Perforin gene expression in T lymphocytes correlates with disease activity in immunoglobulin A nephropathy.

1. We studied perforin gene expression in T lymphocytes obtained from 26 patients with IgA nephropathy and from 15 healthy age-matched control subjects. 2. The majority of patients with IgA nephropathy (96%) had elevated perforin mRNA expression, whereas no perforin mRNA expression was detected in the T lymphocytes of normal control subjects. 3. A positive correlation was noted between perforin mRNA expression and urinary protein excretion. 4. Perforin mRNA expression correlated also with the histopathology in the renal tissue of patients with IgA nephropathy. 5. Sixty per cent of patients with grade III or IV histopathology had high perforin mRNA expression in T lymphocytes [more than (++)]. 6. These studies suggest that disregulation of perforin gene expression in T lymphocytes may be associated with the progression of IgA nephropathy and could be used as an indicator of disease activity.     

Aggregation of erythrocyte sodium/lithium countertransport activity in families of patients with immunoglobulin A nephropathy.

1. We evaluated the inheritance of erythrocyte Na+/Li+ countertransport activity in IgA nephropathy by assessing this parameter in 19 patients with biopsy-proven IgA nephropathy and in their 53 relatives (32 parents and 21 siblings). The possible use of erythrocyte Na+/Li+ countertransport activity as a marker of poor prognosis was also evaluated. 2. A significant correlation was found between 'familial' and proband Na+/Li+ countertransport activity, but not between that of spouses. 3. Mean blood pressure, although within the normal range, and Na+/Li+ countertransport activity were significantly higher in patients with proteinuria than in those without proteinuria. 4. Parents of proteinuric patients had a higher Na+/Li+ countertransport activity than parents of non-proteinuric patients. 5. In IgA nephropathy the inheritance of erythrocyte Na+/Li+ countertransport activity was preserved. Therefore genetic factors could play a role in the non-immunological progression of IgA nephropathy.     

Circulating immune complexes in pulmonary tuberculosis

The presence of circulating Immune complexes (CIC) in sera from patients with pulmonary tuberculosis was demonstrated by 3 techniques (a) latex agglutination (b) 3.5% PEG precipitation and determination of optical density (O.D.) at 280 nm and (c) radioimmunoassay (RIA) of CIC using bovine spermatozoa. 40 normal control sera and 100 T.B. patients sera were included in the study. 12% cases were positive for CIC by all the 3 methods mentioned above, 13% were negative by all the 3 methods and the remaining patients were positive by one or more methods of detection. To correlate the levels of CIC as detected by different techniques with the activity of the disease, patients were broadly grouped as (a) sputum positive and (b) sputum negative. Higher levels of CIC were obtained in sputum positive cases than sputum negative by all the 3 methods studied. While IgG, IgA and IgM were elevated in the CIC of T.B. patients, and IgG and IgA were also present in controls, IgM immunoglobulins were detected only in patients and not in controls. The effect of antitubercular treatment on the levels of CIC was also evaluated and it was found that the levels of CIC remained unchanged even after prolonged chemotherapy.     

Selective deposition of immunoglobulin A1 in immunoglobulin A nephropathy, anaphylactoid purpura nephritis, and systemic lupus erythematosus.

To further characterize the IgA deposits found in glomeruli of patients with IgA nephropathy, anaphylactoid purpura nephritis, and systemic lupus erythematosus, renal biopsies from patients with these disorders were stained by immunofluorescence with monoclonal anti-IgA subclass reagents, anti-light chain reagents and anti-J chain. The mesangium and peripheral capillary were brightly stained for IgA1 and were negative for IgA2. IgA1 and, to a lesser extent, IgA2 were contained in tubular casts. Both kappa and lambda light chains were found in all deposits. The intensity of J chain staining correlated with the intensity of IgM and not IgA staining. Biopsies brightly stained for IgA but negative for IgM were negative for J chain. These results indicate that glomerular IgA deposits in these disorders consist predominantly of monomers of IgA1.     

