Wolfgang L. Gross

An interdisciplinary approach to the care of patients with Wegener's granulomatosis: Long-term outcome in 155 patients

OBJECTIVE To examine the outcome in 155 consecutive patients with Wegeners granulomatosis (WG) followed up for a median of 7 years. METHODS Treatment was adapted to the activity and extent of disease, with regular evaluation by an interdisciplinary team accompanied by group education about vasculitis. RESULTS The estimated median survival time was 21.7 years (95% confidence interval [95% CI] 15.60-27.86). Twenty-two patients died; 19 deaths were attributable to WG and/or its treatment. Significant predictors of survival at diagnosis were age >50 years (hazard ratio [HR] 5.45, 95% CI 1.97-15.02), kidney involvement with impaired renal function (HR 5.42, 95% CI 1.76-16.68), and lung involvement (HR 3.75, 95% CI 1.26-11.16). At some stage, 142 patients received prednisone and cyclophosphamide (CYC), usually as daily CYC plus mesna as uroprotection, 50 patients received trimethoprim/sulfamethoxazole, and 45 received methotrexate. Complete remission was achieved in 83 of the 155 patients. One or more relapses occurred in 99 patients after either complete or partial remission. CYC-induced cystitis and myelodysplastic syndrome occurred in 17 and 11 patients, respectively. A cumulative dose of 100 gm or more of CYC resulted in a 2-fold greater risk of CYC-related morbidity than with lower CYC doses. Serious infections occurred in 41 patients. CONCLUSION An interdisciplinary approach to the care of 155 WG patients resulted in a median survival of >21 years. Kidney or lung involvement at diagnosis was predictive of a >3-fold higher mortality. Although CYC remains essential in the treatment of WG, it was administered as briefly as possible and under close surveillance to avoid permanent CYC-related morbidity, which can lead to serious therapeutic problems in chronic relapsing WG.

Pulse Versus Daily Oral Cyclophosphamide for Induction of Remission in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis A Randomized Trial

