GRANULOMATOSI DI WEGENER E VASCULITI NECROTIZZANTI

Aims: To investigate the predictive value of ANCA in 55 patients with systemic WG included in a randomized trial comparing steroids (CS) and oral vs pulse cyclophosphamide (CY).

Patients: After a CY pulse given at diagnosis, patients were randomized to receive either pulse (0.7 g/m2) or oral CY (2 mg/kg/d) independently of ANCA status. ANCA were monitored throughout the study.

Conclusions: ANCA positivity is correlated with occurrence of relapse. Persistence or reappearance of ANCA should be considered a warning signal preceding a relapse. In patients without clinical manifestations, ANCA alone cannot be used to guide treatment.

We assessed the cost-effectiveness of three days a week TMP-SXT as prophylaxis against PCP in patients with SLE or WG on immunosuppressive therapies.

We constructed a decision analysis tree comparing the TMP-SXT strategy to the no-prophylaxis strategy and subjected each strategy to Markov analysis using DATA™ 3.0 software (TreeAge). We ascertained the occurrence of PCP in our target populations, the morbidity and mortality of PCP infection in those populations, and the costs associated with both prophylaxis and infection with PCP from available data and review of prior studies. Medical outcomes included death from PCP, death from the underlying inflammatory disease, or death from all other causes adjusted for age.

PCP occurs in up to 4.27% of SLE patients and 6.25% of patients with WG on immunosuppressive therapy. The mortality for those infected with PCP in these populations is 33.72%. Three times a week TMP-SXT prophylaxis prevents virtually all cases of PCP and increases the mean life expectancy of SLE patients by 1.28 quality adjusted life years (QALY) and by 1.7 QALYs in WG patients. The cost effectiveness of TMP-SXT prophylaxis in patients with SLE is $27.57 per QALY and $34.82 per QALY in patients with WG. We subjected model parameters to a series of one way sensitivity analyses over a wide range of values and found the results to be very stable to a variety of assumptions including rates of PCP infection as low as 0.427% for SLE and 0.625% for WG.

These results demonstrate that PCP prophylaxis with TMP-SXT three times a week in patients with SLE and WG on immunosuppressive therapies is extremely cost effective and should become the standard of care.

Disease activity in systemic vasculitis can be accurately measured using a clinical tool (Birmingham Vasculitis Activity Score - BVAS) which distinguishes worsening or new features of vasculitis (BVAS-1) from continuing low grade disease activity (BVAS-2). A marker of clinical damage in vasculitis has also been developed (Vasculitis Damage Index - VDI). We have evaluated the relationship between these assessments in a cross-sectional study of patients with systemic vasculitis. Non-parametric statistics (Mann Whitney U and Spearman's rank correlation) were used.

BVAS and VDI are of value in the assessment of WG, MPA and CSS. There is a direct relationship between chronic/grumbling disease activity (BVAS-2) and damage but no correlation between new/worse disease activity (BVAS-1) and current VDI. These measures are of practical value in assessing patients with systemic vasculitis.

In conclusion, TNFR I and II are up-regulated in active WG and tend to normalize when the disease is in remission.

Primary systemic vasculitides are multisystem diseases, renal impairment due to glomerulonephritis is a common feature. Previous studies from tertiary referral centres suggest that the annual incidence of biopsy proven pauciimmune, crescentic glomerulonephritis (GN) is approximately 8/million. Our institution is a central referral hospital for a stable population of 415 000 adults, from which we have previously reported the annual incidence of Wegener's Granulomatosis (WG), microscopic polyangiitis (mPA), Churg Strauss syndrome (CSS) and Henoch Schonlein Purpura (1988-93). The aim of this study was to estimate the incidence of primary vasculitis with renal involvement using clinical diagnoses and histological definitions for comparison with previous studies.

Patients with primary vasculitis were identified from renal biopsy reports and our prospective vasculitis register over 66 months (January 1992-June 1997 inclusive). Case notes were reviewed and clinical and laboratory data extracted. Patients were classified using American College of Rheumatology (ACR) criteria 1990 for WG, HSP, CSS and PAN and the Chapel Hill Consensus Conference Definition for mPA.

