephrotic syndrome

Earlier studies have demonstrated a strong association of steroid-responsive nephrotic syndrome (SRNS), atopy, and elevated serum IgE levels. Interleukin (IL-13) gene expression is significantly increased in children with SRNS in relapse. As interferon (IFN)-, IL-13, and IL-4 have regulatory effects on IgE synthesis, we examined the relationship between intracellular cytokine production and serum IgE levels in children with SRNS, in order to further define the reported association with atopy. The median serum IgE levels in nephrotic patients in relapse with (492 U/ml) or without atopy (561 U/ml) were significantly higher than those in remission (221 U/ml, P<0.002 or 90 U/ml, P<0.001, respectively) and non-atopic controls (177 U/ml) (P<0.001). The percentage of CD3+ IL-13-producing cells was significantly higher in nephrotic children in relapse, and correlated with the serum IgE levels during the active phase of the disease (r=0.90, P<0.001). These data suggest that the elevated serum IgE levels during relapses of SRNS were the result of upregulation of IL-13. This probably reflects some common immune activation following various stimuli, rather than a direct association with atopy.     

检测微小病变型肾病综合征患者腺苷脱氨酶的临床意义

目的：检测微小病变型肾病综合征（MCNS）患者血清及淋巴细胞内腺苷脱氨酶（S-ADA,L-ADA）活性的变化,以评估其细胞免疫状态,并探讨临床意义。方法：选取初发的MCNS患者30例,应用免疫学技术测定不同的病程阶段患者的S-ADA及L-ADA活性,比较ADA活性变化。结果：激素治疗前,激素敏感组（SS）、激素依赖组（SD）和激素抵抗组（SR）两两比较无统计学意义（P〉0.05）,经激素足量治疗4周后,SS组、SD组S-ADA和L-ADA活性均下降,SS组下降最明显（P〈0.01）,而SR组无变化（P〉0.05）;短期缓解期组S-ADA活性降至对照组水平,L-ADA活性也下降,但与对照组相比,其水平仍高（P〈0.05）;长期缓解期组S-ADA及L-ADA活性与对照组相比,无统计学差异（P〉0.05）;S-ADA及L-ADA活性有显著的相关性（r=0.811,P〈0.01）。结论：MCNS患者存在着细胞免疫的紊乱,检测S-ADA及L-ADA活性可作为判断临床治疗效果的指标之一。