CRIOGLOBULINEMIA E MISCELLANEA

OLIGOCLONAL, NOT MONOCLONAL B-CELL EXPANSION IS THE MAJOR FEATURE OT TYPE II MIXED CRYOGLOBULINEMIA
S. De Vita, V. De Re, M. Ballarè, D. Sansonno, B. Pivetta, D. Gasparotto, A. Monteverde, F. Dammacco, E. Bartoli, G.F. Ferraccioli, M. Boiocchi
Rheumatology Unit, DPMSC, University of Udine; Dept. of Experimental Oncology 1, C.R.O, Aviano; Dept. Gastroenterology, Hospital of Novara; DIMO, University of Bari, Italy

Background: Type II mixed cryoglobulinemia (MC-II) is characterized by a monoclonal rheumatoid factor in the serum. Histopathology and IgMk restriction in the bone. marrow (BM) often support the diagnosis of a low-grade B-cell non Hodgkin's lymphoma (B-cell NHL; REAL Classification, 1994), but patients rarely develop an overt NHL during long-term follow-up.

Patients and Methods: We then analyzed B-cell clonal expansion in the MC syndrome at the molecular level, i.e., by IgH VDJ-PCR amplification of BM DNA from 3 groups of patients. Group 1 (3 MC-II cases) included BM low-grade B-cell NHL biopsies obtained at baseline (i.e., at the diagnosis of MC syndrome) in patients who developed a nodal, clinically overt B-cell NHL after 3-0 years. Group 2 (3 MC-II cases) included baseline BM low-grade B-cell NHL from patients who lacked subsequent overt NHL evolution. Group 3 included baseline benign BM lesions from 4 patients with either type II or type III MC (mean follow-up in Groups 2 and 3: 7 years).

Results: Oligoclonal BM B-cell expansion was demonstrated in all the Groups. One dominant clone was overexpanded in 4/6 BM lesions from Groups 1 and 2. However, in 2/2 patients from Group 1, DNA sequence analyses and clonospecyfic oligoprobe hybridization studies (De Vita et al. Arthritis Rheum 1997; 40: 318) revealed that such a dominant clone was not the same subsequently evolving into an overt NHL. Overt NHL originated from a minor BM B-cell clonal population present at baseline.

Discussion: The present findings, in conjunction with recent molecular results in the liver of MC-II patients (Sansonno et al. J Immunol 1998; 160: 3594) indicate that type II MC is characterized by an oligoclonal, rather than monoclonal, B-cell expansion. In contrast with BM pathologic findings, both the clinical course and the molecular findings in target tissues argue against a B-cell malignancy in MC-II patients at disease onset.

ESTABLISHMENT OF HUMAN MONOCLONAL ANTI-MYELOPEROXIDASE ANTIBODIES PRODUCED BY EBV-TRANSFORMED B CELL LINES FROM PATIENTS WITH VASCULITIS
Jun Koide, Tatsuya Ito, Keiko Saito, Yuko Inoue, Koichi Amano, Tohru Abe
Saitama Medical Center, Saitama Medical School, Kawagoe, Saitama 350-8550, Japan