The incidence of crescentic and pauciimmune GN is similar to previous studies but the overall incidence of renal vasculitis is much higher.

Etoposide (VP16) is a podophyllotoxin derivative used in both myelo- and lympho-proliferative disorders. Etoposide has been reported to be efficient in cyclophosphamide-resistant WG, although only in single cases.

In an open study, we treated 4 patients with biopsy-proven, c-ANCA positive generalized WG. Second-line treatment with etoposide aimed at: a) induction of remission (case 1) in a severe cyclophosphamide-resistant flare, b) achieving complete remission in smoldering WG, with orbital pseudotumor (case 2) or pauci-symptomatic persistent alveolar hemorrhage (case 3) and c) replacement of methotrexate treatment after life-threatening pneumocystis pneumonitis (case 4). All received a sequential oral (100 mg/day for 1 week every month) VP16 regimen.

Case 1 2 3 4

Age at WG onset 49 53 39 51

WG duration (mo) 28 204 108 60

Previous treatments iv-cyp, o-cyp, prd iv-cyp, mtx, prd iv-cyp, prd mtx iv-cyp, mtx, prd

Concomitant therapy prd prd prd prd, smz

Duration of VP16 treatment (mo) 15 15 26 38

cyp: cyclophosphamide (intravenous: iv, oral:o); mtx: methotrexate; mo, months; prd: prednisone smz: sulfametoxazole.

Etoposide treatment was well tolerated. It was associated with improvement of clinical status for a protracted period in all patients although complete remission was obtained in one patient only (case 4). However, WG relapsed in all cases after a mean time of 23.5 ± 11 months, leading to VP16 withdrawal. We conclude that etoposide has no clear-cut effect for induction or maintenance of complete remission in WG. Considering the risk of secondary leukemia, increasingly recognized in children treated with VP16, and since "safe" levels of VP16 exposure are currently unknown, its use should be limited to life-threatening, cylophosphamide-resistant active GW.

Proteinase-3 (PR-3) is a neutral serine proteinase present in the azurophil granules of human polymorphonuclear leukocytes. It consists of 228 amino acids with a molecular weight of 29 kDa. Many different physiological and pathological properties have been reported. It is identical to the target autoantigen (C-ANCA) associated with Wegener's granulomatosis. The diagnosis and classification of Wegener's granulomatosis and related vasculitides were advanced by characterization of these antibodies.

Performing northern blot technique we demonstrated the expression of PR-3 in lung tissue as one of the mainly affected organs in Wegener's granulomatosis. In situ hybridisation experiments for PR-3 revealed positive signals in distinct locations of the tissue. The intensities of the PR-3 signals and the number of cells expressing PR-3 mRNA did change in some region of the lung. The PR-3 mRNA expression was focused to regions which were also infiltrated by inflammatory cells. Nearly all PR-3 mRNA positive cells were located at the alveolus covering cell-layer. To characterize the PR-3 positive cells we used immunohistochemical double labeling with anti-CD 68 (macrophages) and anti-CD 15 (neutrophils). Some PR-3 positive cells were also positive for CD 15 and CD 68, indicating that these cells were neutrophils or macrophages. But the majority of PR-3 positive cells were negative for CD 15 and CD 68. With regard to the histomorphological appearance, theses cells are most likely pneumocytes.

In summary, the tissue expression of PR-3 as a target for circulating ANCA is not restricted to hematopoetic cells and may therefore contribute to lung damage in patients with WG via direct interaction.

Genotype: NA1/1 NA1/2 NA2/2 Allele freq. NA1 NA2

Control (n = 190) 25 (13%) 82 (43%) 83 (44%) Control 0.35 0.65

Renal-WG (n = 91)* 19 (21%) 46 (51%) 26 (29%) Renal-WG† 0.46 0.54

Renal-SLE (n = 81) 12 (15%) 30 (37%) 39 (48%) Renal-SLE 0.33 0.67

Purpose: Ophthalmic involvement may be present in up to 58% of patients with Wegener's granulomatosis (WG) and in some instances ocular manifestations constitute the major symptoms, or are the presenting clinical manifestation of the disease. In a previous investigation into corneal autoimmunity, we demonstrated that a putative autoantigen, a protein of 66kD, present in bovine corneal epithelium, binds circulating autoantibodies in 50% patients with WG and PUK (v normal controls p < 0.05, OR = 10). The aim of this study was to purify and identify this 66 kD protein.

