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International audience; A new method for the detection by flow cytometry of anti-double-stranded DNA antibodies and of circulating immune complexes (IC) containing endogenous DNA (IC-eDNA) is described. From each serum sample, two samples were taken, one was used to detect IC-eDNA. The other to detect anti-DNA antibodies was incubated with calf thymus DNA. ICs were isolated by polyethylene glycol precipitation or by cryoprecipitation, after which immunoglobulins were labeled with FITC-conjugated anti-human globulin. Serum samples from 63 systemic lupus erythematosus (SLE) patients, 32 incomplete lupus, and 87 control patients were tested. Detection of anti-dsDNA antibodies by flow cytometry had a diagnostic sensitivity and specificity almost comparable to routine tests, the fluorescent enzyme immunoassay EliA™-dsDNA test, and the ultrasensitive Crithidia luciliae indirect immunofluorescence test. In 21 (33%) out of 63 SLE serum samples, IC-eDNA was detected. In these samples, free anti-dsDNA antibodies were hardly detectable or undetectable by flow cytometry or by routine tests. When anti-DNA antibodies are neutralized by endogenous DNA and can no longer be detected by routine tests, the serologic diagnosis and the follow-up of relapses in patients with SLE is compromised. To overcome this obstacle, we propose an accessible solution: the detection of circulating IC-eDNA by flow cytometry.

Background: Anaphylactic reactions during anesthesia are mainly the result of muscle-relaxant (MR) drugs. Skin tests, serologic detection of specific IgE, and in vitro leukocyte histamine release are used to investigate MR allergy. Objective: We describe a new assay that is based on the detection by flow cytometry of the altered expression of plasma membrane molecules of MR-activated basophils. Methods: For this assay, which we have named the BASIC assay, basophils are incubated in vitro with MR, after which they are fixed and then triple labeled with fluorescein-conjugated anti-CD63, tandem dye R-phycoerythrin-cyanin 5.1 conjugated anti-CD45, and R-phycoerythrin conjugated anti-IgE. The resulting B asophils' A ltered S urface I mmunofluorescence is detected by flow C ytometry (BASIC). Results: Forty-one patients who had an allergic reaction during general anesthesia and 23 control subjects without such a history were studied. All included subjects' basophils were tested in the BASIC assay with at least 4 MR: suxamethonium, gallamine, vecuronium, and pancuronium. After reaction of the basophils of the MR-allergic patients with MRs, increased surface expression of CD63 and CD45 and decreased expression of IgE were detected. Increased expression of CD63 was observed most frequently and it was stronger than the alteration of the 2 other markers. Cross-reactivity between MRs commonly occurred. MRs diluted 10 –1 activate the basophils of the control subjects, suggesting that at relatively high concentrations MRs are also nonspecific basophil activators. Conclusion: In the diagnosis of MR allergy, the BASIC assay has a good specificity but a low sensitivity, and it correlates strongly with skin test results. It is currently appraised for the diagnosis of anaphylactic reaction induced by other classes of drugs. (J Allergy Clin Immunol 1999;104:411-8.)

The specificity and sensitivity of anti-SSA (Ro) antibody detection by double immunodiffusion, ELISA, western blot and a new method named CAPE blot were studied. Using 79 normal sera, 61 sera positive for anti-SSA (Ro) antibodies (double immunodiffusion), and 39 sera without anti-SSA (Ro) antibodies but containing anti-Sm, anti-RNP, anti-SSB (La), anti-Scl 70 and anti-PCNA antibodies, we compared ELISA, western blot and CAPE blot with the immunodiffusion assay. The sensitivities of the three methods were 100, 62 and 100%, respectively. Sera from 196 patients with systemic lupus erythematosus (SLE) were tested. The prevalence of anti-SSA (Ro) antibodies in this group was scored as 26% by double immunodiffusion, 47% by ELISA, 25% by western blot and 38% by CAPE blot.

Immunodeficiency related to HIV may increase the incidence of EBV-associated lymphomas, by altering EBV-specific immune control and consequently favoring EBV reactivation. The aim of the present study was to assess the relationship between the decrease of EBV-specific cellular immunity and the increase of EBV reactivation in a prospective cohort of 72 unselected HIV-infected individuals. EBV-specific immunity was evaluated by a highly sensitive IFN-gamma ELISPOT assay using 22 peptides mimicking latent and lytic antigens, and circulating mononuclear (PBMC) EBV DNA load was quantified by real-time quantitative PCR. The mean circulating cell-associated EBV DNA load was higher in HIV-infected patients (639 copies/10(6) PBMC) than in healthy controls (21, n = 14) ( P = 0.005) and was higher in patients with CD4(+) T-cell count below 350/microL than that in patients harboring higher CD4(+) T-cell count (1112 vs. 389, P = 0.003). The mean intensity of EBV-specific cellular responses was lower in HIV-infected patients than in controls ( P = 0.001), even in patients with CD4(+) T-cell count above 350/-microL ( P = 0.007). The number of EBV peptides recognized was lower in HIV-infected patients than in controls (frequency: 0.44 vs. 0.67; P = 0.02), indicating reduced polyclonality in HIV-infected patients. The polyclonality was 1.5-fold lower in HIV-infected patients with CD4(+) T-cell count below 350/-microL ( P =0.007). For EBV load >1000 copies/10(6) PBMC, the levels of cell-associated EBV DNA and those of EBV-specific cellular immunity, either in intensity or in polyclonality, or both, were inversely correlated. These findings demonstrate early impairment of the EBV-specific cellular immune control with progressive increase of EBV reactivation in the course of HIV infection. These observations likely provide a basis for appreciating the risk to develop non-Hodgkin's lymphomas in HIV-infected individuals.

