John L. Niles

A disease-specific activity index for Wegener's granulomatosis: Modification of the Birmingham Vasculitis Activity Score

OBJECTIVE To refine and validate the Birmingham Vasculitis Activity Score (BVAS) as a disease-specific activity index for Wegeners granulomatosis (WG). METHODS Sixteen members of the International Network for the Study of the Systemic Vasculitides (INSSYS) revised the BVAS, with 3 goals: to reduce the redundancy of some component items, to enhance its ability to capture important disease manifestations specific to WG, and to streamline the instrument for use in clinical research. We defined the items and weighted them empirically as either minor (e.g., nasal crusting = 1 point) or major (e.g., alveolar hemorrhage = 3 points). We then validated the new, disease-specific BVAS/WG in 2 simulation exercises and a clinical case series that involved 117 patients with WG. RESULTS We removed 38 items from the original BVAS, revised 9 items, and added 7 new items. Correlations between the scores on the BVAS/WG and the physicians global assessment (PGA) of disease activity were high, even when patients in remission were excluded. In the clinical case series, Spearmans rank correlation coefficient between the BVAS/WG and the PGA was r = 0.81 (95% confidence interval 0.73-0.87). The interobserver reliability using intraclass (within-case) correlation coefficients in the 2 simulation exercises was r = 0.93 for the BVAS/WG and r = 0.88 for the PGA in the first and r = 0.91 for the BVAS/WG and r = 0.88 for the PGA in the second. There was no significant observer effect in the scoring of the BVAS/WG or the PGA. The discriminant validity of the BVAS/WG was good: r = 0.73 (95% confidence interval 0.43-0.83). CONCLUSION The BVAS/WG is a valid, disease-specific activity index for WG. Tested in simulation exercises and in actual patients, the BVAS/WG correlates well with the PGA, is sensitive to change, and has good inter- and intraobserver reliability. The INSSYS will use the BVAS/WG to assess the primary outcome in a phase II/III trial of etanercept in WG.

Drug-associated antineutrophil cytoplasmic antibody-positive vasculitis: prevalence among patients with high titers of antimyeloperoxidase antibodies.

OBJECTIVE The triggers that induce antineutrophil cytoplasmic antibody (ANCA)-positive vasculitis (APV) are largely unknown. However, there have been reports suggesting that hydralazine, propylthiouracil, and several other drugs may cause some cases of APV, and the majority of these cases have been associated with antimyeloperoxidase (anti-MPO) ANCA. Our experience led us to hypothesize that cases of high titers of anti-MPO antibodies are often drug-associated. METHODS In this study, we determined the prevalence of exposure to hydralazine, propylthiouracil, and other drugs previously implicated in APV among 30 patients with vasculitis and the highest titers of anti-MPO antibodies newly detected in our laboratory between 1994 and 1998. The clinical, histologic, and other serologic features of these 30 patients were also examined. RESULTS The 30 study patients accounted for 12% of the 250 new patients with APV and anti-MPO who were tested during the study period. All 30 study subjects had anti-MPO titers that were more than 12 times the median titer of the 250 patients. Ten (33%) of the 30 patients had been exposed to hydralazine and 3 (10%) had been exposed to propylthiouracil. An additional 5 patients (17%) had been exposed to 1 of the other previously reported candidate drugs: 2 to penicillamine, 2 to allopurinol, and 1 to sulfasalazine. One of the patients exposed to hydralazine had also been exposed to allopurinol. In all cases, the clinical and histologic findings were typical of APV. There was a strong association between the presence of antielastase and/ or antilactoferrin antibodies and exposure to candidate drugs. CONCLUSION These data suggest that a sizable proportion of cases of APV with high titers of anti-MPO antibodies are drug-associated, especially following exposure to hydralazine or propylthiouracil. We recommend that the use of these drugs should be sought in cases of anti-MPO-positive vasculitis, particularly among patients with high titers of these antibodies.

