Laminin-521 is a Novel Target of Autoantibodies Associated with Lung Hemorrhage in Anti-GBM Disease.

BACKGROUND: Antiglomerular basement membrane (anti-GBM) disease is characterized by GN and often pulmonary hemorrhage, mediated by autoantibodies that typically recognize cryptic epitopes within α345(IV) collagen-a major component of the glomerular and alveolar basement membranes. Laminin-521 is another major GBM component and a proven target of pathogenic antibodies mediating GN in animal models. Whether laminin-521 is a target of autoimmunity in human anti-GBM disease is not yet known. METHODS: A retrospective study of circulating autoantibodies from 101 patients with anti-GBM/Goodpasture's disease and 85 controls used a solid-phase immunoassay to measure IgG binding to human recombinant laminin-521 with native-like structure and activity. RESULTS: Circulating IgG autoantibodies binding to laminin-521 were found in about one third of patients with anti-GBM antibody GN, but were not detected in healthy controls or in patients with other glomerular diseases. Autoreactivity toward laminin-521 was significantly more common in patients with anti-GBM GN and lung hemorrhage, compared with those with kidney-limited disease (51.5% versus 23.5%, P=0.005). Antilaminin-521 autoantibodies were predominantly of IgG1 and IgG4 subclasses and significantly associated with lung hemorrhage (P=0.005), hemoptysis (P=0.008), and smoking (P=0.01), although not with proteinuria or serum creatinine at diagnosis. CONCLUSIONS: Besides α345(IV) collagen, laminin-521 is another major autoantigen targeted in anti-GBM disease. Autoantibodies to laminin-521 may have the potential to promote lung injury in anti-GBM disease by increasing the total amount of IgG bound to the alveolar basement membranes.

Accuracy of Anti-GBM Antibodies in Diagnosing Anti-Glomerular Basement Membrane Disease: A Systematic Review and Meta-Analysis.

BACKGROUND: The sensitivity and specificity of anti-glomerular basement membrane (GBM) antibodies have not been systematically analyzed. In this systematic review, we aimed to evaluate the diagnostic accuracy of anti-GBM antibodies for anti-GBM disease. SUMMARY: Potential studies were searched using MEDLINE, Embase, the Cochrane Library, and the International Clinical Trials Registry Platform based on the index test and target condition. The inclusion criteria were prospective or retrospective cohort studies or case-control studies assessing the sensitivity and specificity of anti-GBM antibodies, and the reference standard was clinical diagnosis including biopsy results. The exclusion criteria were review articles, case reports, animal studies, and in vitro studies. Quality assessment was conducted based on the Quality Assessment of Diagnostic Accuracy Studies-2. The pooled estimates of sensitivity and specificity were calculated using a bivariate random-effects model. The overall quality was evaluated using the Grades of Recommendation, Assessment, Development, and Evaluation. Six studies (1,691 patients) and 11 index tests were included in our systematic review. A high risk of bias and concerns regarding the applicability of patient selection were noted because of the case-control design in 67% of the included studies. The pooled sensitivity and specificity were 93% (95% CI: 84-97%) and 97% (95% CI: 94-99%), respectively. The certainty of evidence was low because of the high risk of bias and indirectness. Key Messages: Anti-GBM antibodies may exhibit high sensitivity and specificity in the diagnosis of anti-GBM disease. Further cohort studies are needed to confirm their precise diagnostic accuracy and compare diagnostic accuracies among different immunoassays.

Atypical anti-glomerular basement membrane disease with anti-GBM antibody negativity and ANCA positivity: a case report.

BACKGROUND: Anti-glomerular basement membrane (anti-GBM) disease is an organ-specific autoimmune disease that involves the lung and kidneys and leads to rapid glomerulonephritis progression, with or without diffuse alveolar hemorrhage, and even respiratory failure. Classic cases of anti-GBM disease are diagnosed based on the presence of the anti-GBM antibody in serum samples and kidney or lung biopsy tissue samples. However, atypical cases of anti-GBM disease are also seen in clinical practice. CASE PRESENTATION: We herein report the rare case of a patient with atypical anti-GBM disease whose serum was negative for the anti-GBM antibody but positive for the myeloperoxidase (MPO) anti-neutrophil cytoplasmic antibody (p-ANCA) and another atypical ANCA. Laboratory test results showed severe renal insufficiency with a creatinine level of 385 µmol/L. Renal biopsy specimen analysis revealed 100% glomeruli with crescents; immunofluorescence showed immunoglobulin G (IgG) linearly deposited alongside the GBM. Finally, the patient was discharged successfully after treatment with plasmapheresis, methylprednisolone and prednisone. CONCLUSION: This patient, whose serum was negative for the anti-GBM antibody but positive for p-ANCA and another atypical ANCA, had a rare case of anti-GBM disease. Insights from this unusual case might help physicians diagnose rare forms of glomerulonephritis and treat affected patients in a timely manner.