Methods: The 66 kD autoantigen was purified from a bovine corneal epithelial protein extract using DE52 ion exchange chromatography. Purified protein was used to raise polyclonal antibodies from rabbits. These antibodies were used to screen a bovine corneal epithelial cDNA expression library. Positive clones where purified and sequenced. Clones were identified by DNA sequence homology searches of the Genbank DNA database.

Results: A cDNA clone which showed strong binding to both the rabbit polyclonal antibody and patients sera, showed 85% homology to rabbit cytokeratin type 3 (K3). K3 is a basic cytokeratin specific to corneal epithelium. No bovine DNA sequence for K3 is available. However, bovine keratin 3 is larger than rabbit K3, with a molecular weight of 66 kD. The PUK associated autoantigen has biophysical properties similar to K3. Immunofluorescence using both patient sera and the rabbit antibody shows a cytoplasmic binding pattern on both human and bovine cornea consistent with the antigen being K3.

Conclusion: This evidence suggests that the 66 kD autoantigen associated with PUK in WG is cytokeratin 3 and this forms the basis for further clinical investigation.

Thirty-two patients with newly diagnosed ANCA+ Wegener's granulomatosis were randomized to receive 6 courses of plasma exchange (PE) with high-dose prednisolone and cyclophosphamide (CY) or high-dose prednisolone and CY alone for induction of remission. After 3 months patients were randomized to either replace CY with cyclosporin-A (CyA) or to continue with CY for 9 months. The patients (8 in each group) were comparable with respect to age, sex, ANCA titres, biopsy findings, pulmonary involvement and kidney function. Six patients in the +PE group and 5 patients in the non-PE group had plasma creatinine levels >0.3 mmol/l.

After 1 month, 0/16 patients receiving PE vs. 6/16 patients in the non-PE group had progressive disease (Fisher's test; P = 0.03) and none in the PE group vs. 4 patients in the non-PE group were on hemodialysis (P < 0.05). Similar results were noted after 3 months: +PE group 0/16 patients with progressive disease, non-PE group 5/16 patients with progressive disease. Twenty late relapses were observed, 12 among CyA-treated and 8 among CY-treated patients, 18 patients responded to increased doses of prednisolone. After 12 months, 4 CyA-treated and 5 CY-treated patients had still progressive disease.

PE is beneficial for induction of remission in WG regardless of kidney function. More studies are needed to determine whether CyA may replace CY for maintenance of remission.

Objectives: The aim of this study was to identify factors predicting the outcome of systemic WG.

Methods: We studied 52 patients with systemic WG diagnosed between October 1990 and March 1994 who participated in a prospective therapeutic trial comparing intravenous versus oral cyclophosphamide (CY) in combination with corticosteroids. Survival was recorded and an attempt was made to correlate it with demographic, clinical, biological and immunological findings at diagnosis, and with occurrence of infectious complications during follow-up. Survival curves were calculated using the Kaplan-Meier method. Potential prognostic factors were investigated using log-rank test.

Results: The mean follow-up was 1.9 yr; 19/52 (36.5%) patients died. According to univariate analyses, ENT involvement was correlated with a better prognosis (p = 0.01). No significant prognostic value could be attributed to other organ involvements (e.g., lung, p = 0.85; kidney, p = 0.57), age (>55 years), sex, number of involved organs (>3 involved organs), ANCA positivity, erythrocyte sedimentation rate (>80 mm/1st h), serum creatinine level (>250 µmol/l) or infectious complications.

Conclusion: Independently of treatment, only ENT involvement alone is predictive of a better become.

Purpose: To characterize c-ANCA autoantibodies in patients with Wegener's granulomatosis by using a expression system for conformationally intact recombinant human proteinase 3.

