Serial measurements of antineutrophil cytoplasmic autoantibodies in patients with systemic vasculitis.

PURPOSE: To assess the value of serial determinations of antineutrophil cytoplasmic autoantibodies (ANCA) for monitoring disease activity in patients with systemic vasculitis. PATIENTS AND METHODS: Forty-three patients with histologically proven vasculitis (21 with Wegener's granulomatosis, 17 with microscopic polyangiitis, and 5 with renal-limited vasculitis) were studied for a median follow-up of 22 months. Disease activity was prospectively assessed and quantified by the Birmingham Vasculitis Activity Score. A total of 347 sera were analyzed for ANCA determination. RESULTS: Relapses occurred in 23 (54%) of 43 patients. Diagnostic category (Wegener's granulomatosis vs micropolyangiitis and renal-limited vasculitis), severity of initial symptoms (mean vasculitis activity score, mean number of organs involved), and ANCA pattern [cytoplasmic-ANCA (c-ANCA) vs perinuclear-ANCA (p-ANCA)] did not significantly differ between relapsers and nonrelapsers. Lung involvement was more frequent at onset among relapsers [16 of 23 (70%) vs 6 of 20 (30%); P = 0.02]. Relapses were slightly, but not significantly, more frequent in patients with Wegener's granulomatosis or a c-ANCA pattern. The percentage of relapsers was greater in patients with persistently positive ANCA than in patients with negative or decreasing ANCA titers (86% vs 20%, P = 0.0001). However, the predictive value of an increase in ANCA titers for the occurrence of a subsequent relapse was only 28% (4 of 14) for c-ANCA, 12% (2 of 17) for anti-proteinase 3-ANCA, and 43% (6 of 14) for anti-myeloperoxidase-ANCA. An increase in ANCA occurred before or during relapse in 33% (10 of 30) of cases for c-ANCA/anti-proteinase 3 antibodies, and 73% (11 of 15) of cases for anti-myeloperoxidase antibodies. CONCLUSION: The persistence of ANCA positivity is strongly associated with relapses. However, an increase in ANCA titers has a poor value for the early prediction of a subsequent relapse and should not be used as a sole parameter for therapeutic intervention. In addition, our results suggest that serial anti-myeloperoxidase determination may be useful as a prognostic marker in patients who are p-ANCA positive.

Anti-neutrophil cytoplasmic auto-antibodies (ANCA) in ulcerative colitis (UC): no relationship with disease activity.

The relationship between anti-neutrophil cytoplasmic auto-antibodies (ANCA) and disease activity in inflammatory bowel diseases remains controversial. The aim of this study was to highlight the relationship between ANCA presence or titers and disease activity in ulcerative colitis (UC). Three groups of patients with UC were studied: 1) group A included 39 patients who had not undergone colectomy, 2) group B, 43 patients with subtotal colectomy and ileo-rectal anastomosis, 3) group C, 98 patients with proctocolectomy and ileo-anal anastomosis, including 88% with pouchitis and 12% without pouchitis at the time of the study. Determination of ANCA was performed using the standardized indirect immunofluorescence assay. ANCA were positive in 59%, 65%, and 54% of patients from groups A, B, and C, respectively (NS). No relationship between ANCA presence or titers and UC activity could be detected within groups A and B. In group C, 45 of 86 patients (52%) without pouchitis and 8 of 12 patients (67%) with pouchitis, were ANCA positive (NS). These results do not support a relationship between ANCA and UC activity in this cohort of 180 patients.

Candida albicans blastoconidia in peripheral blood smears from non-neutropenic surgical patients.

An 80 year old woman developed fever 11 days after volvulus surgery. A peripheral blood smear showed numerous yeast cells--both extraleucocytic and intraleucocytic--as well as leucoagglutination. The fungal elements included blastospores, pseudohyphae, and germ tubes. Two days later, blood cultures yielded Candida albicans, Enterobacter aerogenes, and Staphlococcus aureus. The patient had no medical history of immunodeficiency. Several reports indicate that fungal elements may be detected in peripheral blood smears from patients who have a severe intestinal disease.

Amplification of BCR-ABL rearrangement in atypical Philadelphia chromosome: a new variant detected by fluorescence in situ hybridization in one case of acute lymphoblastic leukemia.