Comparison of the performance of a chemiluminescence assay and an ELISA for detection of anti-GBM antibodies.

Objective: Autoantibodies to the α3 chain noncollagen 1 domain of type IV collagen (α3(IV)NC1) are a serological hallmark in the diagnosis of anti-glomerular basement membrane (GBM) disease. The objective of our study was to compare the performance of anti-glomerular basement membrane (GBM) antibody detection by chemiluminescence immunoassay (CIA) and by enzyme-linked immunosorbent assays (ELISAs).Methods: Sera from outpatients who were suspected to have anti-GBM disease and 31 patients with biopsy-proven anti-GBM disease were collected. Thirty normal controls were also included. All samples were tested for anti-GBM antibodies by CIA and commercial ELISA. The anti-GBM antibody-positive samples were confirmed by a homemade ELISA coated with recombinant human α3(IV)NC1.Results: Compared with detection of anti-GBM antibodies with ELISA, detection of anti-GBM antibodies with CIA showed a positivity agreement of 70% and a negativity agreement of 98.6%. Among the 4 patients with different results, the anti-GBM antibody detection by CIA was in agreement with the homemade ELISA coated with recombinant human α3(IV)NC1 and the clinical diagnosis. In 31 patients with anti-GBM disease, good agreement was achieved in the detection of anti-GBM antibodies with CIA, commercial ELISA and the homemade ELISA (100%, 100%). The AUC for CIA and commercial ELISA was 0.987 and 0.966, respectively.Conclusions: The detection of anti-GBM antibodies with CIA demonstrated good sensitivity and specificity and was in good agreement with our homemade ELISA, which seems better than the commercial ELISA in suspected anti-GBM disease patients. The three assays performed in parallel in the diagnosis of anti-GBM disease patients.

The treatment of ANCA-associated rapidly-progressive glomerulonephritis and Goodpasture syndrome with therapeutic apheresis.

Therapeutic plasma aphresis (plasmapheresis) is one form of treatment that is frequently used in practice of Nephrology. Plasmapheresis is the most important part of the therapies for Goodpasture's syndrome and anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis which are causes of rapidly progressive glomerulonephritis. The reason why the effectiveness of plasmapheresis therapy cannot be clearly demonstrated in renal involvement in these diseases is that it does not appear to be possible to recruit an adequate number of patients and plasmapheresis is not effective in advanced disease if early treatment is not initiated.

T cell responses to peptides of Goodpasture autoantigen in patients with anti-glomerular basement membrane disease.

AIM: Cell-mediated autoimmunity, especially autoreactive T cells, is crucial in the initiation of anti-glomerular membrane (GBM) disease. Epitopes for T cells on Goodpasture autoantigen are not fully defined. This study investigated T cell epitopes in anti-GBM patients, aiming to identify the epitopes and their clinical significance. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from 13 patients with anti-GBM disease. Twenty-four overlapping linear peptides were synthesized covering the whole sequence of human α3(IV)NC1. PBMC response to each peptide was detected by proliferation assay. Their associations with clinical features were further analyzed. RESULTS: Peripheral blood mononuclear cells proliferative responses to linear peptides on α3(IV)NC1 could be detected in all patients. Five major epitopes were identified as stimulatory in over half of the patients: α3(IV)NC1127-148 (P14) (69.2%), α3(IV)NC1159-178 (77.8%), α3(IV)NC1179-198 (55.6%), α3(IV)NC1189-208 (P19) (75.0%) and α3(IV)NC1141-154 (57.1%). P14 and P19 were highly recognized in patients comparing with healthy controls (69.2% vs. 0.0%, P = 0.011; 75.0% vs. 0.0%, P = 0.021, respectively). CONCLUSION: T cell proliferation to linear epitopes was detected in human anti-GBM disease. α3127-148 was a mutual T and B cell epitope, implying its initial role in epitope spreading process.

