JulieAnne G. McGregor

Anti–LAMP-2 Antibodies Are Not Prevalent in Patients With Antineutrophil Cytoplasmic Autoantibody Glomerulonephritis

Lysosomal membrane protein 2 (LAMP-2) is a target of antineutrophil cytoplasmic autoantibodies (ANCA) in addition to the more commonly known targets proteinase 3 and myeloperoxidase. The prevalence of anti-LAMP-2 antibodies and their relationship to disease in ANCA glomerulonephritis are not well described. We measured anti-LAMP-2 reactivity in 680 sera samples (two academic centers) from patients with ANCA glomerulonephritis (n=329); those with ANCA-negative glomerulonephritis (n=104); those with fimbriated, gram-negative Escherichia coli urinary tract infection (n=104); disease controls (n=19); and healthy volunteers (n=124). With levels in healthy controls used to define a reference range, anti-LAMP-2 reactivity was present in 21% of ANCA sera from two of the centers; reactivity was present in 16% of the control group with urinary tract infection. Western blotting and immunofluorescence microscopy did not verify positivity. Titers of anti-myeloperoxidase and anti-proteinase 3 antibodies were 1500-fold and 10,000-fold higher than anti-LAMP-2 titers, respectively. There was no correlation between anti-LAMP-2 antibodies and disease activity. Furthermore, Wistar Kyoto rats injected with anti-LAMP-2 antibodies did not develop glomerulonephritis. In conclusion, antibodies that react with LAMP-2 may exist at very low titers in a minority of patients with ANCA disease. These data do not support a mechanistic relationship between anti-LAMP-2 antibodies and ANCA glomerulonephritis.

ANCA-associated Vasculitis Patients Have Defective Treg Function Exacerbated by Presence of a Suppression-Resistant Effector Population

OBJECTIVE The development of pathogenic antineutrophil cytoplasmic antibodies (ANCAs) can result in systemic small vessel vasculitis. However, the breakdown in immune tolerance that results in the induction and persistence of ANCAs is not well understood. We undertook this study to test our hypothesis that abnormal T cell regulation is central to disease pathogenesis in patients with ANCA-associated vasculitis (AAV). METHODS Peripheral blood samples were obtained from 62 patients with AAV and 19 healthy controls for flow cytometric analysis of CD4+ T cell populations. Functional T cell studies were performed with fluorescence-activated cell sorted CD4+ T cell populations stimulated with anti-CD3/anti-CD28. RESULTS We demonstrated two separate abnormalities in T cell regulation in patients with AAV. First, we showed that the Treg cell frequency was increased in the peripheral blood of patients with active disease, but Treg cells from patients with AAV had decreased suppressive function. Treg cells from patients with active disease disproportionately used a FoxP3 isoform lacking exon 2, which might alter Treg cell function. Second, we identified a CD4+ T cell population with increased frequency that was resistant to Treg cell suppression, produced proinflammatory cytokines, and was antigen experienced. CONCLUSION AAV is associated with disruption of the suppressive Treg cell network and with increased frequency of a distinct proinflammatory effector T cell subset that comprises the majority of peripheral CD4+ T cells.

Glucocorticoids and Relapse and Infection Rates in Anti-Neutrophil Cytoplasmic Antibody Disease

BACKGROUND AND OBJECTIVES The optimal course of glucocorticoid therapy in anti-neutrophil cytoplasmic autoantibody (ANCA) disease is unknown. This cohort study evaluates effects of glucocorticoid therapy duration on patient outcomes and adverse events. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This study assessed 147 patients diagnosed between January 1, 2000 and January 1, 2009 who were treated with glucocorticoids and cyclophosphamide. Patients with end stage kidney disease at presentation, treatment resistance, or who had died within 6 months were excluded. Patients were divided into three groups: 0, 5, or >5 mg prednisone daily at 6 months after therapy initiation. The latter two groups were combined for assessment of adverse events. Wilcoxon rank sum, Kruskal-Wallis, or Fishers exact tests were used for between-group comparisons. Time to relapse was evaluated by the Kaplan-Meier method with log-rank test for comparison. RESULTS There were no differences between groups in ANCA specificity, serum creatinine, frequency of risk factors for relapse, or length of therapy with immunosuppressants. Length of glucocorticoid therapy had no impact on time to relapse (hazard ratio, 0.69 [95% confidence interval (CI), 0.23-2.02]; 1.01, [95% CI, 0.57-1.81] for the 5-mg and >5-mg groups, respectively), relapse-free survival, end stage kidney disease, or death. Patients receiving glucocorticoids beyond 6 months had significantly higher incidence of infections (0.64 infections per person-year versus 0.39, P<0.0001) and a marginally significant higher frequency of new-onset diabetes mellitus (odds ratio, 2.03; 95% CI, 0.94-4.38). CONCLUSIONS Glucocorticoid therapy beyond 6 months is associated with a significantly greater risk of infections but not a significantly decreased risk of relapse.