Prevalence of Antineutrophil Cytoplasmic Antibodies in a Large Inception Cohort of Patients with Connective Tissue Disease

Antineutrophil cytoplasmic antibodies (ANCA) are strongly associated with the spectrum of vasculitis that includes Wegener granulomatosis, microscopic polyangiitis, the Churg-Strauss syndrome, idiopathic necrotizing and crescentic glomerulonephritis, and related or overlapping forms of vasculitis [1-3]. Other forms of vasculitis, including Takayasu arteritis, Henoch-Schonlein purpura, and cryoglobulinemia, are not associated with the presence of ANCA. Different assays have been used to test for ANCA, including indirect immunofluorescence and immunoassays that use either crude or highly purified preparations of specific antigens. Although several ANCA antigens have been described [2, 4], only antiproteinase 3 antibodies (anti-PR3) and antimyeloperoxidase antibodies (anti-MPO) have been shown to be of value in the diagnosis of vasculitis [1-3]. When used to stain ethanol-fixed, cytocentrifuged, normal human neutrophils by indirect immunofluorescence, anti-PR3 produce a cytoplasmic pattern of staining (C-ANCA) and anti-MPO produce a perinuclear or nuclear pattern (P-ANCA). At presentation, the clinical features of patients with Wegener granulomatosis, microscopic polyangiitis, and the Churg-Strauss syndrome may include glomerulonephritis, alveolar hemorrhage, tracheobronchitis, sinusitis, palpable purpura, arthritis, ocular inflammation, and neuropathy. Patients with connective tissue diseases may also display many of these features. Therefore, testing for ANCA, if highly specific, could be of great importance in the initial diagnostic evaluation of patients with a differential diagnosis that includes both connective tissue disease and vasculitis. Determination of the specificity of tests for ANCA in the diagnosis of vasculitis is crucial because the decision of whether to pursue biopsies or initiate potentially toxic immunosuppressive therapy may be made on the basis of results of such testing. We report the results of a blinded, controlled study to determine the prevalence of ANCA in a unique group of patients with various connective tissue diseases who were followed for as long as 5 years. A standard testing system, including indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA) for anti-PR3 and anti-MPO, was used to determine the prevalence of ANCA. Methods Patients The Early Undifferentiated Connective Tissue Disease project, a multicenter study funded by the National Institutes of Health through the Cooperative Systematic Studies of the Rheumatic Diseases Program, was designed to develop and prospectively follow a large cohort of patients with rheumatologic disease early in their clinical course. All patients were enrolled within 1 year of the onset of signs, symptoms, or serologic abnormalities that suggested connective tissue disease. Patients were evaluated at study entry and at years 1, 3, and 5. More than 800 clinical and laboratory variables were recorded for each patient according to a standardized protocol. Details of the original project and other study results have been published elsewhere [5-8]. Enrollment began in 1982 and was completed in June 1987. Patients with systemic lupus erythematosus, rheumatoid arthritis, inflammatory myositis, polymyositis or dermatomyositis, or scleroderma had to meet standardized criteria for the diagnosis of these diseases [9-12]. Early undifferentiated connective tissue disease (EUCTD) was diagnosed if patients did not meet criteria for the other connective tissue diseases and met specific criteria that have been described elsewhere [5]. We used serum samples that had been collected from the study patients at baseline. The original study enrolled 410 patients; for 386 (94%) of these, enough serum was available so that the patients could be included in our study. Final diagnoses were determined at the last visit and were therefore based on the cumulative data that had been collected. All analyses and results were based on the final diagnosis; as a result, patients were separated into the following diagnostic groups: systemic lupus erythematosus (n = 70), rheumatoid arthritis (n = 70), scleroderma (n = 45), polymyositis (n = 36), and EUCTD (n = 165). Within the original group, a subgroup of patients who had the Sjogren syndrome was identified. The Sjogren syndrome was defined by the presence, at any time during the study, of xerophthalmia (as determined by positive results on a Schirmer test); xerostomia; and positive results for any one of the following tests: antinuclear antibodies, rheumatoid factor, anti-Ro (anti-SS-A) antibody, or anti-La (anti-SS-B) antibody. All patients in the subgroup with the Sjogren syndrome also had a diagnosis of a primary connective tissue disease as outlined above. Forty-four patients met our definition for the Sjogren syndrome; these patients were drawn from all five diagnostic groups: systemic lupus erythematosus (n = 6), rheumatoid arthritis (n = 9), scleroderma (n = 5), polymyositis (n = 1), and EUCTD (n = 23). Serum specimens from 33 patients who were known to have the antiphospholipid syndrome [13, 14] with medium-to-high titers of IgG or IgM anticardiolipin antibodies (provided by EN Harris) were also studied. Serum samples from 200 random blood donors were collected through the Massachusetts General Hospital Blood Transfusion Service; these donors served as a control group. Serum samples were also collected from 52 patients with Wegener granulomatosis, microscopic polyangiitis, or related forms of vasculitis who had positive results on tests for ANCA; these patients were selected as positive controls for the ANCA assays. This control group of patients with vasculitis included 26 patients with anti-PR3 and 26 patients with anti-MPO; patients with high, low, and intermediate antibody titers were included. Serum Serum samples were both stored and shipped at 20C. All 671 samples, each of which had a unique identifier based on its original source, were assigned new, randomized, study identification numbers and were redivided and relabeled. The laboratory investigators who did the ANCA assays were thus blinded to the diagnosis for each patients sample. The code for the serum samples was not revealed until all data were collected and the analysis was ready to begin. Indirect Immunofluorescence for Antineutrophil Cytoplasmic Antibodies Indirect immunofluorescence was done as described elsewhere [15]. The results of staining were classified as having one of four patterns: C-ANCA (cytoplasmic), P-ANCA (perinuclear), atypical (neither cytoplasmic nor perinuclear), or negative. Because of the subjective nature of scoring the results of immunofluorescence for ANCA, each sample was stained twice and interpreted independently. Results of the first round of staining were interpreted by one observer, and results of the second round were interpreted by this observer and a second observer; both observers had considerable experience in interpreting the results of immunofluorescence staining of ANCA. If all three readings were the same, the interpretation was considered final. If the interpretations differed, a third slide was prepared and reexamined by the two observers. If at least three of the five interpretations matched, the results were considered final; if not, the staining results were considered to be atypical ANCA. Both observers were blinded to the previous results of immunofluorescence and ELISAs. Testing for Antineutrophil Cytoplasmic Antibodies by Enzyme-Linked Immunosorbent Assay We used direct antigen-specific ELISAs to detect anti-PR3 and anti-MPO, as described elsewhere [15-17]. A sandwich ELISA was also done on each sample. In the sandwich ELISA, monoclonal antibody 1E8 [18] was adhered to the wells of microtiter plates and used to bind proteinase 3. Subsequent steps were the same as those of the direct ELISA. An additional control in the sandwich ELISA for anti-PR3 was performed with selected serum specimens. To control for antibodies to the monoclonal catching antibody, additional wells were coated with monoclonal anti-PR3 catching antibody 1E8 but were not subsequently incubated with cytoplasmic extract of granulocytes. The reactivity of the serum to the monoclonal 1E8 alone was then subtracted from the reactivity to the 1E8-PR3 complex. The result is the titer for a revised sandwich ELISA for anti-PR3. Final Interpretation of Results of Testing for Antineutrophil Cytoplasmic Antibodies A final interpretation of ANCA test results was determined for each patient by using the results of immunofluorescence and ELISA. The set of decision rules used for the final interpretation of testing for anti-PR3 is outlined in Figure 1. A final interpretation for the presence of anti-MPO was considered positive only if samples were positive on immunofluorescence for P-ANCA or atypical ANCA patterns and on direct ELISA for anti-MPO. This is the same system that we use to provide a final interpretation for clinical samples submitted to our laboratory. Figure 1. Testing algorithm used for the final determination of the presence of antiproteinase 3 antibodies (anti-PR3). Statistical Analysis Comparisons between groups were analyzed by the Fisher exact test for categorical variables using a two-tailed significance level of 0.05. All data were stored on a SUN SPARC-5 workstation (Sun Microsystems, Mountain View, California) and analyzed using SAS software (SAS Institute, Cary, North Carolina) for UNIX. The 95% CIs for test specificity were determined using the methods described by Collett [19]. Results Indirect Immunofluorescence for Antineutrophil Cytoplasmic Antibodies The final results of immunofluorescence are shown in the (Table 1). None of the study patients or controls had C-ANCA by immunofluorescence staining. The rate of P-ANCA positivity by immunofluorescence was low for all study groups except patients with systemic lupus erythematosus, who had a rate of 31%. However, atypical patterns of ANCA immunofluorescence we

