[Antiphospholipid antibodies in practice]. / Anticorps antiphospholipides en pratique.

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by the occurrence of thrombotic or obstetrical events associated with the presence in the serum of patients of antibodies that are associated with thrombosis. For the diagnosis of APS, the presence of either lupus anticoagulant, anticardiolipin or anti-ß2-glycoprotein1 antibodies of IgG or IgM isotype is required through laboratory testing. Other autoantibodies such as antiphosphatidylethanolamin or antiphosphatidylserin/prothrombin complex antibodies may be interesting in the diagnosis of APS when common antiphospholipid antibodies are missing. These autoantibodies are still under evaluation for their diagnostic contribution. Despite numerous attempts, the assays that are available for the identification of antiphospholipid antibodies have not been standardized yet, which leads to high variability between reagents and laboratories. Thus, to optimize the biological monitoring of APS syndromes, it is mandatory to have consecutive samples analyzed in the same laboratory.

High prevalence of antibodies to platelet factor 4 heparin in patients with antiphospholipid antibodies in absence of heparin-induced thrombocytopenia.

Seventy-two patients with antiphospholipid antibodies (aPL), with or without antiphospholipid syndrome (APS), were studied for detection of heparin-PF4-induced antibodies (HPIA) using a commercial kit (Asserachrom HPIA) PF4-dependant enzyme-linked immunoassay (ELISA) test. None of the patients had a medical history of heparin induced thrombocytopenia (HIT). Eleven percent of patients were positive for HPIA. Plasma from 40 of the 72 patients (seven positive and 33 negative), was also tested with the other available HPIA ELISA (GTI) kit. Five patients were positive with both ELISA kits, two were highly positive only with Asserachrom HPIA and four only with GTI. None of the positive patients had severe thrombocytopenia. Two patients have never received heparin treatment. No relationship was found between HPIA presence and patients' age, sex, aPL levels or presence of lupus anticoagulant. No significant difference in HPIA presence was observed in patients with primary APS, secondary APS or aPL without APS. We found a poor correlation between the two commercial ELISA showing that, on the same blood sample, a patient could be highly positive with one technique and negative with the other. The PF4-dependant enzyme-linked immunoassay, which is often the first test used for the diagnosis of HIT, should be interpreted cautiously in patients with aPL since there is a danger of overdiagnosis and overtreatment.

Testing for anti-nucleosome antibodies in daily practice: a monocentric evaluation in 1696 patients.

The objective was to report our experience of the detection of anti-nucleosome (anti-Nuc) antibodies (Ab) in a large series of consecutive patients, and to compare these results with those of anti-nuclear and anti-dsDNA Ab. In total, 1696 consecutive patients with suspected or confirmed autoimmune disease were tested over a two-year period. The biological investigation included detection of anti-nuclear, anti-dsDNA and anti-Nuc Ab. Among 1696 sera, 382 (23%) were negative for all Ab tested (anti-nuclear, anti-dsDNA and anti-Nuc) and 1314 (77%) were positive for at least one Ab. Anti-Nuc Ab were positive in 350/1314 patients. In this group, 249/350 (71%) also had positive anti-nuclear and anti-dsDNA, 97/350 (28%) had only positive anti-nuclear Ab without anti-dsDNA Ab and 4/350 (1%) had both anti-dsDNA and anti-Nuc Ab without anti-nuclear Ab. No patient had 'isolated' anti-Nuc Ab. Clinical data were available for 307/350 anti-Nuc positive patients. Systemic lupus erythematosus (SLE) was diagnosed in 240/307 (78%) patients, including 43 SLE patients with negative anti-dsDNA Ab. In conclusion, this study extends the relevance of anti-Nuc Ab to routine use for the diagnosis of connective tissue diseases, mainly anti-dsDNA Ab negative SLE.

IgA antineutrophil cytoplasmic antibodies in cutaneous vasculitis.