Decreased CD5+ B Cells in Active ANCA Vasculitis and Relapse after Rituximab

BACKGROUND AND OBJECTIVES B cell significance in ANCA disease pathogenesis is underscored by the finding that ANCA alone can cause disease in mouse models and by the effectiveness of rituximab as therapy in ANCA-small vessel vasculitis (ANCA-SVV). To avoid infections and adverse events from therapy, clinicians require improved markers of disease activity and impending relapse to guide immunosuppression strategies after rituximab treatment. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS The B cell phenotype was investigated in patients with active ANCA-SVV and in remission. From 2003 to 2009, 54 patients were followed longitudinally for 4-99 months and compared with 68 healthy controls. In a subset of 19 patients, the B cell immunophenotype was examined in samples after rituximab therapy. RESULTS Patients with active ANCA-SVV had lower %CD5(+) B cells, whereas %CD5(+) B cells from patients in remission were indistinguishable from healthy controls. After rituximab, median time to relapse was 31 months in patients maintaining normalized %CD5(+) B cells, with or without maintenance immunosuppression. Among patients whose B cells repopulated with low %CD5(+) B cells or had a sharply declining %CD5(+) B cells, those who were on low or no maintenance immunosuppression relapsed sooner (median 17 months) than patients who were maintained on high levels of oral maintenance immunosuppression (29 months; P=0.002). CONCLUSIONS The %CD5(+) B cells, as a component of the human B regulatory cell phenotype, is a useful indicator of disease activity, remission, and future relapse, and thus may guide remission maintenance therapy after rituximab treatment.

Reduced CD5+CD24hiCD38hi and interleukin-10+ regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies

Pathogenesis of anti‐neutrophil cytoplasmic autoantibody (ANCA)‐associated vasculitis is B cell‐dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (Bregs), play a role in immunological tolerance via interleukin (IL)‐10. Putative CD19+CD24hiCD38hi and CD19+CD24hiCD27+ Bregs were evaluated in addition to their CD5+ subsets in 69 patients with ANCA‐associated vasculitis (AAV). B cell IL‐10 was verified by flow cytometry following culture with CD40 ligand and cytosine–phosphate–guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5+CD24hiCD38hi B cells and IL‐10+ B cells compared to patients in remission and healthy controls (HCs). As IL‐10+ and CD5+CD24hiCD38hi B cells normalized in remission within an individual, ANCA titres decreased. The CD5+ subset of CD24hiCD38hi B cells decreases in active disease and rebounds during remission similarly to IL‐10‐producing B cells. Moreover, CD5+ B cells are enriched in the ability to produce IL‐10 compared to CD5neg B cells. Together these results suggest that CD5 may identify functional IL‐10‐producing Bregs. The malfunction of Bregs during active disease due to reduced IL‐10 expression may thus permit ANCA production.

Renal Survival in Patients with Collapsing Compared with Not Otherwise Specified FSGS

BACKGROUND AND OBJECTIVES Idiopathic collapsing FSGS has historically been associated with poor renal outcomes. Minimal clinical data exist on the efficacy of immunosuppressive therapy. Our study sought to provide a comprehensive description of renal survival in patients with collapsing and not otherwise specified FSGS after controlling for factors affecting renal prognosis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We performed a retrospective analysis of an inception cohort study of patients diagnosed between 1989 and 2012. All potential patients with collapsing FSGS fulfilling the inclusion criteria were identified and compared with patients with not otherwise specified FSGS (approximately 1:2 ratio) on the basis of biopsy report and record availability. Time to ESRD was analyzed using Cox proportional hazards models. RESULTS In total, 187 patients were studied (61 collapsing and 126 not otherwise specified), with a mean follow-up of 96 months. At baseline, patients with collapsing FSGS had higher median proteinuria (12.2 [5.6-14.8] versus 4.4 [2.3-8.1] g/d, respectively; P<0.001), lower median albuminemia (2.4 [1.9-3.0] versus 2.9 [1.8-3.7] g/dl, respectively; P=0.12), and lower median eGFR (48 [26-73] versus 60 [42-92] ml/min per 1.73 m2, respectively; P=0.01) than patients with not otherwise specified FSGS. The proportion of patients with remission of proteinuria was similar in patients with collapsing FSGS and patients with not otherwise specified FSGS (65.7% [23 of 35] versus 63.2% [72 of 114], respectively; P=0.84). The overall renal outcome (ESRD defined as eGFR<15 ml/min per 1.73 m2, dialysis, or transplantation) of patients with collapsing FSGS was not poorer than that of patients with not otherwise specified FSGS in multivariate analyses after adjusting for baseline characteristics and immunotherapy (hazard ratio, 1.78; 95% confidence interval, 0.92 to 3.45). CONCLUSIONS Compared with not otherwise specified FSGS, idiopathic collapsing FSGS presented with more severe nephrotic syndrome and lower eGFR but had a similar renal survival after controlling for exposure to immunosuppressive treatment. These results highlight the importance of early diagnosis and institution of immunosuppressive therapy in patients with collapsing FSGS.