Use of rituximab as an induction therapy in anti-glomerular basement-membrane disease.

BACKGROUND: Anti-glomerular basement-membrane (anti-GBM) disease (or Goodpasture disease) is characterized by severe kidney and lung involvement. Prognoses have improved with treatments that combine plasma exchange and immunosuppressive drugs. However, patients with severe renal involvement can have poor renal outcomes and cyclophosphamide can cause significant complications. Anti-GBM antibodies have a direct pathogenic effect on the disease: thus, therapeutics that can decrease their production, such as rituximab, could be a good alternative. METHODS: The medical files of five patients that had received rituximab as a first-line therapy (instead of cyclophosphamide), plus plasma exchange and steroids, were reviewed. All patients had severe disease manifestations. RESULTS: Four patients required dialysis at diagnosis and remained dialysis-dependent over the mean follow-up of 15 months. Three patients had pulmonary involvement, but recovered even though mechanical ventilation was required. Anti-GBM antibodies became rapidly undetectable in all patients. One infectious and two hematological complications were observed. CONCLUSIONS: We report the outcomes of five patients with Goodpasture disease and treated with rituximab as a first-line treatment. This strategy was effective at treating pulmonary manifestations and was associated with a good biological response with no major serious adverse events. However, renal outcomes were not significantly improved.

The pathogenicity of T cell epitopes on human Goodpasture antigen and its critical amino acid motif.

Goodpasture antigen, the non-collagenous domain of α3 chain of type IV collagen [α3(IV)NC1], is the target antigen of anti-glomerular basement membrane (GBM) antibodies. The pathogenicity of T cell epitopes is not elucidated clearly. In this study, we aim to define the nephritogenic T cell epitopes and its critical amino acid residues. Twenty-four overlapping linear peptides were synthesized covering the whole sequence of human α3(IV)NC1. Wistar-Kyoto rats were immunized with linear peptides, and experimental autoimmune glomerulonephritis was evaluated. Critical amino acid was identified by the loss of nephritogenic function after each amino acid substitution by alanine. Of the 24 peptides, P14 (α3127-148 ) could induce 90.5% (19/21) of WKY rats developing anti-GBM glomerulonephritis with proteinuria, elevated serum urea and creatinine, IgG linear deposit on GBM and substantial (in average 82.4 ± 5.6%) crescent formation in glomeruli. Lymphocytes of immunized rats proliferated in response to α3127-148 and α3(IV)NC1 in vitro. Sera of these rats recognized α3127-148 and later on together with intact human α3(IV)NC1. Antibodies towards α3127-148 and intact α3(IV)NC1 could also be detected from the kidney elutes. These antibodies showed no cross-reaction with each other, which implies intramolecular epitope spreading during disease progress. After sequential amino acid substitution, the α3127-148 with substitution of tryptophan136 , isoleucine137 , leucine139 or tryptophan140 lost its nephritogenicity. Human α3127-148 is a nephritogenic T cell epitope in WKY rats, with the critical amino acids as W136 I137 xL139 W140 . These findings might facilitate future investigation on microbial aetiology and potential specific immunotherapy of anti-GBM disease.

Patients double-seropositive for ANCA and anti-GBM antibodies have varied renal survival, frequency of relapse, and outcomes compared to single-seropositive patients.

Co-presentation with both ANCA and anti-GBM antibodies is thought to be relatively rare. Current studies of such 'double-positive' cases report small numbers and variable outcomes. To study this further we retrospectively analyzed clinical features and long-term outcomes of a large cohort of 568 contemporary patients with ANCA-associated vasculitis, 41 patients with anti-GBM disease, and 37 double-positive patients with ANCA and anti-GBM disease from four European centers. Double-positive patients shared characteristics of ANCA-associated vasculitis (AAV), such as older age distribution and longer symptom duration before diagnosis, and features of anti-GBM disease, such as severe renal disease and high frequency of lung hemorrhage at presentation. Despite having more evidence of chronic injury on renal biopsy compared to patients with anti-GBM disease, double-positive patients had a greater tendency to recover from being dialysis-dependent after treatment and had intermediate long-term renal survival compared to the single-positive patients. However, overall patient survival was similar in all three groups. Predictors of poor patient survival included advanced age, severe renal failure, and lung hemorrhage at presentation. No single-positive anti-GBM patients experienced disease relapse, whereas approximately half of surviving patients with AAV and double-positive patients had recurrent disease during a median follow-up of 4.8 years. Thus, double-positive patients have a truly hybrid disease phenotype, requiring aggressive early treatment for anti-GBM disease, and careful long-term follow-up and consideration for maintenance immunosuppression for AAV. Since double-positivity appears common, further work is required to define the underlying mechanisms of this association and define optimum treatment strategies.