Methods and Results: COS cells were transfected with pME18S-human proteinase 3 expression vector. The transfected COS cells were reacted with c-ANCA positive sera, followed by detection with immunocytochemical staining. We found majority of the c-ANCA positive sera stained the transfected COS cells. Recombinant proteinase 3 has proteinase activity, because the recombinant hydrolyzes a peptidyl-MCA substrate. We next studied whether anti-proteinase 3 autoantibodies modulate proteolytic activity of purified proteinase 3. We found that anti-proteinase 3 autoantibodies can be categolized into 3 classes; the autoantibodies which enhance proteolytic activity of proteinase 3; the autoantibodies which deminish proteolytic activity of proteinase 3; the autoantibodies which do not affect proteolytic activity of proteinase 3.

Conclusion: It is suggested that anti-proteinase 3 autoantibodies can be categolized by their effects on proteolytic activity of proteinase 3 into three classes.

Objective: To examine our experience with methotrexate (MTX) and daily prednisone (PRED) as the initial treatment of Wegener's granulomatosis (WG) in patients without life-threatening disease.

Methods: Between November 1992 and November 1997, we treated 19 patients with non-life threatening WG with the combination of oral weekly MTX (starting at 7.5-10.0 mg/week) and daily PRED (median starting dose, 40 mg/day; range: 20-60). Before treatment with this regimen, none of our patients had received previous treatment for WG. The MTX dose was increased to 15 mg/week by the end of the first month, and then by 2.5 mg/week until the disease was controlled. We attempted to taper PRED to 20 mg/day by the end of the second month, but did not use alternate day corticosteroids.

Results: At presentation, the average number of organ systems involved per patient was 3.6. Nine of the 19 patients (47%) had glomerulonephritis, but none had a serum creatinine greater than 1.2 mg/dl at presentation. Only 37% of the patients were hospitalized at presentation. Seventeen of 19 patients (89%) improved with treatment, and 14 (74%) achieved a remission. However, half of those who achieved remission suffered relapses, and only 1 patient (5%) achieved a durable, complete remission (disease-free status off all medications). Fifteen patients (79%) were able to taper prednisone to less than 10 mg/day. Six of 7 disease relapses responded to increases of MTX and/or PRED. Only 1 patient developed glomerulonephritis while on treatment and required a change of therapy to cyclophosphamide.

Treatment with MTX and PRED was well-tolerated: only 2 (11%) of the patients stopped the treatment because of side-effects (major liver function test abnormalities in both cases). No patient suffered permanent morbidity from MTX treatment There were no deaths in this series, either from WG itself or from the treatment regimen.

Conclusions: In selected patients with WG, the combination of MTX and daily PRED effectively controls the disease. However, chronic disease courses are the rule with this treatment regimen, and the likelihood of disease relapse is high. In our experience, the use of MTX and PRED in WG was safer than previously described, despite the use of daily corticosteroids.

Objectives and Methods: To evaluate patient (pt) perceived effects of WG on health, function, income and personal relationships, a self-administered questionnaire, designed by the authors and subsequently revised with a pt focus group, was completed by 60 pts.

Results: Pts had WG for a median period of 5 yrs. Pt-reported responses for organ system involvement, surgeries and disease activity were comparable to physician-recorded data from a WG registry. As previously reported from physician-generated data, a prolonged delay in diagnosis (mean = 16.8 mos) and the need for multiple consultations prior to therapy contributed to medical morbidity. Although 73% of pts perceived WG to be in remission following therapy, 78% required continued immunosuppressive treatment many years after diagnosis. Normal activities of daily living were compromised in 80% of pts. Half of those employed prior to WG required job modification or were totally disabled (31%). A 28.5% reduction (mean) in income occurred within one year after diagnosis. The effects of WG on relationships with pt's spouse, family and friends varied considerably. Pts with WG suffer substantial medical and functional morbidity and incur significant socioeconomic losses.

Conclusions: For most pts, medical advances have transformed WG from a disease with high potential for short-term mortality to a chronic illness. This is the first study of pts' assessments of medical, socioeconomic and quality of life effects of WG and its treatment. The effects of morbidity, mortality, disability and out-patient medical expenses indicate that the financial impact of WG substantially exceeds prior estimates of $30 million/yr for just hospitalizations in the U.S.

Objective: In this multicenter prospective trial we tried to evaluate the efficacy of corticosteroids (CS) alone in PAN and MPA without poor-prognostic factors at onset.