Circulating anti-myeloperoxidase (MPO) antibodies are associated largely with pauci-immune glomerulonephritis and microscopic polyarteritis. Anti-MPO antibodies may interact with MPO when it is present on the membrane of pre-primed neutrophils, resulting in degranulation and the production of cell-damaging free radicals in vitro. The purpose of this study was to produce and analyse newer human anti-MPO monoclonal antibodies (mAbs). EBV-transformed B cell lines (BCL) were isolated from three patients with MPO-anti-neutrophil cytoplasmic antibodies-associated systemic vasculitis. Three mAbs, IK-1F9, SH-2D3 and OA-B5, of the human IgG were screened and cloned from these BCL, and strongly reacted with an MPO antigen, but not with proteinase-3 and other autoantigens by direct binding ELISA. We confirmed these antigenic specificities by several inhibition studies. These mAbs showed a perinuclear staining pattern on ethanol-fixed human neutrophils. Flow cytometry studies were performed to determine that the MPO antigen recognized by three mAbs was expressed on rTNFa-primed neutrophils, but not on non-primed neutrophils. Western blotting analysis demonstrated that the only native MPO antugen corresponded to a single band of 120 kD, suggesting that 1 F9 and B5 mAbs reacted with conformational antigens found on the native molecule. In contrast, 2D3 mAb recognized both 75-kD and 60-kD bands when MPO was boiled and reduced. Pre-incubation of neutrophils with 1 F9 and B5 mAbs could increase the adhesion molecule expression of CD18 and CD31 on the cell surface of primed neutrophils. When supernatant nitric oxide (NO) levels were measured using a chemiluminescence analyser, only 1F9 mAb was capable of stimulating NO production from neutrophils. In addition, incubation of neutrophils with three mAbs caused H2O2-generation by detection with flow cytometric analyses. Oxidative product formation by neutrophils occured as a significant response to membrane stimulation by 1F9 mAb alone. Furthermore, affinity-purified 1F9 mAb by protein A-column, was observed to increase the activity of small GTP-binding protein-dependent phospholipase D in the lysates prepared from membranes of neutrophils by Western blot analysis. Therefore, we established three EBV-transformed BCL secreting mAbs to MPO derived from three patients with vasculitis. We conclude that all three mAbs can bind to the cell surface of primed neutrophils, and one of them may both induce a respiratory burst and degranulation, and modulate neutrophil activation.

HIGH PREVALENCE OF DRUG-ASSOCIATED VASCULITIS IN PATIENTS WITH HIGH TITERS OF ANTI-MYELOPEROXIDASE ANCA
Hyon K. Choi, Peter A. Merkel, John L. Niles
Massachusetts General Hospital, Boston, MA 02114, USA

Introduction: The etiology of ANCA-positive vasculitis (APV) is unknown in most cases. However, there have been reports suggesting that certain drugs, including hydralazine, propylthiouracil, and penicillamine caused vasculitis in some patients. In our experience, patients with drug-associated APV often have extremely high titers of myeloperoxidase (MPO) ANCA. We studied the prevalence of drug-associated APV among patients presenting with vasculitis and high titers of anti-MPO ANCA.

Methods: Among 252 patients with anti-MPO ANCA detected in our laboratory between 1994 and 1998, the 30 with the highest titers were studied. Data collected for each patient included all drugs used for at least 6 months preceding disease onset, clinical manifestations, histologic findings, treatment, and outcome. Two patients found to be ANCA-positive 2 years earlier were excluded from analysis.

Results: Among 28 patients presenting with high-titer MPO ANCA, 14 (50%) were on a candidate drug for at least 6 months preceding the diagnosis of vasculitis; 9 on hydralazine, 3 on PTU and 2 on penicillamine. Clinical details are outlined in the table. Histologic findings were similar in the two groups.

Conclusion: These data suggest that a sizable proportion of APV with high-titer anti-MPO ANCA is medication-associated. Major organ involvement is a common feature of drug-associated APV. In patients with APV, medications suspected of causing the vasculitis should be discontinued and appropriate immunosuppressive therapy considered.

PREVALENCE OF HEPATITIS B- AND C-ASSOCIATED SYSTEMIC NECROTIZING VASCULITIS AT AN URBAN TEACHING HOSPITAL, 1992-1998
Kristin Thomas, David Hellmann, John Stone
Johns Hopkins Univ., Baltimore, MD 21205, USA

Purpose: The frequency of Hepatitis B as a cause of systemic necrotizing vasculitis/polyarteritis nodosa (SNV/PAN) has been said to be diminishing. The prevalence of Hepatitis C among SNV/PAN patients is poorly described. We reviewed our experience with SNV/PAN to determine the prevalence of hepatitis B and C infection among patients with at an urban teaching hospital from 1992-8.

Methods: We reviewed the medical records of 61 patients with the ICD-9 diagnosis of "polyarteritis nodosa" seen at our institution during the period of interest. Patients who had diagnostic angiograms and/or histopathologic proof of SNV/PAN from internal organs were considered to have PAN. Six patients with other defined types of vasculitis, including ANCA-associated vasculitides, were excluded. Of the 55 remaining patients, 47 were assessed for the presence of either Hepatitis B or C (37 were tested for both). The 8 untested patients lacked both identifiable risk factors and clinical findings suggestive of viral hepatitis. Hepatitis B- or C-associated SNV/PAN was diagnosed in the setting of coincident acute viral infections, chronic active hepatitis, or chronic carrier states.