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.

Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis

Anti-neutrophil cytoplasmic antibody-associated (ANCA-associated) small vessel necrotizing vasculitis is caused by immune-mediated inflammation of the vessel wall and is diagnosed in some cases by the presence of myeloperoxidase-specific antibodies (MPO-ANCA). This multicenter study sought to determine whether differences in ANCA epitope specificity explain why, in some cases, conventional serologic assays do not correlate with disease activity, why naturally occurring anti-MPO autoantibodies can exist in disease-free individuals, and why ANCA are undetected in patients with ANCA-negative disease. Autoantibodies from human and murine samples were epitope mapped using a highly sensitive epitope excision/mass spectrometry approach. Data indicated that MPO autoantibodies from healthy individuals had epitope specificities different from those present in ANCA disease. Importantly, this methodology led to the discovery of MPO-ANCA in ANCA-negative disease that reacted against a sole linear sequence. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice. The confounder for serological detection of these autoantibodies was the presence of a fragment of ceruloplasmin in serum, which was eliminated in purified IgG, allowing detection. These findings implicate immunodominant epitopes in the pathology of ANCA-associated vasculitis and suggest that autoantibody diversity may be common to other autoimmune diseases.

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.

Anticoagulation in continuous renal replacement therapy.

Continuous renal replacement therapies (CRRTs) allow for gradual solute and fluid removal. In very sick patients with acute renal failure, they may be better tolerated than hemodialysis. The major drawback to CRRTs is the need for anticoagulation to maintain filter patency. The patients who are likely to benefit from CRRTs are also at higher risk for bleeding from systemic anticoagulation. The most commonly used form of anticoagulation for CRRTs, low-dose heparin, causes bleeding in 10-50% of patients. Regional anticoagulation using protamine may reduce the risk of bleeding, but it is difficult to use. Low molecular weight heparin and prostacyclin both may partially reduce bleeding, but are difficult to dose. Regional anticoagulation with citrate is easy to use and has been shown to prolong filter life without systemic anticoagulation. It is the anticoagulant of choice for most patients on CRRT.

Contaminated Cocaine and Antineutrophil Cytoplasmic Antibody-Associated Disease

BACKGROUND AND OBJECTIVES Approximately 70% of illicit cocaine consumed in the United States is contaminated with levamisole. Most commonly used as a veterinary antihelminthic agent, levamisole is a known immunomodulating agent. Prolonged use in humans has been associated with cutaneous vasculitis and agranulocytosis. We describe the development of a systemic autoimmune disease associated with antineutrophil cytoplasmic antibodies (ANCA) in cocaine users. This complication appears to be linked to combined cocaine and levamisole exposure. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Cases were identified between March 2009 and November 2010 at Massachusetts General Hospitals ANCA laboratory. Cocaine exposure was identified from patient history in all cases. Medical records were reviewed for clinical presentation and for laboratory and diagnostic evaluation. RESULTS Thirty cases of ANCA positivity associated with cocaine ingestion were identified. All had antimyeloperoxidase antibodies and 50% also had antiproteinase 3 antibodies. Complete clinical and laboratory data were available for 18 patients. Arthralgia (83%) and skin lesions (61%) were the most frequent complaints at presentation. Seventy-two percent of patients reported constitutional symptoms, including fever, night sweats, weight loss, or malaise. Four patients had biopsy-proven vasculitis. Two cases of acute kidney injury and three cases of pulmonary hemorrhage occurred. From the entire cohort of 30, two cases were identified during the first 3 months of our study period and nine cases presented during the last 3 months. CONCLUSIONS We describe an association between the ingestion of levamisole-contaminated cocaine and ANCA-associated systemic autoimmune disease. Our data suggest that this is a potentially life-threatening complication of cocaine use.