Background: The prevalence of antibodies to cardiolipin (a-CL) in patients with Raynaud's phenomenon (RP) and/or related disorders (rD) is not known. Objective: The purpose of this study was to determine the prevalence of these antibodies. Methods: We assayed IgG a-CL in 230 consecutive patients with RP/rD and compared the results with those in a series of 230 control blood donors. Results: Sixteen percent of patients were a-CL positive versus 7.8% of the control donors ( p = 0.014). The prevalence of a-CL positivity was 8.7% for primary RP, 10.5% for secondary RP, 8% for chilblains, 25% for essential acrocyanosis, 20% for connective tissue diseases, and 17% for undifferentiated connective tissue diseases. Among patients with digital necrosis, 24% were a-CL positive. Conclusion: The prevalence of abnormal a-CL titers is higher in patients with RP/rD than in control donors, especially in patients with a connective tissue disease. (J Am Acad Dermatol 1998;38:555-8.)

The silicone implant controversy wavers between reassuring epidemiological studies and about 300 case reports of patients developing a definite or incomplete/atypical connective tissue disease (CTD) after receiving a silicone gel-filled breast implant (SBI). Since Hashimoto's thyroiditis (HT) is rarely reported in this context, we report here two new cases of HT associated with a history of bilateral cosmetic SBIs. The first patient was a 45-year-old white woman who had SBIs in 1976. In 1991 she developed HT, evolving to thyroid deficiency which was compensated with levothyroxine treatment. In addition, the patient complained of fatigue, arthralgia, morning stiffness and developed a sicca syndrome necessitating artificial tears. The 1995 evaluation disclosed the presence of antinuclear antibodies at a titre of 1/640, and high level anti-thyroid microsomal antibodies (1/256,000). Gamma globulins rose to 22.6%. Thyroid ultrasonography showed an enlarged thyroid gland with a diffusely hypoechogenic pattern. The implants were painful, and in 1996 they were removed. Microscope examination of the fibrous capsule surrounding the prostheses showed extremely dense connective tissue with fibrosis. The second patient was a 55-year-old white woman who had SBIs in 1984. In 1995, she developed HT with clinical pain and tenderness of the thyroid gland, with mild hyperthyroidism and positive antithyroglobulin antibodies, and was given corticosteroid treatment for 5 months. In 1996, the implants were again painful and the patient developed positive antinuclear antibodies with a titre of 1/200. Ultrasonography showed a heterogeneous thyroid gland, and implant removal was advised. Hashimoto's thyroiditis is recognized as a subset of chronic auto-immune thyroiditis, and its association with SBI is rare. In these 2 observations, an association without relation is possible, but a future survey of similar cases seems warranted.

International audience; OBJECTIVES: Anticardiolipin antibodies belong to the group of antiphospholid antibodies, and may be seen in association with endothelial damage and recurrent vascular thrombosis. The aim of our study was to determine in patients with Crohn's disease the frequency of anticardiolipin antibodies, and to correlate their presence with clinical activity and treatment of the disease. METHODS: One hundred and thirty-eight sera from patients with Crohn's disease and 118 from age-matched controls were tested for IgG anticardiolipin antibodies. In the Crohn's disease group, we determined whether the patients had a past history of vascular thrombosis, a clinically active intestinal disease, or a current immunosuppressive therapy (steroids or azathioprine). RESULTS: Anticardiolipin antibodies were found significantly more often in patients with Crohn's disease than in controls: 11.0% versus 2.5%, P < 0.02. Three patients with Crohn's disease had a past history of vascular thrombosis, but none of them had anticardiolipin antibodies. The presence of anticardiolipin antibodies was not correlated with the fact that patients had a clinically active disease (P = 0.77), or a current immunosuppressive therapy at the time of the serological test (P = 0.95). CONCLUSIONS: There is a significantly high prevalence of patients with anticardiolipin antibodies during Crohn's disease. The positivity of the test does not seem to be correlated to the existence of a past history of vascular thrombosis, nor to the clinical activity of the disease.