Immunoregulatory studies in patients with IgA nephropathy

Numerous studies have evaluated T cell subsets and in vitro IgA synthesis in patients with IgA nephropathy. These reports have resulted in the hypothesis that defective regulation of IgA synthesis is important in the pathogenesis of IgA nephropathy. Baseline immunologic measurements were performed in a clinically well characterized group of 19 pediatric and 13 adult (greater than or equal to 18 years) patients with no macrohematuria or intercurrent infection at the time of study. Mean percentages of OKT3 and OKT4 subsets were significantly decreased for the patients as compared to healthy adult controls. Mean T4:T8 ratios were similar for control and patient groups although 7 patients had T4:T8 ratios greater than 2 SD above the control mean. Unstimulated and pokeweed mitogen stimulated in vitro IgA synthesis was similar for patients and controls. During six episodes of macrohematuria in five patients no significant changes occurred for T cell subset percentages, while mean T4:T8 ratios decreased from baseline. Mean serum concentration of IgA increased during these episodes, although in vitro IgA synthesis remained normal. Our data fail to demonstrate a consistent immunoregulatory abnormality for patients with IgA nephropathy.     

Detection of anionic sites and immunoglobulin A deposits in the glomerular capillary walls from patients with IgA nephropathy

A study of anionic sites in the glomerular basement membrane (GBM) from patients with immunoglobulin A (IgA) nephropathy is described. The relationship between the deposition of IgA and the detection of glomerular extracellular components, i.e., noncollagenous (NC-1) domain of Type IV collagen, in the glomerular capillary walls was examined by double immunofluorescence. Renal biopsy specimens from patients with IgA nephropathy were immersed in polyethyleneimine (PEI) as a cationic probe and then examined by electron microscopy. Renal specimens were also incubated with mouse monoclonal anti-NC-1 domain of Type IV collagen and then stained with fluorescein isothiocyanate (FITC)-labelled goat antimouse Ig antiserum. After these reactions, sections were stained with rhodamine-labelled rabbit antihuman IgA antiserum. GBM subepithelial anionic sites marked by PEI were altered by the deposition of electron-dense deposits (EDD) in patients with IgA nephropathy and there was a significant correlation between the levels of proteinuria and the incidence of EDD in the GBM in such patients. Marked proteinuria was observed in patients who showed loss of anionic sites in the GBM by electron microscopy. By double immunofluorescence, IgA was shown to be focally deposited outside the NC-1 domain of Type IV collagen-detected regions in the same patients. The authors concluded that high levels of proteinuria might be due to alterations of the size barrier and/or anionic sites of GBM in the moderate stage of IgA nephropathy.     

病理程度不同IgA肾病患者血清代谢的差异性研究

目的寻找诊断IgA肾病潜在的生物标志物,并探讨其代谢途径。方法原发性IgA肾病患者35例(IgA肾病组),其中低危组23例,高危组12例,同期体检健康者23名为对照组,采用基于质谱核磁共振代谢组技术结合模式识别分析各组血清代谢物的差异。结果对照组与IgA肾病组血清代谢物比较差异有统计学意义(P<0.05);低危组和高危组血清代谢物比较差异无统计学意义(P>0.05);共筛选出24种IgA肾病的特征代谢物。结论应用质谱核磁共振代谢组技术成功建立了IgA肾病血清代谢诊断模型。     

Polymorphism of immunoglobulin heavy chain switch region in IgA nephropathy]

We examined the relation between IgA hyperproduction and polymorphism of the immunoglobulin heavy chain switch (S) region in patients with IgA nephropathy. The frequency of the heterozygous phenotype of IgA2 switch region was significantly increased in these patients. The patients showed a significant increase in the amount of serum IgA, IgA bearing cells and levels of proteinuria. We also compared two reports on the S region in Europe, which showed different results, and found different frequencies in the phenotype of the S region. These findings suggest that IgAN patients in various countries show heterogeneity in the S region and that this polymorphism might be associated with IgA hyperproduction and the development of proteinuria in Japanese patients.     