Outcomes of patients with Goodpasture syndrome: A nationwide cohort-based study from the French Society of Hemapheresis.

The overall and renal outcomes of patients with Goodpasture syndrome (GS), a rare autoimmune disorder characterized by circulating anti-GBM antibodies and rapidly progressive glomerulonephritis and/or pulmonary hemorrhage, have mostly been reported in small-sized cohorts or by aggregating patients receiving a variety of therapies that include aggressive (i.e., combined plasma exchanges, corticosteroids, and cyclophosphamide) and less aggressive (i.e., either plasma exchanges or immunosuppressive drugs, or no treatment). To address the prognosis of GS patients with relatively homogeneous management including plasma exchanges, we conducted a multicenter retrospective study on GS patients included in the registry of the French Society of Hemapheresis. 122 patients were included (kidney alone (n = 28), lung alone (n = 5), or combined involvement (n = 89)). All 122 patients received plasma exchanges (median number of sessions: 13 [9-17]), either alone (n = 8) or associated with combined corticosteroids and oral or IV cyclophosphamide (n = 101) or with corticosteroids alone (n = 12) or cyclophosphamide alone (n = 2). One-year survival was 86.9%. 7/16 patients died from severe infection. In multivariate analyses (Cox's regression model), being aged <60 years, and number of plasma exchanges were correlated to overall survival. The use of alternative immunosuppressive drugs (because of refractory or relapsing GS) was correlated to mortality at one year. Superiority of oral cyclophosphamide compared to intravenous intake was close to significant. Using a logistic regression model, renal survival in patients alive at 1 year was only predicted by serum creatinine <500 µmol/L at presentation. This large series describes the predictive factors for overall and renal survival of GS patients treated by plasma exchanges. Interventional studies that compare oral and intravenous cyclophosphamide, as well as testing new immunosuppressive therapies, are warranted.

Monoclonal IgG1κ anti-glomerular basement membrane disease: a case report.

We report a case of anti-glomerular basement membrane (anti-GBM) nephritis with indolent course, monoclonal IgG1κ (immunoglobulin G, subclass 1, κ light chain) linear staining of the GBM, and multifocal GBM breaks but without crescents or detectable serum anti-GBM antibody in a patient followed over 9 years. Atypically, anti-GBM nephritis follows an indolent course. A very small fraction of patients with anti-GBM nephritis lack detectable circulating anti-GBM antibodies, and rare reports of monoclonal anti-GBM nephritis exist. We report what is to our knowledge the first case manifesting all 3 of these rare variations. Our patient initially presented with asymptomatic decreased kidney function following an upper respiratory tract infection. He was found to have microhematuria and subnephrotic proteinuria with mild diffuse endocapillary proliferative and exudative glomerulonephritis with linear IgG1κ staining of the GBM. He was treated with an induction regimen of intravenous cyclophosphamide and corticosteroids followed by maintenance monotherapy with mycophenolic acid. Nine years later, repeat kidney biopsy for worsening kidney function after an upper respiratory tract infection showed persistent monoclonal staining of the GBM and acute glomerulonephritis with increased chronicity, including a single fibrocellular crescent. Despite extensive clinical investigations spanning nearly a decade, no circulating anti-GBM antibody or monoclonal protein has been detected. In this case report, we explore the unique features of this monoclonal IgG1κ-associated anti-GBM nephritis.

Circulating anti-glomerular basement membrane antibodies with predominance of subclass IgG4 and false-negative immunoassay test results in anti-glomerular basement membrane disease.

