Persistent high prevalence of thyroid antibodies after immunosuppressive therapy in subjects with glomerulonephritis. A prospective three-year follow-up study.

The prevalence of thyroid antibodies, indicating an autoimmune thyroiditis, has been shown to be significantly increased in patients with autoimmune diseases. A 3-year prospective follow-up study of 42 patients with biopsy-confirmed glomerulonephritis is presented. Although the majority of patients had been treated with immunosuppressants, the prevalence of thyroid peroxidase antibodies was unchanged in both females and males, 47 and 15% respectively, at follow-up. Likewise, the prevalence of thyroglobulin antibodies was unaffected as was that of antinuclear antibodies (ANA) when analysing males and females together. However, for males there was a trend to higher prevalence for ANA at follow-up. On the other hand, the prevalence of antineutrophil cytoplasmic antibodies declined. Furthermore, thyroid antibodies were not restricted to membranous nephropathy, and notably found in 4 out of the 8 patients with vasculitis.

Relapse rate, renal survival, and cancer morbidity in patients with Wegener's granulomatosis or microscopic polyangiitis with renal involvement.

Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) are both frequently associated with antineutrophil cytoplasmic autoantibodies (ANCA). Immunosuppressive treatment has dramatically improved outcome for these patients, but today we have to deal with the problems of relapses, cases refractory to treatment, and long-term side effects of therapy. This study comprises a consecutive series of 123 patients with WG (n=56) or MPA (n=67) with biopsy-confirmed renal involvement, followed up for a median of 55 mo (range, 0.1 to 273.2 mo). ANCA was detected by enzyme-linked immunosorbent assay in 97% of patients. Nearly half of the patients (46%) relapsed. There was no statistically significant difference in overall relapse rate according to type of ANCA. Renal survival was 78% in patients alive at the end of follow-up. Three variables seemed important for renal survival: serum creatinine, the titer of proteinase 3-ANCA measured by capture enzyme-linked immunosorbent assay, and B thrombocyte count, at time of referral. Cancer incidence data were obtained from the population-based South Swedish Regional Tumor Registry. Standardized morbidity ratio was calculated using expected values from the health care region. We found an 11-fold increase in risk for bladder cancer in patients treated with cyclophosphamide for at least 12 mo. Skin carcinoma had the strongest relationship with azathioprine use for at least 12 mo and with corticosteroid therapy for at least 48 mo. In addition, four patients developed myelodysplastic syndrome and five had carcinoma in situ of the skin. Because the therapeutic regimen used today is not efficient enough to prevent relapses and is associated with a host of side effects, of which the risk for cancer is by far the most important, improved therapy and medical care are needed for patients with WG and MPA.

Clinical evaluation of a capture ELISA for detection of proteinase-3 antineutrophil cytoplasmic antibody.

Detection of antineutrophil cytoplasmic antibodies (ANCA) has become a useful tool in the diagnosis of Wegener's granulomatosis and microscopic polyangiitis. However, the results obtained with indirect immunofluorescence (IIF) and by ELISA for ANCA demonstration do not always correlate. A possible explanation for this finding could be that proteins are denatured during the process of antigen purification or during coating onto the solid phase. To avoid this possibility, a monoclonal antibody to PR3 that is precoated on the plate can be used. In the present study we have used the monoclonal antibody (MoAb) 4A3 for the capture of PR3 in an ELISA, and a clinical evaluation of the diagnostic properties of the new capture ELISA has been made. The sensitivity of the capture PR3-ANCA ELISA was 85% in a material of c-ANCA positive sera. A specificity of 90% was obtained in analyses from patients having various forms of glomerulonephritis. There was a significantly higher diagnostic sensitivity of the capture PR3-ANCA ELISA (85%) compared to c-ANCA by IIF (58%) in patients with Wegener's granulomatosis with renal involvement. Capture PR3-ANCA and direct ELISA for MPO-ANCA together gave a diagnostic sensitivity of 98%, versus 75% using IIF. In conclusion, the capture PR3-ANCA ELISA seems to be a valuable tool in the diagnosis of Wegener's granulomatosis with renal involvement. Preliminary data suggest that the technique may have an advantage over direct ELISA for PR3-ANCA, as well as in the follow-up of c-/PR3-ANCA associated vasculitides. However, further prospective studies are needed to clarify this premise.