Context Because cyclophosphamide has many adverse effects, dosing regimens that maintain efficacy but improve safety would be welcome. Contribution In this randomized comparison of pulse and daily oral cyclophosphamide regimens for treatment of ANCA-associated vasculitis, equal proportions of patients had remissions, but the pulse regimen seemed safer, mainly because it caused less leukopenia. Caution Patients and providers were not blinded to the intervention, and the study was not powered to detect differences in relapse rate. Implication The efficacy of pulse cyclophosphamide for treatment of ANCA-associated vasculitis seems no different from that of daily oral treatment and may be safer. The Editors Wegener granulomatosis, microscopic polyangiitis, and the renal-limited variant of microscopic polyangiitis are all associated with antineutrophil cytoplasmic antibodies (ANCAs) and are therefore referred to collectively as ANCA-associated vasculitis. The justification for grouping these diseases together as a single clinical entity goes beyond ANCA seropositivity; they cause similar histologic changes in the kidney, are associated with similar pathogenic autoantibodies, and respond similarly to induction immunosuppressive treatment. However, they also differ in important respects; for example, granuloma formation and relapse after treatment are more common in Wegener granulomatosis (1, 2). Outcomes for these previously fatal diseases improved dramatically with the introduction of daily oral cyclophosphamide therapy (3, 4). However, cyclophosphamide has significant adverse effects that influence long-term morbidity and mortality (5, 6). Strategies to reduce these adverse effects include reducing the duration of cyclophosphamide use to 3 to 6 months (maximum, 9 months) (2) and switching to an alternative immunosuppressive regimen after induction of remission and using methotrexate instead of cyclophosphamide in patients without generalized disease and significantly impaired renal function (7). For many patients, however, cyclophosphamide remains the mainstay of therapy for inducing remission and treating relapse, so regimens that maintain efficacy while minimizing cyclophosphamide dose and maximizing safety would be welcome. Previous studies (8) suggest that pulse cyclophosphamide regimens are safe and provide less cumulative cyclophosphamide exposure than daily oral cyclophosphamide regimens. However, small study sizes and variations in treatment regimens, including the use of treatments alongside cyclophosphamide, make the findings preliminary. We designed this trial to test the hypothesis that a regimen of pulsed intermittent cyclophosphamide would be as effective but less toxic than daily oral cyclophosphamide for inducing remission in patients with generalized ANCA-associated vasculitis with active glomerulonephritis. Methods Trial Design and Participants Our trial was an open-label, multicenter, randomized, controlled trial conducted over 18 months. Patients, providers, and the investigators who assessed trial outcomes were not blinded to treatment assignment. Our inclusion criteria were newly diagnosed Wegener granulomatosis, microscopic polyangiitis, or renal-limited microscopic polyangiitis (diagnostic criteria adapted from the 1992 Chapel Hill consensus conference [9] and our groups previous studies [2, 7, 1012]); renal involvement attributable to active vasculitis (as defined by at least 1 of the following: serum creatinine level >150 mol/L [>1.7 mg/dL] and 500 mol/L [5.7 mg/dL], biopsy demonstrating necrotizing glomerulonephritis, erythrocyte casts, or hematuria [>30 erythrocytes per high-power field] and proteinuria [>1 g/d]); and confirmatory histology or ANCA positivity. Our exclusion criteria were coexistence of other multisystem autoimmune disease; hepatitis B or C virus or HIV infection; serum creatinine level greater than 500 mol/L (>5.7 mg/dL); previous cancer; pregnancy; or age younger than 18 or older than 80 years. We conducted our study according to the Declaration of Helsinki. Informed consent was obtained from each participant, and each participating center reviewed the trial protocol and granted ethical approval. Random Assignment Random assignments were computer-generated and performed centrally by permuted blocks of 4, stratified by country and disease. Patients were enrolled by their treating physician and registered with the central trial coordinating office by fax submission of a form that contained information on center, date of birth, sex, disease, and creatinine level. We randomly assigned patients on a 1:1 basis to receive pulse or daily oral cyclophosphamide. Data were collected in record books, entered into a central computerized database, and validated against the record books before analysis. Eleven patients withdrew before random assignment; we randomly assigned 149 patients. Interventions We designed the pulse cyclophosphamide regimen by investigator consensus, on the basis of published experience with pulse cyclophosphamide in ANCA-associated vasculitis. Patients received 3 intravenous pulses of cyclophosphamide, 15 mg/kg, given 2 weeks apart, followed by pulses at 3-week intervals (15 mg/kg intravenously or 5 mg/kg orally on 3 consecutive days, at the physicians discretion) until remission, and then for another 3 months. The maximum dose per pulse was 1.2 g. We reduced the cyclophosphamide dose by 2.5 mg/kg per pulse for persons age 60 to 70 years, 5 mg/kg per pulse for persons older than 70 years, and 2.5 mg/kg per pulse for persons with a serum creatinine level of 300 to 500 mol/L (3.4 to 5.7 mg/dL). At minimum, blood counts were checked on day 10 and 14 after each pulse and immediately before the next pulse. We reduced the dose of the subsequent pulse by 20% for patients with a leukocyte nadir of 2 to 3109/L and 40% for those with a nadir of 1 to 2109/L. The daily oral cyclophosphamide group received cyclophosphamide, 2 mg/kg per day, until remission, followed by 1.5 mg/kg per day for another 3 months. The maximum oral dose was 200 mg, and we reduced the dose by 25% for persons older than 60 years and 50% for those older than 70 years. At minimum, blood counts were checked weekly for the first month, twice-weekly for the second month, and monthly thereafter. We withheld cyclophosphamide for persons with a leukocyte count less than 4109/L, then resumed therapy at a dose reduced by 25 mg/d when their count increased to greater than 4109/L. Both groups continued the cyclophosphamide regimens for 3 months after remission, after which all patients received azathioprine, 2 mg/kg per day orally, until month 18 for remission maintenance. The maximum daily oral dose of azathioprine was 200 mg. Both groups also received prednisolone, 1 mg/kg orally, tapered to 12.5 mg at the end of month 3 and to 5 mg at the end of the study (month 18). 2-Mercaptoethanesulfonate sodium was optional in both groups. No patients received plasmapheresis. We recommended prophylaxis for Pneumocystis jiroveci for all patients. Treatment was allowed to follow local practice for patients who did not achieve remission at 9 months. We collected data on these patients but censored them for purposes of this analysis. For more details on the protocol, see Appendix 1. Outcomes and Follow-up We defined outcomes by using the Birmingham Vasculitis Activity Score (BVAS) index, which measures manifestations of active vasculitis during the 28 days before the date of assessment (13). Our primary outcome was time to remission, defined as the absence of new or worse signs of disease activity on the BVAS and no more than 1 item indicating persistent disease activity (BVAS 1). Secondary outcomes included the proportion of patients who achieved remission at 6 and 9 months and the proportion with major and minor relapses. We defined major relapse as the recurrence or first appearance of at least 1 BVAS item indicating threatened vital organ function attributable to active vasculitis. We defined minor relapse as the recurrence or first appearance of at least 3 other BVAS items related to nonvital organs. An investigator classified patients as achieving remission or having relapse, and an independent observer validated these classifications retrospectively. Additional secondary outcomes were death; change in renal function; adverse events, including leukopenia and infection; and the cumulative dose of cyclophosphamide and prednisolone, which we calculated as the total cumulative drug dose at each time point in the study (3, 6, 9, 12, 15, and 18 months) divided by the number of patients in the study at that point. For each time point, we considered only the dose of drug for those patients still in the study. Unless otherwise noted, we assessed these outcomes at baseline; at 1.5, 3, 4.5, 6, 7.5, 9, 12, 15, and 18 months after baseline; and at relapse, on the basis of standard recommendations. Clinical assessments included BVAS measures at every visit and measures of cumulative damage from any cause since disease onset, as scored by the Vasculitis Damage Index (14), at baseline and every 3 months. Laboratory assessments included measures of full blood count, C-reactive protein, alanine transaminase, serum creatinine, and glucose, as well as dipstick urine analysis. We calculated glomerular filtration rate at entry, remission, and study end by using the Modification of Diet in Renal Disease method (15). Statistical Analysis We determined the sample size for the trial by clinical rather than statistical considerations. We set a recruitment goal of 160 patients; we considered that number ambitious, given the rarity of these conditions (12 per 1 million persons) and the need to recruit patients and conduct the trial within a period (5 years) that was reasonable for our resources. We performed analyses by intention to treat. To account for censoring, we compared remission and survival by using survival methods instead of relat