Autoantibodies against a constituent of the glomerular basement membrane (GBM), the α3-chain of type IV collagen, can cause both rapidly progressive glomerulonephritis and alveolar hemorrhage, referred to as anti-GBM disease or Goodpasture disease. Anti-GBM antibodies generally are of immunoglobulin G subclass 1 (IgG1) and can in most cases readily be detected in the circulation using enzyme-linked immunosorbent assays (ELISAs). We report 4 cases in which anti-GBM ELISA yielded negative or borderline results despite life-threatening disease. All 4 patients had positive results by IgG4 anti-GBM ELISA and all had undetectable antineutrophil cytoplasmic antibody. All cases were confirmed with kidney biopsy. Two of the patients showed higher signal in anti-GBM ELISA when using a nondenaturing coating buffer. All 4 were young women with severe alveolar hemorrhage and favorable renal outcome, suggesting that patients with predominance of IgG4 autoantibodies may constitute a distinct subgroup of anti-GBM disease. We conclude that patients with idiopathic alveolar hemorrhage can have anti-GBM disease detected by only IgG subclass-specific tests or kidney biopsy.

Comparison of double filtration plasmapheresis with immunoadsorption therapy in patients with anti-glomerular basement membrane nephritis.

BACKGROUND: Double filtration plasmapheresis (DFPP) and (IA) are both used to clear antibody. However, the clinical efficacy and safety of DFPP in patients with anti-glomerular basement membrane (anti-GBM) disease are unclear. METHODS: The 28 enrolled patients diagnosed serologically and pathologically with anti-GBM disease from 2003 to 2013 included 16 treated with DFPP and 12 with IA, with all patients administered immunosuppressive agents. DFPP consisted of an EC50W filter for plasma separation and an EC20W filter for plasma fractionation. A double volume of plasma was processed, and each patient received a 30-40 g human albumin supplement during each session. IA consisted of 10 cycles per session, with 8-10 sessions performed daily or every other day and each session regenerating 30-60 L of plasma. Serum anti-GBM antibodies and IgG were measured, and urinary and blood tests were performed, before and after each procedure. Renal function and outcome were determined. RESULTS: The 28 patients consisted of 13 males and 15 females, of median age 44.5 years (range, 22.5-57 years). Six patients had pulmonary hemorrhage and 18 had serum creatinine concentrations >500 umol/L. The average serum creatinine concentration at early onset of disease was 525 umol/L while the peak concentration was 813 umol/L. All patients showed progressive increases in serum creatinine and required CRRT during the course of disease. Pathological examination showed an average 73.9% of crescents (range, 54.6-95.4%).The clinical and pathological features of the DPPP and IA groups were similar. Efficacy of clearing anti-GBM antibody was similar in the two groups (59.0 vs. 71.2%, P = 1.00), although fewer patients in the DFPP group experienced reduced IgG (62.7 vs. 83.5%, p = 0.002). One patient each had a pulmonary hemorrhage and a subcutaneous hemorrhage during treatment, but there were no other serious complications. At the end of follow-up, patient survival and renal survival were similar in the DFPP and IA groups. CONCLUSION: DPPP plus immunosuppressive therapy efficiently and safely removed anti-GBM antibodies. The fewer plasma-associated side effects and reduced loss of IgG suggest that DFPP may be a better treatment choice for anti-GBM disease, especially in patients with insufficient plasma.

The distribution of IgG subclass deposition on renal tissues from patients with anti-glomerular basement membrane disease.

BACKGROUND: Renal injury of anti-glomerular basement membrane (GBM) disease is defined by the linear deposition of IgG along GBM and rapidly progressive glomerulonephritis. To date, the distribution of anti-GBM IgG subclasses on renal tissue is still unclear. In the current study, we investigated the deposition of the four IgG subclasses using immunohistochemistry in the renal biopsy specimens from 46 patients with anti-GBM disease. RESULTS: All four IgG subclasses can be detected within the GBM. Anti-GBM IgG3 was detected in all patients (100%), with 39 (84.8%) patients presenting with weak segmental staining and 7 (15.2%) patients with strong linear deposition. Anti-GBM IgG2 was detected in 22 (47.8%) patients, with 20 (90.9%) patients having weak segmental deposition and 2 (9.1%) patients presenting strong linear staining. Anti-GBM IgG1 and IgG4 were detected in 9 (19.6%) and 7 (15.2%) patients, respectively. IgG deposition along tubular basement membrane (TBM) was also detected in 31 (67.4%) patients. Among them, the IgG subclass distribution was similar to that of the deposition within the GBM: IgG1 6.5% (2/31), IgG2 45.2% (14/31), IgG3 100% (31/31) and IgG4 9.7% (3/31). We observed increased inflammatory cell infiltration into the interstitium in patients with increased anti-TBM IgG3 deposits (P=0.031). CONCLUSIONS: Anti-GBM IgG3 predominantly deposits along GBM and TBM on renal biopsy specimens from patients with anti-GBM disease, which may be involved in the development of renal injury of the disease.