In order to investigate the clinical value of anti-mitochondrial antibodies type 5 (anti-M5), we carried out a retrospective study on 48 patients with these antibodies. Seventeen of these 48 patients (35%) satisfied at least 4 criteria of the revised American Rheumatism Association classification of SLE. Twenty-nine (61%) had at least one clinical manifestation of anti-phospholipid syndrome; thirteen had symptoms consistent with primary anti-phospholipid syndrome; five had isolated recurrent thrombosis; five had Evans' syndrome; four had auto-immune haemolytic anaemia; two had immunologic thrombocytopenia. Two of the 48 patients had no clinical manifestations, but only anti-M5 and a false laboratory test for syphilis (FBTS). Our data confirm that patients with anti-M5 have a high prevalence of: 1) thrombosis (42% had three or more deep thromboses) and fetal loss (21%); 2) auto-immune cytopenia with idiopathic thrombocytopenic purpura (29%) and auto-immune haemolytic anaemia (54%); 3) laboratory markers of anti-phospholipid syndrome (lupus anticoagulant (71%), FBTS (95%) and anticardiolipin antibodies (aCL) (71%). For 32 patients with anti-M5, anti-beta 2 glycoprotein I antibodies were also tested; 12 (38%) were positive, all of whom had IgG aCL, ie none had anti-beta 2GPI antibodies without aCL. There was no association between the presence of anti-beta 2GPI antibodies and recurrent thrombosis among patients with anti-M5. All these findings suggest that anti-M5 is another marker of the antiphospholipid syndrome. Even though the prevalence of anti-M5 is low, especially in SLE, it was the only marker of the anti-phospholipid syndrome in two patients; this appears to justify routine screening for these antibodies.

The aim of this study was to evaluate the prevalence and the clinical signification of non organ specific autoantibodies in chronic hepatitis C.We studied retrospectively 158 consecutive patients (97 with chronic hepatitis C, 24 with chronic hepatitis B, 67 with alcoholic cirrhosis) and 100 blood-donors.The prevalence of anti-nuclear and anti-smooth muscle antibodies was lower in blood donors than in patients (P0.001), but was comparable among the 3 groups of patients. The anti-liver-kidney microsome type 1 antibodies were detected only in patients with chronic hepatitis C (6%). The serum gammaglobulin level was significantly higher in patients with hepatitis C and anti-nuclear antibody titersor = 1/50. The anti-smooth muscle antibodies detected in patients with hepatitis C had no anti-actin specificity. The response to interferon was not related to the detection of non organ specific autoantibodies before treatment.Anti-nuclear or anti-smooth muscle antibodies are not characteristic of hepatitis C virus infection.

An autoantibody to liver cytosol was previously described in childhood autoimmune chronic active hepatitis type 2. The antigen, liver cytosol antigen type 1, was for the first time partially purified using gel filtration and ion exchange chromatography, and it was characterized using immunodiffusion, immunoblot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitate. Immunoblot detected a unique antigenic peptide at 62 kD from human cytosol and at 58 kD from rat cytosol. The same peptides were also detected when immunoprecipitates of liver cytosol antigen type 1 and autoantibodies to liver cytosol antigen were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polymeric structure, probably a tetramer, is suggested for native liver cytosol antigen type 1 because in gel filtration chromatography liver cytosol antigen type 1 was eluted as a protein of a molecular weight between 240 and 290 kD when human liver cytosol was fractionated and between 220 and 270 kD from rat liver cytosol. Liver cytosol antigen type 1 is probably poor in carbohydrates because it was not stained by periodic acid-Schiff stain. The autoantibodies to liver cytosol were frequently found in association with antiliver kidney microsomal autoantibodies type 1, which are directed against the cytochrome P-450 of the IID6 subfamily. Antiliver kidney microsomal autoantibodies type 1 but not antiliver cytosol autoantibodies were found in association with antibodies to hepatitis C virus. Autoantibodies to liver cytosol antigen type 1 seem to be a more specific marker for autoimmune hepatitis type 2 than antiliver kidney microsomal antibodies type 1 autoantibodies.

Recent studies have shown that hepatitis C virus antibodies are present in a large proportion of patients with autoimmune hepatitis type 2. We have studied 83 patients with liver/kidney microsome antibody-positive type 1 hepatitis. Hepatitis C virus antibodies were sought in every case by second-generation tests (hepatitis C virus enzyme-linked immunosorbent assay and recombinant immunoblot assay). Hepatitis C virus RNA sequences were sought in 22 patients (12 with recombinant immunoblot assay-positive results and 10 with recombinant immunoblot assay-negative results) by means of polymerase chain reaction and by use of primers located in the 5′ noncoding region. Sixty-four patients (77%) had positive results for hepatitis C virus antibodies in the enzyme-linked immunosorbent assay test, and 41 (49.3%) were confirmed by recombinant immunoblot assay. Hepatitis C virus RNA sequences were found in all the recombinant immunoblot assaypositive patients but in none of the 10 who were recombinant immunoblot assay-negative. The recombinant immunoblot assay-negative patients were younger than those who were positive (13 ± 11 vs. 50 ± 11 years) and had higher γ-globulin levels and liver/kidney microsome antibody-positive type 1 titers (61% had a titer of 1:1,000 or more, vs. only 17% of the recombinant immunoblot assay-positive patients). On the basis of these results, chronic hepatitis with liver/kidney microsome antibody-positive type 1 can be divided into two subgroups: (a) true autoimmune hepatitis type 2 (mainly observed in young women), with high titers of liver/kidney microsome antibody-positive type 1, and in which direct autoimmune mechanisms appear to be prominent; and (b) hepatitis C virus-associated hepatitis with liver/kidney microsome antibody-positive type 1 (generally affecting older patients, especially men), with low titers of liver/kidney microsome antibody-positive type 1, and in which the autoimmune process could be a consequence of hepatitis C virus infection. (HEPATOLOGY 1992;16:630–636.)