EULAR recommendations for conducting clinical studies and/or clinical trials in systemic vasculitis: focus on anti-neutrophil cytoplasm antibody-associated vasculitis

Objectives: To develop the European League Against Rheumatism (EULAR) recommendations for conducting clinical studies and/or clinical trials in systemic vasculitis. Methods: An expert consensus group was formed consisting of rheumatologists, nephrologists and specialists in internal medicine representing five European countries and the USA, a clinical epidemiologist and representatives from regulatory agencies. Using an evidence-based and expert opinion-based approach in accordance with the standardised EULAR operating procedures, the group identified nine topics for a systematic literature search through a modified Delphi technique. On the basis of research questions posed by the group, recommendations were derived for conducting clinical studies and/or clinical trials in systemic vasculitis. Results: Based on the results of the literature research, the expert committee concluded that sufficient evidence to formulate guidelines on conducting clinical trials was available only for anti-neutrophil cytoplasm antibody-associated vasculitides (AAV). It was therefore decided to focus the recommendations on these diseases. Recommendations for conducting clinical trials in AAV were elaborated and are presented in this summary document. It was decided to consider vasculitis-specific issues rather than general issues of trial methodology. The recommendations deal with the following areas related to clinical studies of vasculitis: definitions of disease, activity states, outcome measures, eligibility criteria, trial design including relevant end points, and biomarkers. A number of aspects of trial methodology were deemed important for future research. Conclusions: On the basis of expert opinion, recommendations for conducting clinical trials in AAV were formulated. Furthermore, the expert committee identified a strong need for well-designed research in non-AAV systemic vasculitides.

Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo

Apart from the diagnostic value of anti‐neutrophil cytoplasmic antibodies (ANCA), their detailed characterisation and that of their corresponding antigens have opened new ways for the exploration of the pathogenesis of primary systemic vasculitis. ANCA arc now thought to play an important functional role via activation of phagocytic cells (e.g. polymorphonuclear neutrophils (PMN)). In this study we examined the mechanisms by which ANCA could gain access to proteinase 3 (PR3) in intact PMN, at two levels: ex vivo by analysing the presence of PR3 on the plasma membrane of PMN from patients with ANCA‐associated vasculitis, and in vitro by stimulation of PMN using different cytokines, including recombinant tumour necrosis factor‐alpha (rhTNF‐α) and two forms of IL‐8 (produced by monocytic and endothelial cells). Using immunocytochemical staining techniques (FACS and immunoelectronmicroscopy) PR3 has been detected on the plasma membrane of PMN from patients with active ANCA‐associated vasculitis. However, this phenomenon is also seen in patients with sepsis who do not have ANCA. In addition. TNF‐α and both forms of IL‐8 act synergistically and induce a translocation of PR3 from the intragranular loci to the cell surface of PMN. These results provide strong evidence for the hypothesis that ANCA are directly pathogenic by binding to PR3 which is expressed on the cell surface of primed/activated PMN.

Cytokine profiles in Wegener's granulomatosis: Predominance of type 1 (Th1) in the granulomatous inflammation

OBJECTIVE To determine whether a specific cytokine pattern (type 1 [Th1] or type 2 [Th2]) predominates in Wegeners granulomatosis (WG), by evaluating interferon-gamma (IFNgamma) and interleukin-4 (IL-4) expression in different compartments of the body (i.e., biopsied nasal mucosal tissue [NBS], bronchoalveolar lavage [BAL] fluid, and peripheral blood [PB]) and comparing the findings with those in disease and healthy control subjects. METHODS Competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to assess IFNgamma and IL-4 expression in T cell clones (TCC), T cell lines (TCL), and polyclonal CD4+ and CD8+ cells derived from NBS, BAL, and PB. RESULTS Patients with WG and chronic rhinitis were found to share in situ production of messenger RNA (mRNA) specific for IFNgamma (Th1). Only 2 patients with WG expressed IL-4, whereas IL-4 mRNA PCR products were found in inflamed tissues of the disease control patients. The granuloma-derived T cells of WG patients produced only IFNgamma, while TCC, TCL, and CD4+ and CD8+ T cells from BAL and PB produced mainly IFNgamma. CONCLUSION Our data indicate that a Thl cytokine pattern predominates in the granulomatous inflammation in patients with WG.

A New Type of Perinuclear Anti-Neutrophil Cytoplasmic Antibody (p-ANCA) in Active Ulcerative Colitis but not in Crohn's Disease*