Prevalence of autoantibodies associated with glomerulonephritis, unaffected after extracorporeal shock wave lithotripsy for renal calculi, in a three-year follow-up.

Extracorporeal shock wave lithotripsy (ESWL) has become a useful tool in the treatment of renal calculi, but side effects may occur. Hitherto, two case reports have been published of an anti-glomerular basement membrane disease resulting in end-stage renal failure following ESWL treatment. In this prospective study of 59 consecutive patients undergoing ESWL for renal calculi, the prevalence of autoantibodies associated with glomerulonephritis was investigated before ESWL and at 3-year follow-up. The prevalences of antinuclear, anti-glomerular basement membrane, anti-neutrophil cytoplasmic and thyroid antibodies were found to be within the respective normal ranges prior to the first ESWL treatment and to be unaffected at follow-up.

Rapid screening assay for anti-GBM antibody and ANCAs; an important tool for the differential diagnosis of pulmonary renal syndromes.

BACKGROUND: Pulmonary renal syndrome is encountered in several diseases such as Goodpasture's syndrome, antineutrophil cytoplasmic antibody (ANCA) associated systemic vasculitis, systemic lupus erythematosus (SLE) and infection-associated or drug-induced glomerulonephritis. To preserve organ function it is of vital importance to make the correct diagnosis and institute adequate therapy early, in the acute phase. METHOD: An enzyme-linked immunosorbent assay (ELISA), specially designed as a rapid screening assay for antiglomerular basement membrane (anti-GBM) antibody, proteinase-3 (PR3-) ANCA and myeloperoxidase-(MPO-) ANCA were evaluated from 1060 serum samples drawn from patients with clinically suspected pulmonary renal syndrome or rapidly progressive glomerulonephritis (RPGN). RESULTS: Of the 1060 serum samples, 142 were positive for anti-GBM antibody (n = 19), PR3-ANCA (n = 60), or MPO-ANCA (n = 73). Of the 142 samples, 10 were double positive. Reanalysis of positive sera with a quantitative ELISA yielded results manifesting good correlation with those of the the rapid screening assay. Of 918 sera found to be negative in the rapid screening assay, 105 were also tested with IIF, 11 being found to be positive. However, these 11 sera manifested no specificity for PR3 or MPO, but some were specific for bactericidal/permeability-increasing proteins, lactoferrin or elastase ANCAs. Two of the patients whose sera yielded negative results in the rapid assay had systemic vasculitis. CONCLUSION: The ELISA thus detects the true antibodies to PR3, MPO, and GBM, whereas IIF detects additional specificities. The findings suggest the rapid assay results to be of high positive predictive value, and the assay to be of high diagnostic specificity and sensitivity and thus useful in the diagnostic workup in suspected cases of RPGN or pulmonary renal syndrome.

Circulating autoantibodies as serological markers in the differential diagnosis of pulmonary renal syndrome.

OBJECTIVES: Pulmonary renal syndrome (lung haemorrhage and glomerulonephritis) is a fulminant condition that warrants a rapid diagnosis and treatment to prevent mortality and preserve renal functions. However, the patients frequently present with non-specific pulmonary symptoms in the early phase of the syndrome and the diagnosis is often missed. Recently, several autoantibodies have been described in association with various forms of glomerulonephritis. We evaluated the association as well as the diagnostic and the prognostic significance of these antibodies in pulmonary renal syndrome. DESIGN: Retrospective clinical study. SETTING: University Hospital. SUBJECTS: Forty consecutive patients with biopsy verified glomerulonephritis and overt haemoptysis or pulmonary infiltrates compatible with lung haemorrhage. INTERVENTIONS: Analysis of proteinase 3 antineutrophil cytoplasm antibodies (PR3-ANCA), myeloperoxidase (MPO)-ANCA, antiglomerular basement membrane (GBM) and anti-entactin antibodies. RESULTS: Thirty-six (90%) patients possessed one or more autoantibodies. Twenty-seven (70%) patients had ANCA (PR3-ANCA, MPO-ANCA or both). The remaining positive patients (n = 9) had anti-GBM antibodies. Only two patients had anti-entactin antibodies, suggesting a poor association of these antibodies with PRS. The majority of patients with anti-GBM antibodies had a very poor clinical outcome (five irreversible renal failure; three deaths). On the other hand, despite no significant difference in clinical features or renal morphology from patients with anti-GBM antibodies, 19 patients (70%) with ANCA recovered completely following treatment. CONCLUSIONS: Our study demonstrated that the presence of autoantibodies is a predominant feature of PRS and that the type of immunologic injury is of paramount importance in determining the course of illness in this syndrome. Analysis of the aforementioned antibodies can help in an early differential diagnosis and thus, in better management of PRS.