19S IgM rheumatoid factor-7S IgG rheumatoid factor immune complexes isolated in patients with rheumatoid arthritis

Sera of 12 patients with rheumatoid arthritis (RA) who had positive IgM-rheumatoid factor (RF) tests were separated by use of immunoabsorbent columns (goat anti-human C3 [alpha HC3] and rabbit anti-human C1q [alpha HC1q]) and polyethylene glycol (PEG) precipitation-protein A affinity chromatography to isolate their immune complexes (IC). The isolated fractions were assayed for 19S IgM RF and 7S IgG RF by enzyme-linked immunosorbent assay (ELISA). The sera were further analyzed by preparative isoelectric focusing (IEF). The alpha HC1q and alpha HC3 columns were sequentially eluted with barbital buffer, 0.02 mol/L EDTA, 0.5 mol/L NaCl, and 1 mol/L propionic acid. All 12 patients had IgM RF and IgG RF in the EDTA fractions from both immunoabsorbent columns, but only IgM RF in the NaCl fractions. The PEG precipitation-protein A preparations were eluted with 0.5 mol/L glycine HCl and 3.5 mol/L MgCl2. All 12 patients had significant titers of 19S IgM RF (greater than or equal to 1:192) and 7S IgG RF (greater than or equal to 1:96) in the acid-eluted fraction. Analysis of the sera by preparative IEF revealed IgM RF with a polyclonal spectrotype pattern with pH of 3.0 to 10.0, but predominantly acidic proteins with isoelectric points of 4.0 to 5.5 IgG RF were found in the same restricted spectrotypic pattern. These studies demonstrated that IC can be detected in the sera of patients with RA by isolation with alpha HC1q and alpha HC3 immunoabsorbent columns and PEG precipitation-protein A affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expansion of Fc receptor-bearing T lymphocytes in patients with immunoglobulin G and immunoglobulin A myeloma.

Lymphocytes obtained from the blood of normal individuals and six patients with newly diagnosed multiple myeloma were separated into T and non-T cell populations by rosette-formation with sheep erythrocytes, and were then assayed for the presence of surface membrane Fc receptors. When compared with normal individuals, four patients with IgG myeloma had a three- to fourfold increase in T cells with IgG receptors (T gamma cells) and two patients with IgA myeloma had a two- to threefold increase in T cells with IgA receptors (T alpha cells). Patients with IgG or IgA myeloma had normal numbers of non-T lymphocytes with surface receptors for IgG and IgA, respectively. The finding that human myeloma is accompanied by elevated numbers of T cells with Fc receptors for the heavy chain class of the myeloma protein: (1) may account for the apparent "monoclonal" lymphocyte population in patients with myeloma; (b) extends to humans similar observations made in mice with secretory plasmacytomas; and (c) is of interest because T cells with Fc receptors are immunoregulatory lymphocytes.     

Profiles of brucella-specific immunoglobulin G subclasses in sera of patients with acute and chronic brucellosis

Serum specimens from patients with acute brucellosis (164), chronic brucellosis (22) and controls (75) were tested by ELISA for brucella-specific IgG, IgM, IgA and subclasses of IgG1 to 4 antibodies, Rose Bengal antigen slide agglutination (RB) and microagglutination (MA) tests. The RB and MA showed similar results and were positive in 100% and 64% of specimens from patients with acute and chronic brucellosis respectively. ELISA IgG and IgA were positive in sera from all patients with brucellosis while IgM was positive in 100% and 32% of specimens from patients with acute and chronic brucellosis respectively. Elevated IgG subclasses to brucella antigen were found in different proportions in the sera of patients with acute and chronic brucellosis. In patients with acute brucellosis, IgG1 was the predominant response (79%) followed by IgG3 (58%), IgG2 (36%) and IgG4 (14%). In contrast, IgG4 was the predominating subclass response (73%) in patients with chronic brucellosis followed by IgG1 (41%), IgG2 and IgG3 (27% each). When considering the most common elevated IgG subclasses, in each serum, either alone or in combination with each other, patients with acute brucellosis showed IgG1+IgG3 (24%), IgG1 (19%), IgG1+IgG2+IgG3 (16%) while patients with chronic brucellosis showed IgG4 (27%) and IgG1+IgG2+IgG3+IgG4 (18%). This study reveals that in addition to the difference in brucella-specific Ig class response in patients with acute (IgG, IgM, IgA) and chronic (IgG, IgA) brucellosis, the profiles of IgG subclasses are different where IgG1 predominates in the acute and IgG4 in the chronic stages of the disease.     