We report on a 67-year-old woman with acute lymphoblastic leukemia (ALL) who was supposed to have a variant Philadelphia (Ph) translocation identified by conventional cytogenetic techniques. Fluorescence in situ hybridization (FISH) analysis demonstrated the presence of an amplification of BCR-ABL rearrangement at locus 22q11. This is the first observation, to our knowledge, of a duplicated BCR-ABL chimeric gene within the derived chromosome 22 in ALL. Our observation supports the possibility of detecting a variant Ph chromosome at the single-cell level by FISH analysis.

Four cases of follicular lymphoma with t(14;18)(q32;q21) and t(3;4)(q27;p13) with LAZ3 (BCL6) rearrangement.

We report four cases of follicular lymphoma with both t(14;18)(q32;q21) and the newly characterized t(3;4)(q27;p13). Molecular investigation confirmed LAZ3 (BCL6) rearrangement for all patients. The 3q27 aberrations have been rarely described in low-grade lymphomas and may represent secondary events whose implication remains to be elucidated.

Analysis of phospholipid transfer during HDL binding to platelets using a fluorescent analog of phosphatidylcholine.

Electron microscopic examination of the interaction of gold labelled HDL with platelets indicates that the internalized lipoprotein becomes closely associated with surface connected canaliculae and endocytic vacuoles. At the same time granule centralization occurs. Using fluorescent derivatives of naturally occurring lipids we have further investigated lipid exchange between HDL and platelets. Analogs of phosphatidylcholine containing fluorescent fatty acids are rapidly transferred from the lipoproteins to the cells and remain at the plasma membrane as long as they are kept at 4 degrees C. However when the platelets are warmed to 37 degrees C, a rapid degradation of the fluorescent lipids occurs, generating fluorescent diacylglycerol as a consequence of the activation of platelet enzymes.

Alpha1-antitrypsin phenotypes and anti-neutrophil cytoplasmic auto-antibodies in inflammatory bowel disease.

OBJECTIVES: Alpha1-antitrypsin (alpha1-AT) is encoded by a highly polymorphic gene with over 75 codominantly expressed alleles at the protease inhibitor (Pi) locus classified as normal, deficient, dysfunctional or null. The aim of this study was to determine in patients with inflammatory bowel disease: (i) the prevalence of anti-neutrophil cytoplasmic auto-antibodies (ANCA) and their antigen specificities; (ii) alpha1-AT Pi phenotypes; and (iii) possible associations between ANCA, disease activity and deficient alpha1-AT alleles. DESIGN: Study of 95 consecutive patients with ulcerative colitis (UC) and 63 patients with Crohn's disease (CD). METHODS: Diagnosis and disease activity were determined by clinical, endoscopic and histological criteria. ANCA by indirect immunofluorescence (IIF) and Pi phenotyping by isoelectric focusing were performed for all patients. Positive IIF sera were tested in antigen-specific enzyme-linked immunosorbent assay: proteinase 3 (PR3), myeloperoxidase (MPO), lactoferrin (LF), lysozyme, human leucocyte elastase (HLE), cathepsin G and bactericidal/permeability increasing protein (BPI). RESULTS: Sixty-one patients with UC (64.2%) and only 11 with CD (17.5%) had ANCA (P < 0.001). Antigen specificities were PR3 (7/61), MPO (3/61), LF (6/61), HLE (1/63) and BPI (10/61) in UC, and PR3 (2/11) and BPI (2/11) in CD. Three PiZ alleles were found, matching the prevalence in the normal French control population. No relationship was found between the presence of ANCA, antibody specificity, disease activity and deficient alpha1-AT alleles. CONCLUSION: ANCA are more frequent in UC than CD and do not correlate with disease activity. ANCA and protease/antiprotease imbalance do not appear to modulate the clinical course of inflammatory bowel disease.

Antineutrophil cytoplasmic auto-antibodies in sera from patients with ulcerative colitis after proctocolectomy with ileo-anal anastomosis.

Presence of antineutrophil cytoplasmic auto-antibodies (ANCA) in ulcerative colitis could be an epiphenomenon related to colonic inflammation and/or may reflect a primitive disturbance of immune regulation. In this regard, study of ANCA status after the whole colorectal mucosa has been removed could favor one of these two hypothesis. We compared the prevalence of ANCA in a first group of 70 patients with non operated UC and in a second group of 32 patients with UC having had a proctocolectomy with ileoanal anastomosis. Perinuclear ANCA (p-ANCA) were found in 34/70 (49%) of the first group as compared to 11/32 (34%) in the second group (NS). Our results further support that the presence of ANCA in UC reflects an immune disturbance not linked to the presence of the target organ.