A 14-year-old girl presented with anti-liver-kidney microsome autoimmune hepatitis preceded by alopecia 3 years earlier. Both pathologies were greatly improved by immunosuppressive therapy. Alopecia is a newly reported extrahepatic manifestation of type 2 autoimmune hepatitis. Its appearance could alert the clinician to an increased risk of autoimmune hepatitis in children.

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).

Indirect immunofluorescent detection of anti-nuclear antibodies was conducted on sections of rat liver and on smears of human Hep 2 tumour cells in the serum of 1017 patients. Overall, the Hep 2 cells gave titres superior by 1 or 2 dilutions. Taking into account this difference, a good agreement was observed between the 2 cellular reagents in 83 per cent of the sera tested. The divergences, which affect almost one half of the positive sera, are largely due to the presence of anti-centromere antibodies, anti-nuclear antibodies giving a patchy "M3" appearance to the Hep 2 cells and, most importantly, anti-nucleolar antibodies. Such differences demonstrate that there are major qualitative or quantitative antigenic differences between the nuclei of rat hepatocytes and those of Hep 2 cells. Although technically rat liver and Hep 2 cells were found to be fairly easy to use and interpret, only a large comparative study of human pathology will be able to determine which is the better reagent for the routine detection of anti-nuclear antibodies.

The authors report a case of Waldenstrom disease in which the IgM kappa immunoglobulin had an anti-smooth-muscle activity at a very high titre (1/100 000). This activity was found in the purified IgM but not in the Fab fragment; nevertheless, the immunofluorescence inhibition by this fragment is evidence in support of the anti-smooth-muscle activity of the IgM. The IgM specificity in this patient is different from the anti-smooth-muscle antibodies already described.

Antinuclear and antinuclear membrane autoantibodies are detected by indirect immunofluorescence in sera of 62 p. 100 of primary biliary cirrhosis patients; when anti-SS-A (Ro) and anti-SS-B (La) autoantibodies were included, 70 percent of patients had at least one type of antinuclear antibody. Of 89 patients with primary biliary cirrhosis, 30 had either Raynaud's phenomenon, Sjögren's syndrome or the CREST syndrome. Some antinuclear antibodies, anticentromere and speckled S1 type, seem to correlate with the associated connective tissue disease. Antibodies showing the S3 pattern (multiple nuclear dots) and antibodies to nuclear membrane may be present independently of an association with connective tissue disease. In the classical technical conditions used to detect anti-tissue and anti-mitochondrial autoantibodies on tissue sections, antinuclear antibodies like anti-centromere or S3 may not be detected and/or identified. Primary biliary cirrhosis patient sera for antinuclear antibodies determination must be screened by at least two assays: indirect immunofluorescence on a human cell line, like HEp-2, and immunodiffusion. The last assay must be performed even if antinuclear antibodies are undetected by immunofluorescence.

The synthesis of blocking antibodies has been demonstrated in ten patients undergoing rush immunotherapy (R-IT) with mite extracts: basophil degranulation performed on washed cells (Allergolam technique) did not differ significantly before and 10.8 (+/- 6.8) months after R-IT. Preincubation of allergen (100, 25, 10, and 5 micrograms/mL) with individual serum collected before R-IT did not decrease degranulation, but degranulation was significantly decreased when the allergen was incubated 30 minutes at 37 degrees C with individual serum collected after R-IT or with the IgG-fraction of a pool of sera collected in 22 treated patients. A linear correlation between the mean of degranulation and the log allergen concentration was observed in the two latter groups. The antigen neutralizing capacity of the IgG-fraction was studied on basophils of 11 "allergic donors." Antigen neutralizing capacity clearly increased with time of incubation with the allergen (15, 30, and 60 min), but was also significant without preincubation, eg, for 10 and 5 micrograms/mL allergen concentrations. These results indicate that the improvement of asthma usually noticed 2 months after R-IT is probably due, essentially, to blocking antibodies, since the sensitivity of the basophils is not modified significantly.

Six patients developed acute, subacute or chronic hepatitis after taking tielinic acid, a new diuretic used in the treatment of hypertension. Two died of acute liver failure. The condition was characterized by marked increase in serum transaminases, parenchymal necrosis and portal and/or lobular inflammatory fibrosis. In addition, the serum of all patients contained high titers of a liver/kidney microsomal antibody, which disappeared either after tienilic acid was discontinued or after prednisolone was introduced. The study shows that tienilic acid may be responsible for acute or chronic hepatitis and suggests that a liver/kidney microsomal antibody could be a sero-immunological marker of drug-induced liver disease.

Total eosinophil count (TEC) was done in 13 normal subjects, in 8 subjects before and 90 mn after a "French" breakfast and in 29 asthmatic patients. The geometric mean is higher (p less than 0,001) in asthmatic than in normal subjects: 304 (231--391) versus 144 (101--197). The breakfast does not interfer with the result of the TEC. In asthmatic subjects, a significant inverse correlation appears (r = 0,57, p less than 0,01) between the logarithm of TEC and the quotient: formula; see text. This correlation persists whatever may be the subject's age, the duration of the asthma or age at which he first contracted the illness. Asthma appeared after 40 years in 4 of the subjects: their TEC was higher (p less than 0,01) than in subjects who had become asthmatic before the age of forty. The respiratory function was lowered more in these 4 patients than in others who had been ill for the same time (p less than 0,001). The TEC is not related to results of skin testine with usuel inhalant allergens (grass pollen, house dust, cat's or dog's dander) or by result of a provocation test (by inhalation of allergen). Eosinophilia and bronchial obstruction are connected by one (or more) factors that may be inhalant allergens in extrinsec asthma and circulating immune complexes in intrinsec asthma.