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) of the IgA isotype have, for the most part, been detected in patients with Henoch-Schönlein purpura (HSP) or inflammatory bowel disease. OBJECTIVES: We have evaluated the prevalence of IgA ANCA in a series of patients with different causes of cutaneous vasculitis. METHODS: Forty consecutive patients with histologically proven leucocytoclastic vasculitis were included in the study: 18 had systemic vasculitis as well as cutaneous lesions, 10 of whom were diagnosed as having HSP, and 22 had only cutaneous vasculitis (with no identified cause in 10 cases). IgA ANCA were sought by indirect immunofluorescence using ethanol-fixed human neutrophil preparations as the substrate. RESULTS: IgA ANCA were detected in six of 40 patients (15%) (one each with HSP, ulcerative colitis, Sjögren's syndrome, hypergammaglobulinaemia associated with Castelman's disease, erythema elevatum diutinum and bacterial endocarditis). Three of these patients also had IgG ANCA whose target antigen remained unidentified. CONCLUSIONS: IgA ANCA are rarely observed in HSP (10%) and can be detected in a wide variety of other cutaneous vasculitides.

Minocycline-induced clinical and biological lupus-like disease.

A 14-year-old girl developed maculopapular rash, myalgias, arthralgias and myocarditis with elevated anti-nuclear and anti-double-stranded DNA antibodies. She was taking minocycline for acne and all symptoms resolved when this treatment was stopped. The patient has no evidence of disease one year after onset of symptoms. Clinicians should be aware of minocycline's responsibility in inducing lupus-like disease.

Complete primary sequences of two lambda immunoglobulin light chains in myelomas with nonamyloid (Randall-type) light chain deposition disease.

We herein report on the first two primary sequences (BOU and RAC) of monoclonal light chains of the lambda type responsible for nonamyloid lambda light chain deposition disease. Both patients were affected with severe forms of myeloma complicated with renal failure. The pathological presentation typically featured Congo red-negative deposits along tubular basement membranes but differed somewhat from the typical "Randall-type" kappa light chain deposition disease: they lacked the prominent glomerulosclerosis pattern often featuring nonamyloid kappa deposits and were associated with cylinders or myeloma casts. Both protein sequences were deduced from those of the corresponding complementary DNAs in the bone marrow plasma cells. For each chain, products of three independent amplifications by polymerase chain reaction were sequenced and found to be identical. BOU and RAC lambda mRNAs had a normal overall structure consisting of Vlambda2 segments rearranged to Jlambda2Clambda2 but displayed a number of unusual features within their primary sequences. These substitutions are likely responsible for changes in light chain conformation that promote their aggregation and deposition along renal tubule basement membranes.

Use of a renal tubule cell line (LLC-PK1) to study the nephrotoxic potential of a kappa-type Bence-Jones protein.

The cytotoxicity of a Bence-Jones protein was assessed using a porcine renal tubule cell line (LLC-PK1), with the aim of developing a model for studying the potential nephrotoxicity of these proteins. The effects of a kappa Bence-Jones protein on cell viability were studied by means of biochemical methods (supravital dye uptake and measurement of cellular enzyme activities) and morphological electron microscopy. After a 24-h-treatment with Bence-Jones protein, a moderate cytotoxicity (about 15%) was noted but only a minor difference compared to treatment with bovine albumin in the same conditions. The morphological study showed a few cells in the process of lysis, but their numbers were insufficient for the demonstration of a clear cytotoxic effect. Immunocytochemical studies showed Bence-Jones protein fixation on some cells, especially on the outer membrane. Labeling of the hyaloplasm and basal pole of a few cells pointed to internalization of protein by LLC-PK1 cells. Although the cytotoxicity of the Bence-Jones protein tested here was only moderate, the use of this model enabled its cytotoxic effect to be distinguished from that of beta-lactoglobulin. This isolate could serve as a "moderate control" for a later study with a BJP having caused acute renal failure.

Electrophoretic study of the physico-chemical characteristics of Bence-Jones proteinuria and its association with kidney damage.