Patients and Methods: Patients with PAN or MPA were enrolled when none of the following 5 factors was present at entry: myocardial, severe GI tract or central nervous system involvement, creatininemia > 120 µmol, proteinuria > 1 g/d. The regimen consisted of CS alone; immunosuppressive agents (oral azathioprine (AZA) or monthly IV cyclophosphamide (CY)) were randomized only in the case of relapse, uncontrolled vasculitis or when it was not possible to taper CS to <20 mg/d. Only patients completing at least 6 months of follow-up were considered for this abstract.

Results: 51 patients (23 men, 28 women, mean age: 57.2 ± 15.6 yr) were included between February 1994 and December 1997. Mean follow-up was 608 ± 353 d (range: 5-1231 d). 26/51 (51%) are in clinical remission (CR) and have not relapsed to date. 23 relapses in 22 patients (43%) were noted, and CR could be reobtained in 18/22 (81.8%): 7/9 with the adjunction of AZA, 5/7 with the addition of CY, and 6/6 with higher doses of CS or IVIg addition. At this time, 44/51 (86.3%) patients are in CR. 3 patients died during follow-up: 1 aspiration pneumonia (d 5), 1 mesenteric infarct occurring after a short period of remission (d 90), and 1 refractory polyneuropathy which progressed to neurovegetative dysfunction (d 376).

Conclusion: These preliminary results show that, in PAN and MPA without manifestations of poor prognosis, a CR can be obtained with CS alone in 51%. Immunosuppressors are useful to achieve CR when CS alone cannot. These encouraging results will be further assessed at the end of the study.

Substantial morbidity occurs in SNV, despite remission induction with standard cyclophosphamide (Cy) therapy, due to scars/damage from persistent disease activity, relapse and drug toxicity. Less than 50% of cases enter full remission within 6 months and early damage is disease related, indicating a therapeutic window for early intensive therapy. We propose intensive Cy therapy for SNV at presentation or initial relapse will rapidly control disease activity and prevent early damage.

An open label safety trial of pulsed IV Cy at 2.5-0.6 g/m2 was conducted in eight primary SNV cases (4 PAN, 4WG). Cy dose was reduced for age, renal impairment and previous exposure to Cy. Safety measures included frequent monitoring, antimicrobial prophylaxis for leucopaenia plus G-CSF if prolonged. Patients received 2 identical cycles of 3 pulses in descending doses. Trough neutrophil counts <1 × 109/L were dose-limiting at 2.5 g/m2 Cy. Toxicity was predictable and reversible. High dose Cy produced marked clinical benefit with complete suppression of all new disease activity at 3 months and a substantial improvement in well being. The damage observed at 6 months was minimal compared to conventional regimes.

This study suggests that intensive Cy for induction is well tolerated, induces earlier remission and diminishes the development of early damage. It appears a useful induction regime.

In primary systemic vasculitis (PSV) indirect evidence suggests endothelial damage occurs early and persists during inactive disease, whilst severity of the inflammatory disease correlates with subsequent cardiovascular events. We test the hypothesis that endothelial damage is detectable in PSV in vivo in a clinically uninvolved vessel. Blood flow induces endothelial derived nitric oxide (NO), and impaired NO release characterises endothelial damage. Initially we examined this in a cross-sectional study of 13 biopsy-proven PSV with disease activity scored by international criteria. A trained sonographer using standardised high-resolution ultrasound measured brachial artery diameter to determine flow induced NO mediated endothelium dependent vasodilatation (EDV) and GTN induced endothelium independent vasodilatation (EIV). EDV was profoundly impaired irrespective of disease activity or treatment, artery diameter increased 0.8% ± 2.3% in PSV versus 9.2% ± 2.3% in healthy controls (p < 0.0001). All subjects responded to GTN (EIV), artery diameter increased 13.6% ± 4.1% in PSV versus 18.1% ± 4.5% in controls (p = 0.017). Currently, EDV is being compared in limited versus systemic vasculitis.

This study provides novel evidence of profound endothelial dysfunction in vivo in PSV at a clinically uninvolved site in active and inactive disease. These findings in PSV are similar to patients with multiple risk factors for atherosclerosis - where inflammatory mechanisms of endothelial damage are also implicated. This has implications for late development of cardiovascular events in PSV.