Immunoglobulin A nephropathy. Quantitative immunohistomorphometry of the tonsillar plasma cells evidences an inversion of the immunoglobulin A versus immunoglobulin G secreting cell balance.

Primary IgA nephropathy (Berger's disease) is characterized by renal deposits of IgA, the origin of which is still unknown. However, several clinical and biological findings suggest that these immunoglobulins might have a mucosal origin, and that such patients should present mucosal abnormalities. This paper reports the results of the immunohistomorphometrical analysis of tonsillar plasma cells from seven patients suffering from Berger's disease and seven controls also with recurrent tonsillitis. IgG, IgA, and IgM-secreting cells were enumerated after immunofluorescent staining of serial frozen-cut sections from 20 tonsils. In controls, a predominance of the IgG-secreting population, similar to this reported in the literature was observed (65% of IgG secreting cells and 29% of IgA plasma cells), while an inversion in the patients' plasma cells percentages was evidenced (IgG:37%, IgA:56%). This increment in the IgA population was paralleled by an augmentation of the number of dimeric IgA-secreting cells (75% of IgA plasma cells), stained both for cytoplasmic IgA and J chain. In controls, the latter cells were in similar proportions as previously reported by others (45% of IgA plasma cells). These results demonstrate an imbalance in the IgA-producing system of patients with Berger's disease, which is in keeping with the hypothesis favoring a mucosal origin for the mesangial IgA present in their kidneys.     

Determination of human immunoglobulin A and secretory immunoglobulin A in bronchoalveolar lavage fluids by solid phase enzyme immunoassay

Solid phase enzyme immunoassay methods for the determination of secretory immunoglobulin A (IgA) and the total amount of serum and secretory IgA in bronchoalveolar lavage fluids (BALF) were developed. The solid phase was prepared by immobilizing rabbit anti-human IgA. Horseradish peroxidase-conjugated goat anti-secretory component or horseradish peroxidase-conjugated goat anti-human IgA (Fc) were used as labeled antibodies. The minimum detectable amounts of secretory IgA and total IgA were 2 and 0.5 ng/well, respectively. These assay methods were successfully applied to the determination of secretory and total IgA levels in BALF samples obtained from 44 subjects including healthy non-smokers, smokers and patients with the following lung diseases: idiopathic pulmonary fibrosis, sarcoidosis and hypersensitivity pneumonitis. The secretory and total IgA levels in BALF collected from healthy non-smokers (n = 9) were 10.5 ± 3.6 and 25.4 ± 15.5 (S.D.) μg/ml, respectively. In healthy smokers, the secretory IgA concentration was significantly decreased and in idiopathic pulmonary fibrosis, the total IgA was increased. These results indicate that the quantitation of secretory and total IgA may be useful for the investigation of lung disease.     