Glomerular C1q deposition and serum anti-C1q antibodies in anti-glomerular basement membrane disease.

BACKGROUND: Anti-glomerular basement membrane (GBM) disease is a well-known antibody-induced autoimmune disease. A few patients have glomerular C1q deposition, but it is usually absent on renal histopathology. The role of C1q deposition in kidney injury is unclear. Recently, anti-C1q antibodies are demonstrated to be pathogenic in the target organ damage of many autoimmune diseases, by facilitating C1q deposition and enhancing complement activation via classical pathway. In the current study, we investigated the associations between anti-C1q antibodies in sera and C1q deposition in kidney of patients with anti-GBM disease. RESULTS: It was shown that the severity of kidney injury was comparable between patients with and without C1q deposition, including the prevalence of oliguria/auria, the median percentage of crescents in glomeruli and the mean concentration of serum creatinine. Serum anti-C1q antibodies were detected in 15/25 (60%) patients with a low titer. The prevalence of C1q deposition in kidney was comparable between patients with and without serum anti-C1q antibodies (26.7% vs. 30.0%, p > 0.05). No association was found between anti-C1q antibodies and the severity of kidney injury. CONCLUSIONS: The classical pathway of complement may not play a pathogenic role in the kidney injury of human anti-GBM disease. Anti-C1q antibodies could be detected in more than half of patients, which need further investigations.

Serum BAFF and APRIL might be associated with disease activity and kidney damage in patients with anti-glomerular basement membrane disease.

AIM: B cell activating factor belonging to the tumour necrosis factor family (BAFF) and a proliferation inducing ligand (APRIL) are two tumour necrosis factor (TNF)-like cytokines that were found to be elevated in many autoimmune diseases. Anti-glomerular basement membrane (GBM) disease is a typical severe autoimmune disease characterized by raised serum anti-GBM antibodies. In this study we aimed to detect the serum levels of BAFF and APRIL in patients with anti-GBM disease, and their clinical significance was further analyzed. METHODS: Forty-seven patients with anti-GBM disease were enrolled in this study. Forty-eight healthy individuals were used as normal controls. The levels of serum BAFF and APRIL were assessed using commercially available enzyme linked immunosorbent assay kits. The association between the levels of serum BAFF and APRIL, and the clinical and pathological parameters were further evaluated. RESULTS: The serum levels of BAFF and APRIL in patients with anti-GBM disease were significantly higher than that in normal controls (12.3 ± 14.1 ng/mL vs. 0.9 ± 0.3 ng/mL, P < 0.001; 19.1 ± 22.9 ng/mL vs. 1.6 ± 4.6 ng/mL, P < 0.001), respectively. The levels of serum APRIL were correlated with the titres of anti-GBM antibodies (r = 0.347, P = 0.041), and the levels of serum BAFF were associated with the percentage of glomeruli with crescents (r = 0.482, P = 0.015) in patients with anti-GBM disease. CONCLUSION: The levels of serum BAFF and APRIL were raised in patients with anti-GBM disease and might be associated with disease activity and kidney damage.

Pulmonary renal syndrome in a child with coexistence of anti-neutrophil cytoplasmic antibodies and anti-glomerular basement membrane disease: case report and literature review.

BACKGROUND: Pulmonary renal syndrome (PRS), denoting the presence of diffuse alveolar hemorrhage and glomerulonephritis as manifestations of systemic autoimmune disease, is very rare in childhood. The coexistence of circulating anti-neutrophil cytoplasmic antibody (ANCA) and anti-glomerular basement membrane (GBM) disease in children affected by this syndrome is exceptional, with unfavorable outcome in five out of seven patients reported to date. We describe a child with PRS associated with both circulating anti-myeloperoxidase (anti-MPO) ANCA and anti-GBM disease on renal biopsy who was successfully treated with immunosuppressive therapy. CASE PRESENTATION: A 10-year old girl presented with fever, fatigue, malaise, and pallor followed by hemoptysis and severe anemia. Diffuse alveolar hemorrhage was revealed on fiberoptic bronchoscopy. Renal findings consisted of microscopic hematuria, moderate proteinuria, and anti-GBM disease on renal biopsy. ANCA with anti-MPO specificity were present whereas anti-GBM antibodies were on borderline for positivity. Methyl-prednisolone pulses followed by prednisone led to cessation of hemoptysis, marked improvement of lung fuction, and normal finding on chest x-ray within 10 days. An immunosuppressive regimen was then given consisting of prednisone daily for 4 weeks with subsequent taper on alternate day, i.v. cyclophosphamide pulses monthly for 6 doses, followed by mycophenolate mofetil that resulted in normal lung function tests, hemoglobin concentration, and anti-MPO level within four subsequent weeks. During 10-months of follow-up she remained well, her blood pressure and renal function tests were normal, and proteinuria and hematuria gradually resolved. CONCLUSION: We report a child with an exceptionally rare coexistence of circulating ANCA and anti-GBM disease manifesting as PRS in whom renal disease was not the prominent part of clinical presentation, contrary to other reported pediatric patients. A review of literature on disease with double positive antibodies is also presented. Evaluation of a patient with PRS should include testing for presence of different antibodies. An early diagnosis and rapid institution of aggressive immunosuppressive therapy can induce remission and preserve renal function. Renal prognosis depends on the extent of kidney injury at diagnosis and appropriate treatment.