Increased prevalence of thyroid peroxidase antibodies (TPO-Ab) in women with glomerulonephritis.

In a cross-sectional study adjusting for age, gender, and catchment area, the prevalence of thyroid antibodies was assessed in 51 consecutive subjects with biopsy-proven glomerulonephritis and in 112 control subjects admitted for extracorporeal shock-wave lithotripsy treatment for renal stones. Women with glomerulonephritis had both a significantly greater prevalence of thyroid peroxidase antibodies (odds ratio 3.85, 95% confidence interval 1.04-14.3) and an increased prevalence of elevated serum TSH values (P = 0.007). No such difference was found in men. The prevalence of thyroglobulin antibodies did not differ between the groups. It is suggested that the possibility of an autoimmune thyroid disease should be taken into consideration in patients with glomerulonephritis, particularly in women.

Anti-neutrophil cytoplasm antibodies, anti-GBM antibodies and anti-dsDNA antibodies in glomerulonephritis.

The diagnostic potential of assays detecting anti-neutrophil cytoplasm antibodies (ANCA), anti-GBM antibodies and anti-dsDNA antibodies was evaluated by examining sera from time of admission in a consecutive series of 455 patients with biopsy verified primary or secondary glomerulonephritis (GN). ANCA were classified into c- and p-ANCA by indirect immunofluorescence (IIF) and ELISAs using alfa-granule extract, proteinase-3, myeloperoxidase (MPO), elastase and lactoferrin. C-ANCA was virtually confined to 64 patients with systemic small vessel vasculitis, 66-74% being c-ANCA positive. P-ANCA against MPO, seen in 47 patients, segregated through many diagnostic categories of primary and secondary severe GN. ANCA against lactoferrin and elastase were rare. Anti-dsDNA positive patients constituted 57% of the 44 ANA-positive patients with systemic lupus erythematosus. It is concluded that the IIF and ELISAs for anti-proteinase-3, anti-MPO, anti-dsDNA and anti-GBM have an acceptable performance and are useful in the primary diagnostic work-up of patients suspected for secondary GN as the majority of such patients will be classified by these assays.

Autoimmunity and glomerulonephritis.

Autoimmunity is now unequivocally regarded as the predominant pathogenic process underlying most forms of primary and secondary glomerulonephritis in humans. Most of the investigations so far have been focused upon humoral mechanisms. Consequently, the role of cell-mediated immunity in nephritis is still incompletely understood. Nonetheless, as a result of contemporary studies, a number of previously unidentified auto-antibodies in association with glomerulonephritis have been discovered. However, apart from anti-NC1 antibodies in the classical Goodpasture syndrome, the exact role of these auto-antibodies in the pathogenesis of glomerulonephritis yet remains undefined. This fact, however, does not undermine the relevance of exploring these auto-antibodies. They have been of immense help in sub-classifying glomerulonephritis previously thought homogeneous (Figure 3). Besides, analysis of auto-antibodies has assisted tremendously in the early diagnosis of rapidly progressive glomerulonephritis. This, in turn, has aided in early commencement of therapy thus contributing to regression in morbidity and mortality resulting from these disorders. Moreover, investigation of these auto-antibodies is of enormous value for future studies aimed at understanding the pathogenic mechanisms involved in glomerulonephritis.

[Autoantibodies as serological markers in the diagnosis of glomerulonephritis]. / Autoantikroppar som serologiska markörer i diagnostiken av glomerulonefrit.