Antineutrophil cytoplasmic autoantibodies in inflammatory bowel diseases.

Antineutrophil cytoplasmic autoantibodies have been recently reported in sera from patients with inflammatory bowel disease. We report our experience based on 90 patients with ulcerative colitis, 148 patients with Crohn's disease, and 60 controls. Determination of antineutrophil cytoplasmic autoantibodies was performed by the indirect immunofluorescence technique on ethanol fixed leucocytes. The specificities for proteinase 3, myeloperoxidase, and lactoferrin were tested by enzyme-linked immunosorbent assay. Forty-three out of 90 (48%) patients with ulcerative colitis, 10 out of 148 (7%) patients with Crohn's disease, and none of controls were positive by indirect immunofluorescence technique. All patients but two with positive immunofluorescence exhibited a perinuclear staining pattern. Among patients with ulcerative colitis, there was no relationship between the presence of perinuclear antineutrophil cytoplasmic autoantibodies and disease location or activity. Seven out of 20 (35%) patients with ulcerative colitis who had a previous colectomy (including 1 with ileoanal anastomosis) had perinuclear antineutrophil cytoplasmic autoantibodies. Antineutrophil cytoplasmic autoantibody specificity was not directed against myeloperoxidase, proteinase 3 or lactoferrin in sera from patients with inflammatory bowel diseases. Inflammatory bowel diseases are associated with a new subset of antineutrophil cytoplasmic autoantibodies. Among patients with Crohn's disease and ulcerative colitis, the sensitivity and specificity of the presence of perinuclear antineutrophil cytoplasmic autoantibodies for ulcerative colitis was 46% and 93%, respectively. Their presence reinforces the likelihood of underlying immunologic dysregulation in ulcerative colitis. Identification of the autoantigen(s) to which these antibodies are directed might facilitate the understanding of inflammatory bowel disease pathophysiology.

Anti-endothelial cell antibodies in sera from patients with inflammatory bowel disease.

IgG anti-endothelial cell antibodies (AECA) have been described in sera from patients with vasculitis and other immune disorders such as systemic lupus erythematosus. Presence of AECA may be relevant to the hypothesis that Crohn's disease (CD) is a form of intestinal vasculitis. The aim of this study was to search for IgG AECA among 141 patients with CD, 94 patients with ulcerative colitis (UC) and 71 healthy blood donors and to assess the relationship between AECA and demographic or disease data. The cut-off point was defined from the mean OD values + 2 SD obtained from healthy blood donors. Seventeen percent of sera from patients with CD were positive for IgG AECA, whereas 24.5% of sera from patients with UC were positive. Among disease data, only a significant relationship between presence of IgG AECA and CD activity was noticed. These results might reinforce the hypothesis that intestinal vascular injury may be an important event in CD. However, detection of AECA in an almost similar percentage of patients with UC is more suggestive of an immune response to hidden endothelial self-antigen exposed after endothelial cell damage or a further marker of disturbed immunoregulation in inflammatory bowel disease.

[Detection by flow cytometry of T cell subsets secreting IL-2 and UFNy(Gamma): optimalisation for the technic and the establishment of reference values]. / Détection par cytométrie en flux des sous-populations lymphocytaires T sécrétrices d'IL-2 et IFN-gamma : optimisation de la technique et établissement des valeurs de référence.