Sixty-five patients with histologically proven chronic active hepatitis of unknown cause but associated with the antiliver/kidney microsome antibody type 1, confirmed by immunofluorescence and immunoprecipitation, were selected as forming a special entity. This disease was found to be rare with a prevalence of 5/1,000,000. The female to male ratio was 8:1. The condition occurred at all ages but was most common between the ages of 2 and 14 years. In 22 of the 65 cases, the hepatitis was associated with an autoimmune disease, most commonly insulin-dependent diabetes, autoimmune thyroid disease and vitiligo. The same autoimmune diseases were present in first-degree relatives from seven families. In 36 cases, the onset of disease resembled acute viral hepatitis. Serum biochemical tests showed marked elevation in aminotransaminases and hypergammaglobulinemia. Paradoxically, serum and salivary IgA levels were often normal or low. Histologic findings were multifocal hepatic necrosis with bridging in the acute stage, and aggressive hepatitis with mononuclear cell infiltration or macronodular cirrhosis in the late stages. Serologically, apart from the presence of antiliver/kidney microsome antibody type 1, the disease was characterized by the absence of antiactin, antimitochondria and antinucleus antibodies; however, organ-specific autoantibodies were often present. The clinical course was usually severe: six patients in the acute stage presented with fulminant hepatitis, and all, except two, other patients progressed to cirrhosis. Prolonged treatment with corticosteroids and immunosuppressants was usually effective in rendering the cirrhosis inactive. The cumulative survival rate was 51% at 14 years. We propose to call this entity "anti-LKM1 chronic active hepatitis" or "autoimmune hepatitis type II" to differentiate it from classical "lupoid hepatitis" or autoimmune hepatitis type I.

The comparative study of the human tumor cell line HEp-2 and rat liver for the detection of antinuclear and antinucleolar autoantibodies by the indirect immunofluorescence technic in routine screening test demonstrate that, taking titer and staining pattern into account, both substrates are able to separate autoimmune disease (systemic lupus erythematosus, rheumatoid arthritis, scleroderma and primary biliary cirrhosis) patients from healthy subjects. The minimal screening test must include sera diluted 1:20 and 1:80. The capability of the HEp-2 substrate to reveal and to discriminate different speckled nuclear and nucleolar patterns explain its greater performance, notably in detecting anticentromere antibodies highly specific for the CREST syndrome and a speckled nuclear antibody frequently associated with primary biliary cirrhosis, allowing an earlier diagnosis of autoimmune diseases presenting these patterns.

In stimulated cultures, a proportion of human T lymphocytes form multilayer rosettes with sheep red blood cells (SRBC) and they can be easily separated from common E rosettes by velocity sedimentation. We have developed a simplified method for harvesting multilayer rosettes and quantifying them. Practical applications of this technique are discussed.

The differentiation of human B lymphocytes in vitro into Ig secreting cells, as measured by a radioimmunoassay rendered specific to IgG and IgM, in response to a polyclonal stimulant, requires the participation of several cellular types. Upon poleweed-mitogen stimulation, B-cell enriched and monocyte containing cell suspensions prepared by elution of nylon wool adherent cells, when mixed with various concentrations of T cells purified by filtration through nylon wool column and depletion of EAC-rosette forming cells, synthesize 5 to 20 times more Ig than non separated lymphocytes. These experiments which demonstrate the cooperation of certain T cells also suggest that other cells eliminated by nylon wool adherence act as suppressor cells. The cooperation of these T cells is independent of their proliferation and can be obtained with allogeneic T cell.

A new autoantibody was detected by immunoprecipitation in the serum of 21 patients with chronic active hepatitis. The antibody reacted against a soluble cytosolic antigen in liver. The antibody was organ specific but not species specific and was therefore called anti-liver cytosol antibody Type 1 (anti-LC1). In seven of 21 cases, no other autoantibody was found; the remaining 14 cases had anti-liver/kidney microsome antibody Type 1 (anti-LKM1). With indirect immunofluorescence, a distinctive staining pattern was observed with the seven sera with anti-LC1 and without anti-LKM1. The antibody stained the cytoplasm of hepatocytes from four different animal species and spared the cellular layer around the central veins of mouse and rat liver that we have called juxtavenous hepatocytes. The immunofluorescence pattern disappeared after absorption of sera by a liver cytosol fraction. The 14 sera with both antibodies displayed anti-LC1 immunofluorescent pattern after absorption of anti-LKM1 by the liver microsomal fraction. The anti-LC1 was found in the serum only in patients with chronic active hepatitis of unknown cause. Anti-LC1 antibody was not found in sera from 100 patients with chronic active hepatitis associated with anti-actin antibody classic chronic active hepatitis Type 1, 100 patients with primary biliary cirrhosis, 157 patients with drug-induced hepatitis and a large number of patients with liver and nonliver diseases. This new antibody was considered a second marker of chronic active hepatitis associated with anti-LKM1 (anti-LKM1 chronic active hepatitis) or autoimmune chronic active hepatitis Type 2.