Autoimmunity is now regarded as the unequivocally predominant pathogenic process underlying most forms of primary and secondary glomerulonephritis in humans, and a number of autoantibodies occurring in conjunction with glomerulonephritis have been discovered. However, apart from the anti-NC1 antibodies occurring in classic Goodpasture's syndrome, the exact pathogenic role of these autoantibodies in human glomerulonephritis remains to be established, though this in no way diminishes the importance of their study. They have been of enormous value in subclassifying glomerulonephritis, previously thought to be a homogeneous entity. Autoantibody analysis has become a vital aid in the early diagnosis of rapidly progressive glomerulonephritis, which in turn has enabled treatment to be started early, thus contributing to a decline in the morbidity and mortality resulting from these disorders. Moreover, investigation of these autoantibodies will be of immense value in future studies focused on the pathogenic mechanisms involved in glomerulonephritis.

Circulating anti-entactin antibodies in patients with glomerulonephritis.

Sera from 305 consecutive patients in a renal biopsy series were analyzed for the presence of anti-entactin antibodies by ELISA. Of these patients, 59% had primary glomerulonephritis, 21% had secondary glomerulonephritis, while 20% had other nephropathies (noninflammatory conditions like amyloidosis, diabetic nephropathy, nephrosclerosis, etc.). Forty-one of these patients (13.4%) were positive for IgG/IgM antibodies against entactin: 60% of them had primary glomerulonephritis, 35% had secondary glomerulonephritis, while the remaining 3 patients had other nephropathies. Fifteen (70%) of the 23 patients with primary glomerulonephritis had proliferative glomerulonephritis (PGN), whereas 13 (87%) of the 15 patients with secondary glomerulonephritis were due to systemic connective tissue diseases (SCTD): 7 due to SLE, 4 due to SLE like SCTD and two due to other SCTD. There was a peak of incidence corresponding to the group aged 18 to 30 years. A majority of these patients (12 of the total 17) had primary glomerulonephritis and were associated with nephrotic or subnephrotic grade proteinuria, poorly or nonresponsive to immunosuppressive treatment and associated, in several cases, with progressive deterioration of renal function. In addition, there was a tendency to another peak in the age group 51 to 60 years. Most of these patients (6 of the total 8) had glomerulonephritis secondary, mainly, to SLE or SLE like SCTD with milder degree of proteinuria and better preserved renal functions. Anti-entactin antibodies were not found in certain glomerulonephritides like IgA nephropathy and those secondary to systemic vasculitides and in control subjects (healthy subjects, and patients with a variety of non-renal disorders including inflammatory diseases).(ABSTRACT TRUNCATED AT 250 WORDS)

Fibronectin-immunoglobulin complexes in the early course of IgA and Henoch-Schönlein nephritis.

We have previously reported the presence of circulating IgA-fibronectin complexes in adult patients with primary IgA nephropathy. In the present study five children were serially investigated during the early course of IgA nephropathy and Henoch-Schönlein glomerulonephritis. Using affinity chromatography procedures and enzyme-linked immuno-sorbent assay, IgA, IgG and IgM in complex with fibronectin were repeatedly demonstrated during the follow-up period in both groups of patients. Most patients had, at the same time, IgA, IgG, as well as IgM deposits in the glomerular mesangium. The simultaneous presence of IgA and IgG in complexes purified from serum was furthermore demonstrated. The results are thus in contrast to the findings in adults with IgA nephropathy, in whom the immunoglobulin-fibronectin complexes only contained IgA. Whether this reflects different subgroups of patients or a different pathophysiology in children and adults remains to be elucidated.

Circulating autoantibodies in patients with extracapillary glomerulonephritis.

Circulating autoantibodies, namely c-ANCA, MPO-ANCA, anti-Goodpasture (anti-NC1), and anti-entactin antibodies were analysed in sera from 82 consecutive patients with crescentic involvement of more than 50% glomeruli in renal biopsy specimens. Sixty-eight (approximately 83%) patients possessed one or more of these autoantibodies. About two-thirds of all patients had ANCA (c-ANCA, MPO-ANCA or both). Most of the remaining positive patients had anti-NC1 antibodies. Very few patients had anti-entactin antibodies, thereby suggesting a poor association of these antibodies with extracapillary glomerulonephritis (ECGN). Thus two different categories of patients, one possessing ANCA and the other anti-NC1 antibodies, could be recognised. Patients with anti-NC1 antibodies were characterised by linear immune deposits along the glomerular basement membrane and the clinical outcome was invariably grim. On the other hand, despite no significant difference in renal morphology from patients with anti-NC1 antibodies, the disease in patients with ANCA, in general, had a milder course. Among patients with ANCA, those with c-ANCA mainly had systemic small-vessel vasculitis with widespread systemic manifestations, whereas most patients with renal restricted primary ECGN with non-linear immune deposits possessed MPO-ANCA. Furthermore, patients with c-ANCA had a more severe disease than those with MPO-ANCA. These observations indicate that a continuous spectrum of diseases exists between idiopathic small-vessel vasculitides and primary non-linear ECGN. Our study also demonstrates that the presence of auto-antibodies is a dominant feature of severe ECGN and that the type of immunological injury is more important than the extent of crescentic involvement of glomeruli in determining the course of illness in patients with ECGN.