Diagnostic Value of Anti-neutrophil Cytoplasmic Antibodies in Scleritis Associated with Wegener's Granulomatosis

Serum antineutrophil cytoplasmic antibodies (ANCAs) are a sensitive and specific marker for generalized Wegeners granulomatosis. However, ANCA sensitivity and specificity in identifying patients in whom ophthalmic signs constitute the presenting or only definitive manifestation of Wegeners granulomatosis have not been tested. The authors report on 7 patients in whom scleritis was the initial manifestation leading to the diagnosis of Wegeners granulomatosis. Six had the limited form of Wegeners granulomatosis. Results of serum ANCA tests were positive in all these patients. In contrast, the serum ANCA was negative in 54 patients with ocular inflammation due to other disorders; 16 of these patients had scleritis. Serial ANCA titers reverted to normal in only two of the four patients with Wegeners granulomatosis who attained clinical remission. One of the patients who did not revert to normal experienced relapse 2 months after discontinuation of therapy. Antineutrophil cytoplasmic antibodies appear to be both sensitive and specific for Wegeners granulomatosis-associated scleritis, and testing is useful in the evaluation of patients with scleritis.

Correlation of antineutrophil cytoplasmic antibodies with the extrarenal histopathology of Wegener's (pathergic) granulomatosis and related forms of vasculitis.

We studied the histologic findings from extrarenal biopsies (especially of the lung or upper respiratory tract) or autopsies of 68 patients who were tested for serum antineutrophil cytoplasmic antibodies (ANCAs). We used antigen-specific assays to detect antibodies against proteinase 3 (PR3) and myeloperoxidase (MPO), the two types of ANCAs of proven diagnostic value for the spectrum of diseases that includes Wegeners (pathergic) granulomatosis, microscopic polyarteritis (microscopic polyangiitis), Churg-Strauss syndrome, idiopathic necrotizing and crescentic glomerulonephritis, and their variants. Twenty-eight patients had antibodies to PR3 and 16 had antibodies to MPO; no patient had antibodies to both. All 44 patients with ANCAs had histologic evidence of this spectrum of diseases. Thirteen patients without histologic evidence of this spectrum of diseases had negative tests for ANCAs. There were no pathologic features that reliably identified patients with one or the other type of ANCA. Eighteen of 31 patients with lesions of Wegeners granulomatosis had antibodies to PR3, seven had antibodies to MPO, and six had neither. Three of four patients with necrotizing arteries without granulomas had anti-MPO antibodies, but similar lesions were seen, together with extravascular granulomas, in three patients with anti-PR3 antibodies. Of 16 patients with alveolar hemorrhage, nine had anti-PR3 and five had anti-MPO antibodies. Two patients diagnosed clinically as having Churg-Strauss syndrome had anti-MPO antibodies. In 16 of the 25 patients with ANCAs and a histologic diagnosis of Wegeners granulomatosis the diagnosis was made on the basis of extravascular granulomatous lesions alone, which argues against the requirement for vasculitis. Of six patients with negative tests for ANCAs and histologically diagnosed Wegeners granulomatosis, none had evidence of renal involvement. We conclude that in the appropriate clinical setting the presence of anti-PR3 or anti-MPO antibodies provides reliable evidence of the above spectrum of diseases, but that subclassification (to the extent this is possible) depends on the presence of distinctive clinical or pathologic features. In patients with negative tests for ANCAs, interpretation of clinical and histologic findings remains the only definitive method of diagnosis.