Using HEp2 cells to study antinuclear antibodies has resulted in the discovery of the anti-centromere antibody which is thought to separate the CREST syndrome from progressive systemic sclerosis (scleroderma). This antibody seems to be exceptional in healthy subjects and are in patients with connective tissue diseases, except for scleroderma. It has also been found in CREST syndrome associated with other diseases, such as primary cirrhosis and neoplasias. In our study, the sensitivity of the anti-centromere antibody was 89.1% and its specificity 92.3% which shows that it is worth looking for.

The anti-native DNA antibodies were measured by a radioimmunoassay (RIA) type Farr assay in the sera from 648 patients: 108 with active or inactive systemic lupus erythematosus (SLE), 181 with clinical symptoms of another connective tissue disease, 171 with liver diseases, 29 with different pathology and 159 normal sera were obtained from a blood bank. The anti-DNA kit has been calibrated against the first international units/ml. This assay has proved to be sensitive and specific, and appears to be reliable for the diagnosis and follow-up of SLE patients. The authors propose a new reference cut-off level higher than producer's one.

One hundred and two rheumatoid arthritis patients were tested before treatment for the presence of 16 autoantibodies. Low titers of antinuclear factors appeared, but there were no antitissue antibodies. At different intervals during the treatment of a 2nd series of 176 patients, elevations of antinuclear factors were observed that correlated slightly with either autoimmune or toxic side effects. Antitissue antibodies were seen only in pemphigus and myasthenia after the onset of these complications. The practical value of systematic evaluation for autoantibodies in penicillamine-treated rheumatoid patients is questionable.

Twenty sera from patients with systemic lupus erythematosus (SLE) and high titre of IgG anti-cardiolipin antibodies (ACA) were studied in order to evaluate the prevalence of anti-mitochondrial type 5 antibodies (AMA 5). None of these sera were found to be AMA 5 positive but five of 18 were positive for VDRL. Twenty sera from patients with AMA 5 were studied in order to evaluate the prevalence of ACA: only six of 20 were positive for ACA. In contrast to this finding, 15 of the 20 sera positive for AMA 5 were also positive for VDRL (P less than 0.001). The six sera positive for ACA and AMA 5 were absorbed with cardiolipin micelles. This absorption eliminated the ACA activity but not the AMA 5 activity. Despite the clinical similarities between the two groups of patients with AMA 5 or ACA, these data suggest that patients with AMA 5 and patients with ACA belong to two different subsets of SLE or SLE-like syndromes and that AMA 5 antigen is different from cardiolipin.

To evaluate if antimitochondrial type 5 antibodies (AMA5) might be included among antiphospholipid syndrome (APS) markers.In a retrospective study, blood variables of 48 patients with AMA5 were analyzed in relationship with clinical and biological markers of APS and systemic lupus erythematosus (SLE).We observed a high prevalence of false biological test for syphilis (95%), lupus anticoagulant (LAC) (71%), anticardiolipin antibodies (aCL) of IgG (71%) and IgM (75%) isotype, positive direct Coombs' test (54%), thrombocytopenia (52%), anti-B2 glycoprotein I antibodies (38%). Twenty-nine patients (61%) had at least one clinical manifestation of APS; 42% had recurrent arterial and/or venous deep thrombosis and 21% had recurrent fetal loss. But, for 2 patients, AMA5 were the sole detected immunological marker. Moreover, SLE was observed in 35% of the patients. These were different from 100 control patients with SLE with the respect to skin involvement and dsDNA antibodies which were less frequent (p0.01) and aCL, LAC, false biological test for syphilis (p0.001), positive direct Coombs' test and thrombocytopenia (p0.05) which were more frequent.Our data suggests (1) AMA5 is another marker of the APS (2) in patients with SLE, AMA5 seems to be a marker of a subset of SLE. This appears to justify the routine detection of these antibodies.

The sera of 131 patients with anti-liver-kidney microsome antibodies (anti-LKM) detected between 1973 and 1979 in two different laboratories were re-examined. (1) Eighty-six anti-LKM corresponded to the description given by Rizzetto, Swana & Doniach (1973), with a pattern of fluorescence predominating on the 3rd portion of the proximal tubules (P3). This group comprised 45 cases of idiopathic chronic hepatitis or idiopathic cirrhosis and one case of halothane-induced hepatitis. (2) Forty-five anti-LKM gave a different pattern on male mouse liver and male rat kidney: (a) fluorescence was greater on centrolobular than on periportal hepatocytes; (b) the first and second portions of proximal tubules (P1 and P2) predominated over P3; (c) P1 fluorescence was equally intense as P2 and (d) P3 cells were heterogeneous with one cell out of 20 more positive than the rest. Absorption tests confirmed that the corresponding antigen was also present in the liver microsomal fraction. A retrospective clinical study discovered tienilic acid-induced hepatitis in all cases. We suggest naming this new antibody 'anti-LKM2'.