AIM: To identify a physico-chemical criterion, or set of criteria, explaining and possibly predicting the nephrotoxic behaviour of Bence-Jones proteins (BJP). METHODS: The electrophoretic mobility and isoelectric point (pI) of 92 BJP isolates were determined using various electrophoresis procedures on polyacrylamide gel. The proportions of monomers and dimers were determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) in 58 cases. PAGE data for 10 BJP isolates were used to construct Ferguson plots and titration curves. RESULTS: The distribution of electrophoretic mobility and pI values was bimodal and showed a positive correlation when the pI was above 6. The values of these two parameters in 22 patients with renal impairment were not significantly different from those in the patients without renal impairment, and the statistical analysis showed no predictive value for the onset of renal impairment. However, patients excreting the lambda light chain isotype had a 2.8-fold higher risk of developing renal impairment compared with the other patients. Studies of the charge variation of the protein with pH indicated three types of behaviour, suggesting that the charge of BJP is highly variable at physiological pH. CONCLUSION: It is important to study not only the positivity or negativity of the BJP charge at a given pH, but also its intensity. The study of the BJP titration curves in patients with renal impairment suggests that a low charge at physiological urinary pH could predict renal impairment.

Time-resolved immunofluorometric assay of complement C3: application to cerebrospinal fluid.

Complement components have a role in various neurological disorders. Complement C3 can be measured by immunochemical methods, but only radioimmunoassays and electroimmunodiffusion assays (EIDs) are sufficiently sensitive to be applied to biological fluids in which the C3 concentration is low, especially cerebrospinal fluid (CSF). We report a sandwich-type time-resolved immunofluorometric assay (TR-IFMA) for C3 in CSF. The linearity (0.7-3650 micrograms/L) and intra- (CV < 4.8%) and inter-assay (CV < 10.9%) precision were satisfactory and the results agreed with those of EID. The assay is extremely sensitive (< 1 microgram/L) and its analytical range is large and well suited to clinical applications. This simple TR-IFMA is thus a nonisotopic alternative to radioimmunoassay for the quantification of complement C3 in CSF.

Characterization of cryoglobulins by immunoblotting.

We adapted an immunoblotting technique for the immunochemical characterization of cryoglobulins. We first compared the results from 157 samples with results obtained by immunofixation and immunoelectrophoresis and then with clinical observations in 125 cases. Full identification was possible in 98% of the cases by immunoblotting, in 54% by immunofixation, and in 28% by immunoelectrophoresis. Novel microheterogeneity aspects were observed by immunoblotting and immunofixation in 13% and 6% of the cases studied, respectively. Our results confirm the frequency of autoimmune and infectious diseases associated with mixed cryoglobulinemias, whereas a lymphoproliferative syndrome was observed only in cases of cryoglobulinemia with a monoclonal constituent or a microheterogeneity aspect, which was not always revealed by immunofixation or immunoelectrophoresis. The pathophysiological importance and classification of microheterogeneity aspects is not clear. These observations justify using a sensitive and specific method for identifying cryoglobulins, even when present at low concentrations, and call for long-term studies of these patients.

Filtration and local synthesis of lacrimal proteins in acquired immunodeficiency syndrome.

In AIDS the onset of the ocular dry syndrome, characterized by lacrimal hyposecretion and deterioration of the corneal and conjunctival epithelium, generally accompanies the clinical aggravation of immunodepression. The study of serum and lacrimal proteins contributes to our knowledge of the pathophysiology of this syndrome. The lacrimal clearance of albumin indicates changes in the permeability of the haemato-lacrimal and conjunctival barrier. Lacrimal monomeric IgA and IgG are mainly of plasmatic origin, while polymeric IgA and IgM are synthesized in situ. The concentrations of these analytes thus reflect ocular humoral immune status. They show a strong humoral protein response in patients with cytomegalovirus retinitis. Lacrimal concentrations of lactoferrin and lysozyme were found to be significantly decreased in AIDS patients with ocular dryness, reflecting a decrease in the secretory activity of the lacrimal gland. Moreover, ocular lympho-plasmocyte infiltration was observed in several patients, with an increase in lacrimal beta 2-microglobulin concentrations. These various lacrimal proteins could be good markers of the ocular dry syndrome in AIDS.

Study of six antisera used for the immunonephelometric assay of human IgG.