Results: Seventeen out of fifty-five patients (31%) had SNV/PAN secondary to viral hepatitis. Thirteen patients (24%) were Hepatitis C positive, and 9 (16%) were Hepatitis B positive. The results of viral hepatitis testing are displayed in the Table below:

Hep C Hep B

ELISA PCR HepBSAg HepBC

Patients tested 25 15 43 2

Number positive 7 6 8 1

TOTALS 13 9

Five patients (9%) were infected with both Hepatitis B and C. Of the thirteen patients positive for hepatitis C, 8 of 9 patients tested had cryoglobulins. Among the 9 patients with hepatitis-associated SNV/PAN who had mesenteric angiograms, 6 (67%) were positive. Among the 13 who underwent biopsies of involved internal organs, 10 biopsies (77%) were diagnostic.

Conclusions: The prevalence of Hepatitis B- and/or C-associated SNV/PAN was at least 31% at our city teaching hospital between 1992 and 1998. Hepatitis C infections were slightly more common than Hepatitis B infections, but Hepatitis B remains an important cause of SNV/PAN.

ROLE OF ANDROGENS IN HCV-RELATED MIXED CRYOGLOBULINEMIA
C. Ferri, M. Cutolo, A.L. Zignego, G. Longombardo, A. Sullì, D. Cavallaro, M. Giusti, S. Accardo, A. Mazzocca, G. Pasero
Rheumatology Unit, University of Pisa; Div Rheum. Dept Int Med. Dept Endocr Metab Sci, University of Genova; Intern Med Inst, University of Firenze, Italy

Introduction: Sex hormones can play a predisposing and/or modulating role in autoimmune diseases, such as rheumatoid arthritis and systemic lupus, which are more prevalent in females: broadly extrogens exert immunostimolatory effects while androgens are natural immunosuppressors. Mixed cryoglobulinemia (MC) is an immune-mediated systemic vasculitis triggered by Hepatitis C Virus (HCV) infection. The contribution of hormonal factors in the pathogenesis of the MC can be suspected because of its prevalence in women.

Methods: We investigated the possible role of gonadal and adrenal steroid hormones in 21 HCV+ MC pts (M/F 12/9; mean age 59 ± 8 SD yrs) and in sex- and age-matched series of 21 HCV + 3 chronic hepatitis (HCV-CH) without MC (M/F 10/11; 58 ± 9 yrs) and 54 healthy controls (hC) (M/F 28/26; 58 ± 8 yrs). All female individuals were postmenopausal; hormone determinations (RIA) included: dehidroepiandrosterone sulphate (DHEAS), androstenedione (D4), total testosterone (tT), and 17b-estradiol (E2).

Results: Significantly lower levels of adrenal androgen DHEAS were observed in both female and male MC pts when compared to hC (p < .0001); DHEAS was also significantly lower in MC females than in HCV-CH (p < .01). In addition, significantly altered tT levels were detected in female MC pts when compared to hC (p < .01). Interestingly, the androgen D4 was significantly lower in both female and male MC than in HCV-CH pts (p < .003) and hC (p < .003) indicating its altered peripheral metabolism. Significantly higher levels of E2 in MC and HCV-CH pts compared to hC (p < .003) could be related to altered hepatic metabolism in these patients.

Conclusions: These results suggest a role for both adrenal and gonadal androgens in the pathogenesis of MC. The autoreactive lymphocyte expansion in the MC can be amplified by a reduced immunosuppressive activity of androgens. In addition, the possible contribution of hormonal factors in the pathogenesis of HCV-related MC yields new and interesting therapeutic implications.

HEALTH-RELATED QUALITY OF LIFE IN PRIMARY SYSTEMIC VASCULITIDES
Karen Herlyn1, Eva Reinhold-Keller1, Angela Zeidler1, Heiner Raspe2, Joachim Gutfleisch3, Hartmut H. Peter3, Wolfgang L. Gross1
1 Rheumaklinik Bad Bramstedt GmbH/Department of Rheumatology, 24576 Bad Bramstedt; 2 Institute for Social Medicine, University of Lübeck, 23564 Lübeck; 3 Department of Rheumatology/Immunology, University of Freiburg, Germany

Purpose: Measurement of physical, mental and social function is essential for the evaluation of patients (pts) with rheumatic diseases. Purpose of this study is the assessment of health-related quality of life (HRQL) with the generic instrument short-form 36 (SF-36) in patients with primary systemic vasculitides (PSV), the comparison of pts in complete remission (CR) with a healthy population (n = 350) and to confirm that the SF-36 is a valid and reliable outcome measure in PSV.