Seven hundred and eleven sera were simultaneously studied by immunofluorescence (IF), complement fixation test (CFT) and ELISA for the detection of anti-mitochondrial antibodies (AMA). One hundred and nineteen of these sera were also studied by Western-blot techniques, while some of them were examined by two-dimensional gel electrophoresis so as to identify the polypeptides recognized by M2 antibodies. The results indicated that: (1) ELISA is a more sensitive technique for detecting type M2 AMA (27 scored positive in 27 primary biliary cirrhosis (PBC), as compared to 21/27 by IF and 16/27 by CFT). (2) Although ELISA appeared to be a promising screening method, some false positive results were observed that necessitated a double confirmation of positive sera by another technique. (3) Western-blot experiments with rat mitochondrial purified preparation indicated that sera from AMA type 2 could recognize eight different polypeptides and that most of them identified 63-60, 48, 44, and 35-33 kD polypeptides, whereas the 54 and 27 kD were less frequently recognized. A trypsin treatment of antigens confirmed the enzyme sensitivity of most of these antigens. These results suggested some heterogeneity among M2 AMA, though this series of PBC was not large enough to relate the heterogeneous pattern noticed in Western-blot to the clinical and histological patterns observed in PBC.

A total eosinophil count (TEC) and a white blood cell count (WBCC) were performed simultaneously in 213 patients. Discrepancies were found between TEC and WBCC. WBCC did not allow detection of hypereosinophilia (TEC exceeding 300 eosinophils/microliter) in 45 patients. Patients were classified in three groups : controls (n = 27), benign disease (n = 65), and apparently cured cancer (n = 36). There was no significant difference between total eosinophil counts for these three groups (Wilcoxon non-parametric test). The incidence of hypereosinophilia (TEC exceeding 300) was similar in the three groups (11%, 14% and 6%). Average TEC levels were determined in 20 patients with decompensated hepatic cirrhosis, in 31 patients with active cancer, and in 14 patients with infection. These levels were significantly different from those found in each of the three previous groups. (Wilcoxon test). The incidence of hypereosinophilia (greater than 300/microliter) was significantly higher among patients with cirrhosis (50%) or active cancer (58%). The possible mechanisms of this hypereosinophilia and it's eventual prognostic utility are considered.

Vascular abnormalities are frequent in connective tissue disease. In this kind of patients a lot of autoantibodies are observed. To screen them a strategy must be selected according to the connective tissue disease that is suspected. When the diagnosis is unknown the tests must screen a great number of autoantibodies and this is the case for indirect immunofluorescence assays, at the opposite when the diagnosis is known antibodies specific for the disease should be screened. Some antibodies are specific for a disease this is the case for antibodies directed against double stranded DNA, centromere, extractable nuclear antigens Sm, RNP, Scl-70, Pm-Scl, Jo1, neutrophil cytoplasmic antigen proteinase 3, and beta 2-glycoprotein I the cofactor of anti-cardiolipin antibodies. Anti-SS-A(Ro) antibodies are very frequent in autoimmune diseases and they are not specific for anyone of them. Some autoantibodies are frequent in autoimmune and also in non autoimmune diseases and this is the case for antibodies directed against phospholipids, single stranded DNA, histones, rheumatoid factor. The detection of antibodies depends on the assays used for screening, that is why results should mention the assay used for their detection. The standardization of the detection of the autoantibodies especially when quantitative results are requested, is not yet performed enough. So to obtain reproducible results when monitoring a patient the screening must be done by the same assay in the same laboratory.

Skin reactions to heparin are rare, the most frequent manifestation being skin necrosis. Reactions occur either as an isolated manifestation or within a context of heparin-induced thrombopenia. Urticarious and eczematous reaction, either localized at injection sites or with a general distribution, have been recently reported. In all cases, it is mandatory to withdraw heparin therapy. Such accidents can be observed with all types of heparin (standard heparin and low-molecular weight heparin). Cross-reactions between two types of heparin are frequent. Skin tests can be used to guide heparintherapy.

The aim of the study was to investigate the hypothesis that oral contraceptives (OC) lead to antiethinyl-estradiol antibody (AEEA) synthesis which might in turn be responsible for the thrombo-embolic complications observed in women users. The trial included 428 women divided into a) 204 healthy female volunteers as the control group (CONT), b) 139 women who had suffered thrombo-embolic accidents (THR), c) 85 women suffering from recurrent fetal loss (RFL). In each of these three categories, 50% were pill users (OC+) and 50% were not (OC-). Specific immune markers of systemic autoimmunity and anticardiolipin antibodies were looked for, as they are thought to be present in increased amounts in thrombosis or recurrent fetal loss. The AEEA prevalence differed significantly (p less than 0.05) between the CONT OC+ (32%) and the CONT OC- (13%) women. It also differed (p less than 10(-5)) between the RFL OC+ (60%) and the RFL OC- (12%) women. It did not differ between THR OC+ (36%) and THR OC- (39%) women. Within the OC+ women, the difference between the THR and the CONT groups was not found to be significant, whereas it was found to differ significantly between the RFL and the CONT groups (odds-ratio RFL/CONT estimated at 3.20, confidence interval 1.53, 6.69). Within the OC-women, the AEEA prevalence was found to differ significantly between the THR (39%) and the CONT (13%) groups (odds-ratio THR/CONT estimated at 4.40, confidence interval 2.07, 9.38%) but not between the RFL and the CONT groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Purified Fab fragments of ovine anti-digoxin antibodies (Wellcome Foundation) were used to treat a patient who attempted suicide by absorbing 10 mg of digitoxin (serum concentration 265 micrograms/l). The poor prognosis, as assessed clinically and from serum potassium levels (7.5 mEq/l), seemed to warrant such a treatment. The weak (6.85%) cross-reactivity elicited in vitro between the anti-digoxin antibodies and digitoxin was compensated by increasing the doses, but improvement was observed with 3.6 g, i.e. about half the effective dosage initially considered. The criteria of effectiveness were clinical, electrocardiographic (reversal of the ventricular fibrillation), biochemical (simultaneous and opposite changes in extra- and intracellular potassium levels, suggesting that ATPase inhibition by digitalis is a reversible process) and toxicological: there was an increase in digitoxin serum levels suggesting displacement of the drug from tissue sites to plasma and other extracellular compartments where the Fab fragments are distributed, and Fab-bound digitoxin appeared fairly rapidly in the urine, which suggested shunting of the normal hepatic metabolic pathway.