Sera of 64 patients with chronic inflammatory bowel disease (IBD) were screened for antibodies against neutrophil cytoplasmic antigens (ANCA) using an indirect immunofluorescence technique on ethanol-fixed human neutrophil granulocytes. 20 of 34 sera (59%) from patients with ulcerative colitis (UC) produced a fine-granular and perinuclear ANCA staining pattern (p-ANCA) clearly different from the typical diffuse and granular cytoplasmic ANCA fluorescence (c-ANCA, synonym ACPA) seen in active Wegeners granulomatosis (WG). The majority of the 20 p-ANCA positive UC patients had a high inflammatory disease activity. Among the 14 p-ANCA negative UC patients nine were without steroids; five of them had active disease, two were inactive and two had previously undergone colectomy. The remaining five patients still had active disease but received steroids for more than 4 weeks. Only 3 of the 30 sera from patients with Crohns disease (CD) showed positive p-ANCA reactions. To narrow the specificity of the p-ANCA reaction all 64 sera were tested by ELISA for antibodies against anti-proteinase-3 (WG specific) and on HEp-2 cells for antinuclear (ANA) and anticytoplasmic antibodies. Ten p-ANCA positive UC sera were also tested in a myeloperoxidase ELISA. Only one UC serum reacted positively in the proteinase-3-ELISA and another one produced a weakly positive anti-nucleolar ANA fluorescence on HEp-2 cells. None of the tested sera reacted with myeloperoxidase suggesting that the p-ANCA staining pattern of granulocytes is not restricted to anti-myeloperoxidase antibodies as reported in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral Blood and Granuloma CD4+CD28− T Cells Are a Major Source of Interferon-γ and Tumor Necrosis Factor-α in Wegener’s Granulomatosis

To elucidate whether the fraction of CD28− T cells within the CD4+ T-cell population is a major source of Th1-like and proinflammatory cytokine production driving Wegener’s granulomatosis (WG) granuloma formation, we analyzed the phenotype and functional characteristics of peripheral blood CD4+CD28− T cells and of T cells in granulomatous lesions of 12 patients with active WG. Surface markers and intracytoplasmic cytokine and perforin expression were assessed by flow cytometry. Cytokine secretion was measured by enzyme-linked immunosorbent assay. Immunohistological studies demonstrated interferon-γ and tumor necrosis factor-α cytokine positivity attributable to CD4+CD28− T cells in granulomatous lesions. Peripheral blood CD4+CD28− T cells expressed CD57, also found on natural killer cells, and intracytoplasmic perforin. They were generally CD25 (interleukin-2 receptor)-negative. CD18 (adhesion molecule β2-integrin) was strongly up-regulated on CD4+CD28− T cells, whereas only a minority of CD4+CD28+ T cells expressed CD18. CD4+CD28− T cells appeared as a major source of interferon-γ and tumor necrosis factor-α. In contrast, CD4+CD28+ T cells were able to produce and secrete a wider variety of cytokines including interleukin-2. One-quarter of CD4+CD28+ T cells expressed the activation marker CD25, but they lacked perforin. Thus, CD4+CD28− T cells appeared more differentiated than CD4+CD28+ T cells. They displayed Th1-like cytokine production and features suggestive of the capability of CD4+ T-cell-mediated cytotoxicity. CD4+CD28− T cells may be recruited into granulomatous lesions from the blood via CD18 interaction, and may subsequently promote monocyte accumulation and granuloma formation through their cytokine secretion in WG.

Improved outcome in 445 patients with Wegener's granulomatosis in a German vasculitis center over four decades

OBJECTIVE To determine the long-term outcome in patients with Wegeners granulomatosis (WG) over 4 decades in an academic hospital unit specializing in rheumatology. METHODS We included 290 patients, divided them into 2 cohorts, and compared them with the historical cohort of 155 patients. Comparisons were retrospective regarding disease manifestations, therapy, mortality, and incidence of malignancies. The historical cohort (cohort 1) included 155 patients diagnosed between 1966 and 1993, cohort 2 included 123 patients diagnosed between 1994 and 1998, and cohort 3 included 167 patients diagnosed between 1999 and 2002. RESULTS Over time, the interval between first symptoms and diagnosis was reduced by half (from 8 months to 4 months). Organ manifestations were similar in the 3 cohorts, and more than 80% of patients still required cyclophosphamide (CYC); however, the median cumulative dose was reduced significantly (from 67 gm in cohort 1 to 36 gm in cohort 2 and to 24 gm in cohort 3). The standardized mortality ratios (SMRs) declined (from 2.1 in cohort 1 to 1.41 in cohort 2 and to 1.03 in cohort 3), with fewer deaths related to WG and/or therapy (86.4% in cohort 1, 76.9% in cohort 2, 50% in cohort 3), decreasing relapse rates (63.9% in cohort 1, 51.2% in cohort 2, 35.3% in cohort 3), and no increased rate of malignancies. Compared with young females, young males had a considerably higher SMR (8.87 [95% confidence interval 4.05-16.8]) and more frequent renal manifestations (54.4% versus 33.8%). CONCLUSION Mortality of WG patients declined over the last 4 decades, probably due to improved diagnostic and therapeutic procedures and increased awareness of WG, which led to earlier diagnosis and therapy, reduction in relapse rates, and lower cumulative CYC dose with fewer deaths related to therapy.