Gleaning relapse risk from B cell phenotype: decreased CD5+ B cells portend a shorter time to relapse after B cell depletion in patients with ANCA-associated vasculitis

B cell depletion is an effective remission induction and maintenance therapy in patients with antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV).1–6 Rituximab targets both pathogenic effector B cells and protective regulatory B cells. To avoid infections and adverse events from therapy, clinicians require improved markers of disease activity and impending relapse to guide immunosuppression strategies following B cell depletion. We reported that CD5+ B cells, as a surrogate marker of B regulatory cells, are decreased in patients with active AAV and normalise during disease remission.7 After B cell depletion, patients who repopulated with a low or decreasing percentage of CD5+ B cells and were on low maintenance immunosuppression had a shorter time to relapse than patients on similar levels of immunosuppression with normalised CD5+ B cells or patients with similarly low CD5+ B cells but higher immunosuppression. The CD5+CD24hiCD38hi B cell subpopulation correlates inversely with active disease but parallels both interleukin (IL)-10 production and suppression of ANCA.8 CD5 may identify B cells enriched in IL-10 production, the defining cytokine of B regulatory cells.8 ,9 Whether CD5+ B cells can serve as an indicator of time to relapse without considering remission maintenance immunosuppression dose is not known. We sought to address this question and confirm our previous findings in a larger cohort by separating patients solely based on their CD5+ B cells at repopulation. We examined B cell phenotype in 50 patients with AAV following rituximab therapy by flow cytometry (table …

What Everybody is Doing but No One is Talking About: Use of Complementary and Alternative Medicine in the ANCA Associated Vasculitis Population.

The use and impact of complementary and alternative medicine (CAM) for anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) has not been reported. AAV patients seeking care at our center inquired about CAM, prompting a formal study. Study objectives were to discern how many AAV patients used CAM and its perceived helpfulness in disease management. Methods AAV patients completed a CAM questionnaire between July 2011 and May 2012. Patients were 18 years or older and had biopsy proven and/or clinical evidence of AAV. Medical record abstraction supplemented data. Classification detailed CAM type including “Mind” or “Mind-Body”. Perceived helpfulness of CAM was assessed as “very”, “somewhat” or “not at all/don’t know”. Results A total of 107 patients participated and were a mean age of 53 (range: 18–85), 62% female; 48% proteinase 3 (PR3)-ANCA, 44% myeloperoxidase (MPO)-ANCA and 8% ANCA-negative. Top organs involved included kidney (87%), joints (55%), lung (53%) and upper respiratory (53%). At least one type of CAM treatment or self-help practice was reported by 81% of study participants, with the most frequent being prayer (64%), exercise (27%) and massage therapy (19%). Mind-based practices were used by 28% (excluding prayer) and Mind-Body practices by 14%. Most practices were used to improve wellbeing, and Mind and Mind-Body were deemed very helpful by 83% and 87% respectively. Only 24% of study participants discussed CAM with their physician. Conclusion CAM practices were commonly used to improve well-being and found to be beneficial among AAV patients, but more open discussion is needed about CAM between physicians and patients.

The Case: Acute kidney injury in a patient with P. carinii pneumonia.

A 61-year-old man who had undergone remote orthotopic liver transplant was admitted for fever and progressive dyspnea of several days duration. A chest radiograph demonstrated bilateral interstitial infiltrates with diffuse patchy alveolar opacities. The patient was treated empirically for presumed pulmonary infection with broad-spectrum antibiotics. High-dose trimethoprim/sulfamethoxazole was initiated 24 h after admission because of continued fevers and concern for Pneumocystis carinii pneumonia. Bronchoscopy was performed and direct fluorescent antibody staining for Pneumocystis carinii pneumonia was positive, confirming the diagnosis. Before admission, the patient carried a diagnosis of chronic kidney disease because of toxicity from calcineurin inhibitors, which had since been discontinued. Seventy-two hours after presentation and despite ongoing intravenous hydration, the patients creatinine increased from a baseline of 1.6 mg per 100 ml (122 μmol/l) to 3.4 mg per 100 ml (259 μmol/l), representing a decline in estimated glomerular filtration rate of 42–19 ml/min per 1.73 m2. Urine output had also decreased dramatically. Renal ultrasound was unremarkable with no evidence for obstruction. By initial dipstick analysis, the urine had pH of 5.0, specific gravity 1.020, negative blood, trace protein, and negative leukocyte esterase. A microscopic evaluation of the urine sediment demonstrated rare granular casts, no red blood cells, rare white blood cells per high-powered field, and innumerable yellow-brown crystals as depicted (Figure 1). Figure 1 Microscopic urine analysis What is the cause of the patients renal failure based on the findings of the urine sediment?