The role of IgA1 rheumatoid factor in the formation of IgA-containing immune complexes in Henoch-Sch?nlein purpura

The present study was performed to investigate the role of IgA rheumatoid factor (RF) in the formation of IgA-containing immune complexes and to determine the IgA subclass composition of IgA RF in patients with Henoch-Sch?nlein purpura (HSP). Immune complexes were isolated from the sera of 22 children with HSP and 13 controls by means of polyethylene glycol (PEG) precipitation. The percentage of IgG, IgA, and IgM precipitated by PEG was significantly greater in HSP patients than controls (p less than 0.01). There was a strong correlation (r = 0.723, p less than 0.001) between the amount of IgG and IgA in the PEG precipitates from HSP patients, but not controls. HSP patients had significantly higher levels of IgA RF in their serum (p less than 0.05) and in their PEG precipitates (p less than 0.05) compared to controls. There was a strong correlation between IgA RF concentrations in the serum and PEG precipitates in HSP patients (r = 0.910, p less than 0.001). PEG precipitation eliminated IgA RF activity from the serum of 7 of 8 HSP patients tested, and substantially reduced the titer in the remaining patient. IgA RF was recovered in the PEG precipitates from all patients. Testing of HSP patients showed that IgA1 was the predominant IgA subclass of the serum IgA RF (p less than 0.02) and PEG precipitate IgA RF (p less than 0.01). These results indicate that IgA RF is a constituent of IgA-containing immune complexes in HSP, and that IgA RF is composed primarily of IgA1.     

Serial circulating immune complexes and mononuclear phagocyte system function in infective endocarditis

Twenty patients with infective endocarditis were followed prospectively and all had elevated levels of circulating immune complexes (CICs) detected by staphylococcal binding assay. Mean CIC levels declined for the group as a whole (193 micrograms/ml +/- 24 to 100 +/- 17, p less than 0.05) and became undetectable in eight patients (47%) who were cured. Patients who died or had complicated courses had higher mean CIC levels at the start and finish (254 micrograms/ml +/- 24 and 145 +/- 37) of antibiotic therapy than patients with uncomplicated courses (178 micrograms/ml +/- 19 and 38 +/- 24), p less than 0.05. CIC levels did not decline significantly in patients with glomerulonephritis or arthritis, in contrast to patients without these features. Despite elevated CIC levels, 10 patients had enhanced mononuclear phagocyte system (MPS) function as assessed by Fc-dependent IgG-coated red blood cell clearance. These data suggest that CICs probably are pathogenic in endocarditis and may contribute to the development of arthritis and glomerulonephritis. Elevated CICs in infective endocarditis do not appear to be directly related to defective MPS function.     

Mesangial cell autoantigens in immunoglobulin A nephropathy and Henoch-Schönlein purpura.

The autoantigen(s) that we have previously described in human glomeruli, recognized in IgA nephropathy, has (have) been identified as mesangial cell in origin. Cultured mesangial cells expressed 48- and 55-kD components binding IgG isotype autoantibodies (IgG-MESCA) present in sera of patients with both IgA nephropathy and Henoch-Sch?nlein purpura (HSP). IgG-MESCA were not detected in sera of normals, or patients with other autoimmune-mediated glomerulonephritides: anti-glomerular basement membrane disease, Wegener's granulomatosis, or in IgM-mesangial proliferative disease. Binding specificity was proven by F(ab')2 studies in enzyme-linked immunosorbent assay (ELISA) and Western blotting, and there was no significant affinity of IgA or IgM immunoglobulins. Fluorescein isothiocyanate-conjugated IgG from ELISA-positive sera localized to the mesangium and peripheral capillary loops of glomeruli, supporting the belief that the antigen is expressed in normal human renal tissue. However, only about one third of mesangial cells in culture showed affinity for IgG from ELISA-positive sera, suggesting variable expression of the antigen(s) in vitro. The only autoantigen(s) present in glomeruli, and extractable from whole normal glomeruli by the techniques employed, localized on the mesangial cell. In both IgA nephropathy and HSP, autoimmunity was intermittently present, with fluctuating levels of IgG-MESCA detectable in sera. There were positive correlations with the degree of glomerular injury assessed by erythrocyturia and proteinuria in IgA nephropathy, but significance was reached with only the degree of hematuria in HSP. These findings suggest a contributing role in the pathogenesis of the mesangial proliferative lesions and demonstrate autoimmunity common to both IgA nephropathy and HSP.     

Sperm-related antigens, antibodies, and circulating immune complexes in sera of recently vasectomized men.