Intravenous pulse cyclophosphamide in the treatment of interstitial lung disease due to collagen vascular diseases.

OBJECTIVE Substantial toxicity limits the use of daily oral cyclophosphamide (CYC) for the treatment of interstitial lung disease (ILD) due to collagen vascular diseases. We examined whether intravenous (i.v.) pulse CYC can be substituted for daily oral therapy. METHODS Six patients with rapidly progressive ILD due to polymyositis, systemic sclerosis, systemic lupus erythematosus, or primary Sjögrens syndrome received 6-9 cycles of i.v. pulse CYC (0.5 gm/m2 of body surface area), together with an initial course of 50 mg of prednisolone, which was tapered to a maintenance dosage of 5-7.5 mg/day, and their response was measured clinically, by high-resolution computed tomography (HRCT) and by assessment of the bronchoalveolar lavage (BAL) cell profile. RESULTS All patients showed significant improvement in exercise tolerance and lung function. Elevated BAL neutrophils dropped substantially, whereas the response of BAL lymphocytes was inconsistent. Low-attenuation opacities in the HRCT regressed in 4 patients and remained unchanged in 2, but reticular infiltrates remained largely unaffected. Remission was maintained with hydroxychloroquine, azathioprine, or cyclosporin A. CONCLUSION I.v. pulse CYC proved to be an effective and well-tolerated treatment in these patients. Since it appears to target mainly the inflammatory component of the disease, it should be reserved for progressive ILD featuring indices of high inflammatory activity.

Subacute bacterial endocarditis with positive cytoplasmic antineutrophil cytoplasmic antibodies and anti–proteinase 3 antibodies

OBJECTIVE To report a potentially important limitation of antineutrophil cytoplasmic antibody (ANCA) testing: positive results in patients with subacute bacterial endocarditis (SBE). METHODS We describe 3 patients with SBE who presented with features mimicking ANCA-associated vasculitis (AAV) and positive findings on tests for cytoplasmic ANCA (cANCA) by indirect immunofluorescence and for anti-proteinase 3 (anti-PR3)antibodies by antigen-specific enzyme-linked immunosorbent assay (ELISA). We also reviewed the published literature describing infectious diseases with (misinterpreted) positive ANCA results through a Medline search of English-language articles published between 1966 and January 1999. These previously reported cases were reinterpreted using an ANCA scoring system that combines the findings of immunofluorescence and antigen-specific ELISA testing. RESULTS We are now aware of a total of 7 cases of SBE with positive cANCA and anti-PR3 antibodies. We are not aware of any cases of SBE associated with antimyeloperoxidase/perinuclear ANCA. Clinical manifestations mimicking AAV included glomerulonephritis, purpura, epistaxis, or sinus symptoms in 6 of the patients. Streptococcal species were identified in 5 patients, and cardiac valvular abnormalities were demonstrated in 6. All patients except 1, who died of a complication of SBE, recovered with antibiotic therapy. CONCLUSION Findings of tests for anti-PR3/ cANCA antibodies may be positive in patients with SBE. When encountering ANCA positivity in patients suspected of having systemic vasculitis, physicians should take appropriate steps to rule out infectious diseases, including SBE, before committing the patient to long-term, aggressive immunosuppressive therapy.