Entactin: a possible auto-antigen in the pathogenesis of non-Goodpasture anti-GBM nephritis.

It has recently been demonstrated that many patients with various types of glomerulonephritis have antibodies to the 6M guanidine-HCl extract of glomerular basement membrane (Bygren et al, Nephrol Dial Transplant 4:254-261, 1989). In the present study a 150 K protein was isolated from the guanidine extract of bovine glomerular basement membrane utilizing ion exchange and gel filtration chromatographic procedures. Amino acid analysis and size of the isolated protein revealed similarity to that of entactin/nidogen. The identity of this protein as entactin/nidogen was further suggested by its precipitation with two different antibodies in a radioimmunoassay and by its reaction with four different antibodies in a sandwich ELISA. Inhibition of the antibodies to 150 K by bovine entactin, which was isolated separately and sequenced for amino acids, confirmed the identity of the 150 K protein as entactin/nidogen. Furthermore, it was shown that about one third of those patients who show antibodies to the crude guanidine extract have circulating antibodies directed against entactin. This was further confirmed by the competitive inhibition of antibodies to the crude guanidine extract in one of the positive serum by entactin in an ELISA inhibition and by immunoblotting experiments. These observations propose entactin as a possible non-Goodpasture glomerular basement membrane antigen that could be involved in the pathogenesis of certain forms of autoimmune glomerulonephritis (non-Goodpasture anti-GBM glomerulonephritis) in man. Most of these patients have a granular pattern of the immunoglobulin deposition along the glomerular basement membrane. This suggests the possibility that anti-GBM glomerulonephritis in human beings can have non-linear immunoglobulin deposits along the GBM.

An ELISA for the detection of anti-neutrophil cytoplasm antibodies (ANCA).

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of circulating anti-neutrophil cytoplasm antibodies (ANCA), which are defined by a diffuse, granular staining of the cytoplasm of alcohol-fixed human neutrophils by indirect immunofluorescence (IIF). Detection of antineutrophil cytoplasm antibodies has a high sensitivity and specificity for active Wegener's granulomatosis (WG) and reflects the effect of treatment. In the present enzyme-linked assay, immunoplates were coated with the cytoplasmic alpha fraction of neutrophils obtained from apparently healthy human donors by nitrogen bomb cavitation and subsequent Percoll gradient centrifugation. Alkaline phosphatase-labelled anti-human IgG was used as a secondary antibody. Diluted sera from 70 patients with WG and 16 patients with other diseases with anti-myeloperoxidase antibodies (anti-MPO) were examined. It is concluded that the ELISA accurately detects IIF ANCA positive patients with WG, is helpful in detecting WG patients in remission, is not influenced by the presence of anti-MPO and may help in detecting ANCA in cases with granulocyte-specific anti-nuclear antibodies since this IIF pattern obscures the IIF ANCA patterns. The ELISA with titration can be carried out in 3.5 h whereas a rapid test just to detect ANCA can be performed in 30 min.

Specificity of kidney-bound antibodies in Goodpasture's syndrome.