Methods: 303 Pts with PSV of two German vasculitis centers completed the SF-36 at baseline (T0) and at 12 months follow-up (T1). The 36 item-questionnaire measures the dimensions physical functioning (PF), role physical (RF), pain (P), vitality (V), mental health (MH), role emotional (RE), social functioning (SF) and general health (GH). Scales range from 0 to 100 with high numbers indicating high HRQL. To assess convergent validity the disease extent index (DEI) was calculated.

Results: 303 pts (60% WG, 12.3% AT, 7. mean age of 52.4 (range 17-85) and a mean disease duration of 41.8 months participated (45.3% men, 54.7% women). Means of all SF-36 dimensions are presented in the table. DEI significantly correlated with PF, RP, P, GH, V and SF (Pearson correlation coefficients r = -0.17 to r = -0.32, p < 0.001). The internal consistency was high with Cronbach alpha values ranging from 0.83 to 0.94 except for GH (0.61).

Conclusions: HRQL in PSV is reduced in each aspect of the SF-36 at baseline and follow-up. Pts's estimates of their HRQL during complete remission reveal considerable limitations at T0 and T1 compared with a healthy population particularly in general health, physical function and role physical. Further studies are planned to elucidate the relatively low HRQL in pts with complete remission. The SF-36 presents a useful, valid and reliable measure in PSV with moderate convergent validity.

POLYARTERITIS NODOSA TYPE VERSUS SYMPTOMATIC MIXED CRYOGLOBULINEMIA IN HEPATITIS C PATIENTS
P. Cacoub, A. Gatel, T. Maisonobe, V. Thibault, P. Hausfater, J. Servan, L. Musset, O. Blétry, P. Godeau, J.C. Piette
Hôpital La Pitié-Salpêtrière, Paris, France

Objective: To compare the features of hepatitis C virus (HCV) infected patients presenting with different types of vasculitis syndrome.

Methods: We retrospectively compared two groups of HCV patients: 10 patients with biopsy-proved polyarteritis nodosa type systemic vasculitis (PAN, group 1), and 7 patients with mixed cryoglobulinemia syndrome (MC, group 2).

Results: Patients of group 1 presented with different clinical and biological features than those of group 2: lifethreatening systemic vasculitis [10 vs 0 respectively, p < 0.01], malignant hypertension [5 vs 0, p = 0.04], severe multifocal sensory-motor mononeuropathies versus distal moderate sensory polyneuropathies, cerebral angiitis [2 vs 0], ischemic abdominal pain [2 vs 0], kidney and liver microaneurisms [2 vs 0], increased ESR or C reactive protein levels [7 vs 0, p < 0.01], renal insufficiency [5 vs 0, p = 0.04], and HCV genotype 1b (3 vs 6, p = 0.06]. Liver biopsy specimen revealed a lower activity of chronic hepatitis in group 1 than in group 2 [p = 0.02]. Neuromuscular biopsies showed lesions of vasculitis in all patients of both groups but the type of vasculitis was different in group 1 versus group 2 with regard to medium size arteries involvement [7 vs 0, p < 0.01], necrotizing vasculitis [10 vs 0, p < 0.01], mononuclear cells infiltrate in perivascular area [0 vs 7, p < 0.01]. Using prednisone, plasma exchanges and interferon alfa, a complete recovery was obtained in all PAN type patients except one. In patients of group 2, interferon alfa had no effect on the peripheral neuropathy.

Conclusion: Systemic vasculitis of PAN type should be added to the numerous extrahepatic manifestations of HCV infection. Considering its severity, prognosis and requirement for a particular therapeutic approach, HCV-PAN should be distinguished from classical features of symptomatic HCV-MC.