Sera from 35 men were collected before and at timed intervals subsequent to vasectomy and examined for the presence of (a) antibody reactive with human spermatozoa, (b) sperm-related antigen, and (c) circulating immune complexes (CIC). Fewer than 10% of the men examined were ever positive for antisperm antibodies. However, sperm-related antigens were elevated in the sera of 18, 18, and 26% of the mean at 2 wk, 2 mo, and 4 mo postvasectomy, respectively. CIC were detected in the sera of some vasectomized men by three different assays. The CIC in patients' sera were precipitated with polyethylene glycol, dissociated, and the individual CIC components identified by an enzyme-linked immunosorbent assay. Most, but not all, of the CIC contained antigen reactive with antisperm immunoglobulin (Ig)G and some also contained complement components C3 and/or Clq. IgA was identified in some of the CIC positive for IgG and sperm antigen and two men had IgM-containing CIC. Analysis of the CIC by sucrose gradient centrifugation revealed them to be heterogeneous in size.     

Effects of lisinopril administration on blood bcl-2 concentrations in patients with immunoglobulin A nephropathy.

We evaluated blood concentrations of bcl-2, a proto-oncogene that can inhibit apoptotic phenomena, in a group of patients with immunoglobulin A (IgA) nephropathy. Concentrations of bcl-2 were higher in patients with proteinuria than in those without proteinuria. A 6-month course of 5 mg/day lisinopril given to subjects with proteinuria significantly reduced blood bcl-2 concentrations and caused a reduction in proteinuria. Therefore increased blood bcl-2 concentrations may be considered an index of risk in subjects with IgA nephropathy, and the positive effects of angiotensin-converting enzyme inhibitors on proteinuria in patients with IgA nephropathy may be attributed, at least in part, to their effect on the mechanisms that regulate apoptosis. This is of fundamental importance in resolving glomerular hypercellularity in the course of glomerulonephritis.     

Detection of platelets in urinary sediments from patients with “Advanced” stages of immunoglobulin a nephropathy

The presence of platelets in urinary sediments was studied by an immunofluorescence method in patients with immunoglobulin A (IgA) nephropathy. The aim of the present study was to determine if the presence of platelets in urinary sediments is correlated with glomerular injuries in patients with IgA nephropathy. Sixteen patients with IgA nephropathy and eight with diffuse proliferative glomerulo nephritis (DPGN) were examined. This study showed a significant correlation between the number of platelets found in urinary sediments and the severity of glomerular injuries in patients with IgA nephropathy. It was suggested that detection of platelets in urinary sediments is useful in evaluating the degree of histopathological changes and/or prognosis in IgA nephropathy prior to renal biopsy.     

Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity

IgA nephropathy (IgAN) is characterized by circulating immune complexes composed of galactose-deficient IgA1 and a glycan-specific IgG antibody. These immune complexes deposit in the glomerular mesangium and induce the mesangioproliferative glomerulonephritis characteristic of IgAN. To define the precise specificities and molecular properties of the IgG antibodies, we generated EBV-immortalized IgG-secreting lymphocytes from patients with IgAN and found that the secreted IgG formed complexes with galactose-deficient IgA1 in a glycan-dependent manner. We cloned and sequenced the heavy- and light-chain antigen-binding domains of IgG specific for galactose-deficient IgA1 and identified an A to S substitution in the complementarity-determining region 3 of the variable region of the gene encoding the IgG heavy chain in IgAN patients. Furthermore, site-directed mutagenesis that reverted the residue to alanine reduced the binding of recombinant IgG to galactose-deficient IgA1. Finally, we developed a dot-blot assay for the glycan-specific IgG antibody that differentiated patients with IgAN from healthy and disease controls with 88% specificity and 95% sensitivity and found that elevated levels of this antibody in the sera of patients with IgAN correlated with proteinuria. Collectively, these findings indicate that glycan-specific antibodies are associated with the development of IgAN and may represent a disease-specific marker and potential therapeutic target.