Characterization of the renal CD4+ T-cell response in experimental autoimmune glomerulonephritis.

Autoimmunity against the Goodpasture antigen α3IV-NC1 results in antiglomerular basement membrane glomerulonephritis. Although antibodies are central to the pathogenesis, there is good evidence for the participation of T cells in this disease. To define the contribution of T cells, we used the model of experimental autoimmune glomerulonephritis. Immunization of DBA/1 mice with α3IV-NC1 resulted in proteinuria, a biphasic course of the disease, and an eventual loss of kidney function. In the initial phase, the mice developed an α3IV-NC1-specific IgG response, had IgG deposition along the glomerular basement membrane, and steadily increased proteinuria, but only marginal signs of inflammation with limited leukocyte infiltration. After 9-13 weeks, mice proceeded to develop crescentic glomerulonephritis, extensive tubulointerstitial damage, and massive macrophage infiltration. T-cell infiltration was less pronounced, mostly confined to the interstitium, and T cells displayed an activated phenotype with a significant fraction of Th1 or Th17 CD4(+) T cells. Close examination revealed the presence of autoreactive T cells producing IFNγ upon restimulation with α3IV-NC1. Thus, our results suggest that accumulation of effector T cells, including autoreactive T cells, represents a critical step in the progression from mild glomerulonephritis, with limited glomerular damage, to severe crescentic glomerulonephritis accompanied by tubulointerstitial inflammation and loss of kidney function.

Circulating anti-glomerular basement membrane autoantibodies against α3(IV)NC1 undetectable by commercially available enzyme-linked immunosorbent assays.

AIM: Cases with anti-glomerular basement membrane (GBM) disease have been reported with linear deposit of immunoglobulin G (IgG) along GBM, but have undetectable anti-GBM antibodies in circulation by enzyme linked immunosorbent assays (ELISA). We speculated that the structure of the antigens recognized by these antibodies may contribute to the negative results of ELISA. METHODS: Sera from four patients were collected, with typical linear deposit of IgG along GBM but no anti-GBM reactivity by commercial ELISA kits. Circulating anti-GBM antibodies were detected by indirect immunofluorescence. Antigen specificity and its conformational structure was investigated by western-blot analysis, using recombinant human α1-α5(IV)NC1 and chimeric proteins E(A) and E(B) as antigens. RESULTS: The presence of circulating anti-GBM antibodies were confirmed by indirect immunofluorescence with linear deposit of IgG towards cryptic epitopes along GBM on normal kidney sections. These antibodies did not recognize recombinant human α1, α2, α4 or α5(IV)NC1, but could blot α3(IV)NC1 under non-reducing non-boiling conditions on western-blot analysis, when the conformational epitope(s) on α3(IV)NC1 were thought to be preserved. When α3(IV)NC1 was prepared under reducing conditions with ß-mercaptoethanol and/or boiled to destroy the disulfide bonds, the binding with the antibodies disappeared. Moreover, these antibodies recognized neither E(A) nor E(B) , indicating their distinct epitope repertoire. CONCLUSION: Circulating anti-GBM antibodies undetectable by ELISA could recognize cryptic and conformation-dependent epitopes restricted on α3(IV)NC1, distinct from E(A) and E(B) . Indirect immunofluorescence was necessary for antibody detection and treatment monitoring under such circumstances.