A case of idiopathic portal hypertension associated with connective disease resembling systemic lupus erythematosus is described. The patient was a 50-year-old woman with splenomegaly, ascites, esophageal varices, and pancytopenia, but without extrahepatic portal obstruction or cirrhosis of the liver. Electron microscopy of the liver showed perisinusoidal fibrosis. High titers of autoantibodies against proliferating cell nuclear antigen (PCNA) were found in the sera as well as in ascites; anti-DNA antibodies appeared after anti-PCNA antibodies and remained thereafter at a moderate titer. The possibility of an immunological process in the pathogenesis of idiopathic portal hypertension is discussed.

Comparison of hypertensive patients receiving no treatment with subjects of the same age reveals the presence of antinuclear antibodies in twice as many of the former as the latter. Various antihypertensive drugs increase this prevalence. They may induce iatrogenic lupus. Alpha-methyldopa: the frequency of antinuclear bodies is three times greater than in untreated hypertensive patients. However there are no reported cases of clinical symptomatology of induced lupus. Beta-blockers. Practolol, acebutolol, labetalol, atenolol, timolol, metoprolol, pindolol and oxprenolol are suspected of increasing antinuclear antibodies. These drugs may be weak inducers of iatrogenic lupus when compared with major drugs: procainamide, high doses of hydralazine and D-penicillamine.

Anticardiolipine antibodies are a marker of the antiphospholipid syndrome. They are detected by Elisa. Despite its simplicity, the results obtained by this assay are not always reproducible despite several worldwide standardizations and the distribution of calibration sera by EN Harris. On 4 December 1992, the First French Anticardiolipin Antibodies Standardization Workshop was held in Paris. Eight coded standards were sent to 33 laboratories in France, one in Switzerland and one in Luxembourg. Some of them used several assays. Agreement between qualitative results was good for samples with high and moderate level of antibodies. But the results expressed in IgG antiphospholipid units were scattered, the interlaboratory coefficient of variation for each of the eight standards was higher than 70%, though the within-run coefficient of variation had a median value of 10%. Assays using bovine serum for the blocking buffer and for the dilution buffer seemed to give more reproducible results than assays using purified bovine serum albumin (p0.001). For only nine assays out of 38 which expressed results quantitatively, was a linear regression observed between the dilutions of a sample and the results expressed in IgG antiphospholipid units. Absence of linearity is an obstacle which must be overcome if we wish to compare quantitative results obtained by different laboratories.

The means for analysing T-cell activities are very limited. Indeed, the numerical estimation of the changes which take place in human T lymphocytes had to rely solely on the observation of transformed cells (blasts). However, even if various mitogens appear selective for either T of B cells, we know that both types of lymphocytes are ultimately transformed into blasts. This is particularly true in studies conducted on mixtures of T and B cells where stimulated T cells can release substances which act on B cells. On the other hand, studies with purified populations of T or B cells do not express the cellular interactions that occur naturally between these two types of lymphocytes. For all these reasons, it seemed that the estimation of " quanta " of activity unique to T lymphocytes would contribute significantly to our knowledge of this line of cells. We describe a new method of detecting the in vitro stimulation of lymphocytes based on the appearance of cells having the property to cluster several layers of sheep red blood cells (SRBC) around themselves, forming multilayer rosettes (CFC). This formation is temperature-dependent and requires trypanblue-excluding, metabolically active blastoid cells. Non-separated cells as well as purified T cells stimulated with allogeneic leucocytes (MLR), specific antigens or polyclonal mitogens (PWM, ConA) gave rise to CFC. Such multilayer rosettes were not formed by separated B cells. In the MLR, CFC were detected 48 h after beginning of cultures and increased in number thereafter just like thymidine incorporation. The response to ConA and PWM was detected earlier and gave rise to two or three peaks, the first rise in the number of CFC coinciding with the peak of thymidine incorporation, but the maximal increase occurring two or three days later. Treatment of blastoid cells with a serum specific for T cells--but not with an anti-immunoglobulin serum--abolished their ability to form ordinary or multilayer rosettes. When separated, however, on a nylon wool column, CFC were more adherent than the bulk of T cells. It is suggested that CFC form a subpopulation of T cells with distinct characteristics, allowing a direct assessment of membrane changes which take place when T lymphocytes are activated in vitro.