The specificity of kidney-bound antibodies in Goodpasture's syndrome was studied and compared with the specificity of circulating anti-glomerular basement membrane (GBM) antibodies. Antibodies were eluted from kidneys of two patients with Goodpasture's syndrome and from one normal human kidney. Their specificity was studied by indirect immunofluorescence microscopy, ELISA and immunoblotting using purified GBM components as antigens. It was shown that the reactivity of the eluted antibodies was very similar to those of the circulating anti-GBM antibodies. Both showed major reactivity against the M2 subunit of the globular domain of collagen IV. Competitive inhibition of the binding of 125I-labelled serum antibodies from a patient with Goodpasture's syndrome to the Goodpasture antigen by the eluted antibodies and those from their respective sera further supported that the circulating and the kidney-bound anti-GBM antibodies have similar specificity. These observations extend the support to the clinical usage of plasmapheresis and more recently, immunoadsorption using staphylococcal protein A as the effective therapeutic measure for the removal of circulating anti-GBM antibodies in the management of Goodpasture's syndrome.

Circulating anti-neutrophil cytoplasm antibodies in patients with rapidly progressive glomerulonephritis and extracapillary proliferation.

We investigated 39 patients with rapidly progressive glomerulonephritis and extracapillary proliferation for the presence of circulating anti-neutrophil cytoplasm antibodies (ANCA) by indirect immunofluorescence. Patients with antibodies against the glomerular basement membrane, systemic lupus erythematosus or with glomerulonephritis following infection and where renal disease could be ascribed to treatment with drugs were not included. IgG class ANCA producing diffuse cytoplasmic immunostaining were detected in 54% of the patients. With reservation for the difficulties involved in distinguishing between different forms of systemic vasculitis, these autoantibodies were present in patients with Wegener's granulomatosis (12 out of 17 patients), polyarteritis nodosa (4/6), idiopathic extracapillary glomerulonephritis (1/4), and with less distinct syndromes (4/10), but not in Henoch-Schönlein's purpura (0/2). Another 18% of the patients showed granulocyte-specific anti-nuclear antibodies' with perinuclear immunostaining. This pattern was recently reported to indicate the presence of antibody against myeloperoxidase of alcohol-fixed neutrophils. Autoimmune mechanisms, particularly those involving components of neutrophil granulocytes, could play a significant role in development of extracapillary glomerulonephritis.

A rapid assay for circulating anti-glomerular basement membrane antibodies in Goodpasture syndrome.

A rapid ELISA for the detection of circulating anti-glomerular basement membrane antibodies in Goodpasture syndrome is described. The specificity of the test was shown to be highly dependent on the antigens used. Using the purified Goodpasture antigen it was possible to shorten the incubation times to 10 min in a routine assay using alkaline phosphatase-labeled second antibodies and the total assay was complete in 30 min. 200 reference sera, 500 sera from patients with various types of glomerulonephritis and 32 sera from patients with Goodpasture syndrome were analyzed by this rapid assay. The assay was able to discriminate between Goodpasture syndrome and other forms of glomerulonephritis. Using enzyme amplification it was possible to further shorten the incubation times to 1 min and the total time of the assay to 6 min.

Non-Goodpasture anti-GBM antibodies in patients with glomerulonephritis.

The sera of 206 consecutive patients with biopsy-proven glomerulonephritis were tested by ELISA for the presence of Goodpasture and non-Goodpasture anti-GBM antibodies. Antigens were solubilised from human GBM with purified bacterial collagenase and with 6 mol/l guanidine-HCl respectively. Only 12 sera reacted when collagenase-resistant GBM proteins were used as antigens in ELISA. Sera from two of these patients also reacted with the Goodpasture antigen, that is the globular domain of collagen IV, purified from collagenase extracts of GBM. These two patients had classical Goodpasture syndrome with linear crescentic nephritis. The other ten sera did not react with the Goodpasture antigen and immunofluorescence microscopy showed granular glomerular immune deposits. Antibodies against antigens present in 6 mol/l guanidine-HCl extracts of human GBM were much more frequent, particularly in lupus nephritis and IgA nephropathy, but relatively common also in patients with glomerulonephritis associated with systemic connective tissue and systemic vasculitic disorders. In contrast, these non-Goodpasture antibodies were only sporadic in primary forms of glomerulonephritis such as minimal-change nephropathy, membranous glomerulopathy, or acute post-infectious glomerulonephritis. The presence of circulating IgG, IgA or IgM antibodies against 6 mol/l guanidine-HCl extractable GBM antigens correlated with granular deposits of corresponding immunoglobulins in both mesangial and capillary loop regions of glomeruli, indicating a possible pathogenic role for non-Goodpasture anti-GBM antibodies in several forms of glomerulonephritis.