A dysregulation in Th1/Th2 balance has been described for different pathological situations. Knowing the cytokine profile in a given pathology could assist in understanding the disease mechanism and in choosing an immune intervention most effective for the management of this condition. In this work, the production of two Th1 cytokines, IL-2 and IFN- gamma, was analyzed for different T-cell subsets from 20 normal subjects (mean age 33.5 years) and reference values were defined using the flow cytometric analyses. The optimum operating conditions were set as following: mononuclear cells were stimulated with PMA (20 ng/ml) and ionomycin (1 uM) for 6 h in the presence of brefeldin A (10 ug/ml). Cells were fixed with 4% paraformaldehyde and then dually stained, with anti-CD3 or anti-CD4 or anti-CD8 for the membrane and with anticytokine antibody for the intracytoplasma after being permeabilized with 0.5% saponine solution. The frequency determination of cells that produce IL-2 or IFN-gamma revealed large 95% confidence intervals: (CD3-IL-2: 4.60-10.67%, CD8-IL-2: 1.47-23%, CD3-IFN-gamma: 2,97-32,49%, CD4-IFN-gamma: 2.83-21%, CD8-IFN-gamma: 4.60-35.28%). CD4+ lymphocytes produce the majority of IL-2 (85 vs 13% for CD8+). For IFN-gamma, the situation is more balanced, but the CD4+ lymphocytes remain the predominant producer cells (63 vs. 41%).

Involvement of Fcgamma receptors and beta2 integrins in neutrophil activation by anti-proteinase-3 or anti-myeloperoxidase antibodies.

We previously described the requirement of tumour necrosis factor-alpha (TNF-alpha) and the role of beta2 integrins in the Fc-gamma receptor IIa (FcgammaRIIa)-mediated mechanism of neutrophil activation by antiproteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies. In the present study, we assessed the involvement of FcgammaRIIIb by studying the respiratory burst activation of completely FcgammaRIIIb-deficient neutrophils primed by TNF-alpha and exposed to anti-PR3 or anti-MPO. Activation of the NADPH oxidase occurred normally in these neutrophils, which indicates that engagement of FcgammaRIIIb is not essential in our model. Experiments performed with neutrophils from severe leucocyte adhesion deficiency (LAD) patients confirmed that beta2 integrins play a pivotal role in this activation. We next studied whether adhesion per se, beta2-integrin-mediated adhesion, or beta2-integrin ligation without adhesion is necessary or sufficient for this activation. Anti-PR3 or anti-MPO induced an FcgammaRIIa-dependent burst in TNF-primed neutrophils incubated in wells coated with poly-L-lysine, known to induce beta2-integrin-independent adhesion, but this reaction was still inhibited by blocking CD18 antibodies. In a system with granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils, which did not enhance adhesion, we measured a similar activation by anti-PR3 or anti-MPO and inhibition by CD18. We also noticed that treatment with the beta2-integrin-activating CD18 MoAb KIM185 per se is insufficient for neutrophil activation by anti-PR3 or anti-MPO. We therefore conclude that ligation of beta2 integrins rather than adherence per se is essential for this activation, and that TNF-alpha or GM-CSF is needed for priming but not for adherence.

Latex immunoassay of human serum Lp(a+) lipoprotein.

A sensitive latex immunoassay for human serum lipoprotein Lp(a+) is based on direct agglutination by Lp(a+) of latex particles coated with specific antibody. The agglutination is quantified by turbidimetry using a photometer at 360 nm. The stabilization of antibody-coated latex particles by bovine serum albumin occurs under well-defined conditions (pH, concentration of bovine serum albumin, and antibody loading of latex particles). The standard curve of serum lipoprotein Lp(a+) ranges from 0.05 to 1.15 mg/l. Inter- and intra-assay coefficients of variation were less than 8% and 3%, respectively. Results were well correlated with those obtained by electroimmunodiffusion (r = 0.98, n = 108).

Simultaneous measurement of plasma apolipoproteins A-I and B by electroimmunoassay.

We describe a simplified electroimmunoassay for quantification of human apolipoproteins A-I and B on prepared plates. A solution of agarose at 55 degrees C, containing hydroxyethylcellulose and antibodies, is poured onto a plastic film and allowed to gel. Wells are punched in the gels and the plates are dried for storage. Before use, they are rehydrated and buffered. We use succinylated Sudan Black to prestain lipoprotein fractions of plasma. After electrophoresing samples of plasma or standards for 3 h at 4 degrees C at 12.5 V/cm, we measure the peak heights and read the results from a standard curve prepared by using calibrated sera of known apolipoprotein B and A-I content as secondary standard. The within- and between-assay coefficients of variation were less than 4% in all cases. Results correlated well with those obtained by classic electroimmunodiffusion. Subjects with confirmed atherosclerotic lesions had significantly (p less than 10(-9)) lower ratios of apolipoprotein A-I to apolipoprotein B, compared with ratios in controls.