DIAGNOSTIC STRATEGY FOR THE ASSESSMENT OF RHEUMATOID VASCULITIS
Alexandre E. Voskuyl1, Johanna M.W. Hazes1, Aeilko H. Zwinderman2, Ewa M. Paleolog3, Felix J. van der Meer4, Mohammed R. Daha5, Ferdinand C. Breedveld1
Department of 1 Rheumatology; 2 Medical Statistics; 4 Hemostasis; 5 Nephrology of the Leiden University Medical Center, Leiden, The Netherlands
3 Kennedy Institute of Rheumatology, London, UK

Objective: To determine the clinical features associated with histologically proven rheumatoid vasculitis (RV) and the additional diagnostic value of serological markers in an inception cohort of 81 patients with rheumatoid arthritis (RA) suspected of RV.

Methods: The presence and number of recently developed extraarticular manifestations (EAM) and a weighted EAM score, as well as the levels of the serological markers, were compared between 31 RA patients with histologically proven vasculitis and 50 RA patients in whom vasculitis could not be documented hsitologically. The following markers were evaluated: circulating immune complexes, complement components C3 and C4, class-specific rheumatoid factors (IgM-RF, IgG-RF, IgA-RF), anti-neutrophil cytoplasmic antibodies, anti-nuclear antibodies, anti-endothelial antibodies, circulating intercellular adhesion molecule (ICAM) -1 and -3, circulating vascular adhesion molecule (VCAM) and E-selectin, cellular fibronectin, von Willebrand factor antigen, and C-reactive protein. The diagnostic value of these markers, in addition to the clinical features, was evaluated with logistic regression analysis.

Results: Peripheral neuropathy and/or purpura/petechiae were the most important clinical features to discriminate RA patients with and without RV. The presence of a high number of EAMs and a higher weighted EAM score in RA patients suspected of vasculitis were also associated with an increased probability of RV. After adjustment for EAM, only the combination of an increased serum IgA-RF and a decreased serum C3 level appeared to make an additional contribution to the diagnosis RV.

Conclusions: In the diagnostic process of RV the presence of peripheral neuropathy and/or purpura/petechiae or a high weighted EAM score will increase the probability of RV. Of the circulating factors previously suggested to be markers for RV only IgA-RF and C3 further increase the probability of RV and may be useful to guide diagnostic decisions.

PERINUCLEAR ANTINEUTROPHIL CYTOPLASMIC ANTIBODIES (pANCA) IN EROSIVE OSTEOARTHRITIS (EOA)
D. Zauli, M. Fusconi1, C. Descovich, S. Ghetti, G. Casagrande1, C. Berti Ceroni1, F. Giorgetti1, M. Magnani1, F.B. Bianchi, R. Corinaldesi1
Dept. of Internal Medicine, Cardioangiology, Hepatology; 1 Dept. of Internal Medicine and Gastroenterology, University of Bologna, Italy

pANCA, originally described in small vessel vasculitis, have been subsequently observed in both rheumatological (rheumatoid arthritis-RA, systemic lupus) and non rheumatological (primary sclerosing cholangitis-PSC, autoimmune hepatitis-AIH) diseases. It has been suggested that EOA is a unique entity, distinct from non inflammatory osteoarthropaty (OA), characterised by prominent destructive changes and inflammation.

Aim of the present study was to verify the prevalence of pANCA in RA, which seems to vary from 16 to 68%, and to evaluate their occurence in EOA. For this purpose 30 RA, 20 EOA and 30 OA were studied. Seventy PSC and 30 AIH served as positive controls and sera from 50 age and sex matched blood donors as negative ones. Antibody testing was performed by immunofluorescence (IF) on alcohol-fixed neutrophils with sera initially diluted 1:20, according to standard procedures.

pANCA were detected in 13% RA, 21% EOA, 63% PSC, 65% AIH-1 and in none OA and blood donors. Titres were generally lower in RA and EOA than in PSC and AIH. This might, at least in part, account for the different IF pattern of the antibody in the two groups (thick perinuclear staining in the latter, thin perinuclear rim in the former). However, since the target antigen of pANCA in all these conditions has not been identified yet, the role of different antigen specificities cannot be completely ruled out. The present preliminary data further confirm that EOA, sharing serological markers with RA and other inflammatory conditions, is distinct from OA. Although not confirmed yet, it has been suggested that pANCA is a marker of a "vasculitic" subgroup of RA. The significance of pANCA in EOA is still poorly understood and requires further investigation of larger series.