The composition and performance of six commercial antisera for the immunonephelometric assay of IgG were studied. The analysis of the protein content of these reagents revealed significant differences in composition: the total protein contents ranged from 41 to over 130 g/l and gamma-globulins were twice to thirteen times higher than in a normal serum of the species. The antibodies found in these reagents were mostly IgG, with traces of IgM, and no IgA. Precipitation curves yielded by these antisera with a standard nephelometer showed a great diversity of performance. The very different shapes, the span of antigen concentrations and antibody dilutions necessary to obtain comparable quantities of precipitates further demonstrated the wide differences between these reagents. A method previously proposed by other authors was found to be inadequate. The failure of this method is attributed to the different conditions for the antigen-antibody reaction, as determined by the instrumentation used. It is concluded that the fundamental and classical criteria for testing antisera remain valid: specificity and the study of the precipitation curve to define the quantitative limits of the reaction. A simple universal method is inapplicable.

[Contribution of immunoenzymatic technics to the assay of anti-native DNA antibodies in the biological diagnosis of lupus erythematosus disseminatus]. / Apport des techniques immunoenzymatiques au dosage des Ac anti-ADN natif dans le diagnostic biologique du lupus érythémateux disséminé.

Anti native DNA antibodies (anti nDNA Ab), which are a highly specific feature of systemic lupus erythematosus (SLE) were measured by 3 methods: an enzyme linked immunosorbent assay (ELISA), an indirect immunofluorescence test on Crithidia luciliae (IFCL) and the Farr assay (reference test). 114 sera from patients with SLE or another connective tissue disease or without autoimmune rheumatic disease were tested. This study showed that ELISA seemed to be a more sensitive and specific test than IFCL (classical test). ELISA was also as sensitive as the Farr assay. ELISA should replace IFCL for the diagnosis and the follow up of patients with SLE. In other connective tissue diseases, ELISA might give more positive results. Thus these had to be confirmed, especially in the case of low antibodies levels, by using another method (e.g., the Farr assay).

[Oxygen free radicals and inflammatory diseases of intestines]. / Les radicaux libres oxygénés (RLO) et maladies inflammatoires de l'intestin (MII).

Free oxygen radicals (F.O.R.) belong to a very aggressive chemical species derived from molecular oxygen. Their role in inflammation is well established and Polymorphonuclear neutrophils (PMNS) make use of them as antibacterial weapons. Their role has been experimentally demonstrated in numerous ischemia-reperfusion models. Free radical scavengers such as the superoxide dismutase, allopurinol or desferrioxamine can prevent the occurrence of lesions. The essential role of PMNS in these models is demonstrated by the fact that previous depletion of the animal in PMNS also prevents such lesions. Histologically, in these ischemia-reperfusion models, PMNS infiltration may be quantified by assay of myeloperoxidase. In experimental models of inflammatory colitis (acetic acid, bacterial polysaccharides) intestinal wall infiltration by PMNS is a fundamental phenomenon and is also a characteristic of Crohn's disease and exacerbations of Ulcerative Colitis. Thus, it is probable in both disorders that F.O.R. play an important role since steroids inhibit their secretion by PMNS and 5-aminosalicylic acid has been shown to be a F.O.R. scavenger.

[Serum and lacrimal proteins in acquired immunodeficiency syndrome]. / Protéines sériques et lacrymales au cours du syndrome d'immunodéficience acquise (SIDA).

The concentrations of seven proteins [albumin (Alb), immunoglobulins (IgG, A, M), lactoferrin (Lf), lysozyme (Lzm) and beta 2-microglobulin (beta 2 m)] were measured in the tears and serum of 10 patients with acquired immune deficiency syndrome (AIDS) and in 13 normal subjects. In the AIDS group, alteration of the blood-lacrimal barrier was revealed by the high Alb clearance. In this group also, the significantly high IgG, IgA, IgM and beta 2 m concentrations indicated a systemic and local immune response, with passive transfer of these proteins from serum to tears. However, Lf and Lzm concentrations were normal, showing that the secretory activity of the lacrimal gland remained unchanged.

Determination of total protein in human seminal plasma.

Methodological problems are encountered in determinating human seminal protein content since results are influenced especially by techniques which are used, and by modifications due to liquefaction process. In order to test these two points, three methods (Folin, Biuret and Meulemans's reactions) were applied to the same seminal samples and results were compared. The differences seem principally due to the high level of glycoproteins. To determine the modifications of total protein content with time after semen collection, each sample was separated in aliquot fractions and proteolysis was blocked at different times for each fraction. There is an increase of the protein concentration during the first fifteen minutes that follows semen collection. The cause of this phenomenon is discussed.