Mycophenolate mofetil in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis: a prospective pharmacokinetics and clinical study.

Anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) treatment strategy is based on immunosuppressive agents. Little information is available concerning mycophenolic acid (MPA) and the area under the curve (AUC) in patients treated for AAV. We evaluated the variations in pharmacokinetics for MPA in patients with AAV and the relationship between MPA-AUC and markers of the disease. MPA blood concentrations were measured through the enzyme-multiplied immunotechnique (C(0), C(30), C(1), C(2), C(3), C(4), C(6) and C(9)) to determine the AUC. Eighteen patients were included in the study. The median (range) MPA AUC(0-12) was 50·55 (30·9-105·4) mg/h/l. The highest coefficient of determination between MPA AUC and single concentrations was observed with C(3) (P < 0·0001) and C(2) (P < 0·0001) and with C(4) (P < 0·0005) or C(0) (P < 0·001). Using linear regression, the best estimation of MPA AUC was provided by a model including C(30), C(2) and C(4): AUC = 8·5 + 0·77 C(30) + 4·0 C(2) + 1·7 C(4) (P < 0·0001). Moreover, there was a significant relationship between MPA AUC(0-12) and lymphocyte count (P < 0·01), especially CD19 (P < 0·005), CD8 (P < 0·05) and CD56 (P < 0·05). Our results confirm the interindividual variability of MPA AUC in patients treated with MMF in AAV and support a personalized therapy according to blood levels of MPA.

Association of Fcγ receptor IIIA variant with a subset of anti-topoisomerase I-positive patients in systemic sclerosis: a descriptive pilot study.

Hypothesizing a pathophysiological role of anti-topoisomerase I antibodies (anti-topo I) through autoantibody-dependent cell-mediated cytotoxicity (ADCC) and cytotoxic effectors expressing receptors for the Fc portion of IgG in systemic sclerosis (SSc), 267 SSc patients (56 with anti-topo I and 102 with anti-centromere antibodies (ACA)) were genotyped for the functional FCGR3A-V158F polymorphism. A descriptive analysis of patients according to their clinical and immunological status and FCGR3A-158 V/F genotypes was performed using multiple correspondence analysis. This descriptive analysis revealed an association between the FCGR3A-158 VV genotype and the presence of anti-topo I. By contrast, no relationship was found between FCGR3A polymorphism and the presence of ACA. SSc patients with anti-topo I appear to be more frequently homozygous for the high-affinity FcγRIIIA-coding allele, suggesting that some autoantibodies may be pathogenic through ADCC.

Evaluation of a peptide ELISA for the detection of rituximab in serum.

Pharmacokinetic studies of therapeutic monoclonal antibodies necessitate the measurement of their biologically active fraction. The aim of this work was to develop an enzyme-linked immunosorbent assay (ELISA) for rituximab, a chimeric anti-CD20 monoclonal antibody, based on its binding to a 20-mer peptide (P20) derived from the extracellular loop of human CD20 (residues 165-184). Derivatives of P20 were prepared by conjugation to bovine serum albumin (BSA-P20ACM) or biotin (Biot-P20ACM). Interactions of P20 and its derived peptides with rituximab were analyzed by surface plasmon resonance (SPR) and by ELISA. A monoclonal anti-idiotype antibody (MB2A4) was used as the reference in each case. SPR analysis showed that P20 (conjugated or unconjugated) had a lower affinity for rituximab than MB2A4. ELISA methods based on P20 or MB2A4 were both highly accurate and reproducible for rituximab measurement in spiked samples, but the MB2A4-based assay had a lower limit of quantification. Interestingly, discrepant results were obtained with the two ELISA methods when analyzing pharmacokinetic samples, with the rituximab concentrations obtained with the MB2A4-based method being systematically higher than those determined by the P20-based method. Possible interference of circulating CD20 with the P20-based method was supported by competition experiments. Rituximab aggregation in the bloodstream may also account for the bias observed in samples from healthy mice. The P20-based ELISA is far less sensitive than the MB2A-based ELISA, thus limiting its utility for pharmacokinetic studies. However, the discrepancy observed between two different approaches for rituximab measurement indicates that data from different studies should be interpreted with care.

Isolation and characterization of syncytiotrophoblast plasma membrane from human placenta.

Human full-term syncytiotrophoblast plasma membranes isolated by mechanical procedures (sieving and ultrasonic disintegration), purified by phase centrifugation, form a single band of 1.052 +/- 0.002 g/ml density in percoll gradient. The purity of the preparation was assessed by electron microscopy, enzyme analysis and beta 2-microglobulin determination.

Complete saturation of protamine sulphate by dsDNA is necessary in order to obtain a highly sensitive and specific anti-dsDNA ELISA.

A protamine sulphate (PS) pretreated solid phase coated with different amounts of dsDNA has been used to develop a sensitive, specific and reproducible anti-dsDNA ELISA. Using low concentrations of a dsDNA coat 50% of SLE sera were found to be positive and false positive reactivity due to anti-PS reactivity was found in 3/40 patients with other auto-immune diseases (OAID). In contrast, when PS was saturated with higher concentrations of dsDNA 80% of SLE sera were detected, the reproducibility of the results was better and anti-PS reactivity of OAID patients with an anti-PS reactivity disappeared. The sera of three other OAID patients contained low avidity anti-dsDNA, measured after a salt elution step in the ELISA procedure, and 2/60 patients with non-auto-immune disease exhibited a false positive anti-dsDNA reactivity since they reacted with the solid phase even in the absence of PS and dsDNA. Thus an ELISA procedure using a PS pretreated solid phase permits the sensitive, specific and reproducible measurement of anti-dsDNA antibodies only if a high concentration of dsDNA is coated on the PS and appropriate controls are performed.

A balanced brain asymmetry modulates T cell-mediated events.

Partial ablation of the left fronto-parietal cerebral cortex decreases the number of spleen T cells, impairs IgG-alpha SRBC and T mitogen-induced responses, and delays the response to alloantigens. In contrast, these events are increased following a symmetric lesion of the right neocortex. The findings extend previous results showing that the neocortex modulates NK activity and the efficacy of T cell-specific serum factors. B cells and macrophages are not affected. In these assays, mice subjected to ablation of one lateral cerebral neocortex serve as controls for symmetrically lesioned mice, in addition to no surgery or sham-operated controls. The findings suggest that brain lateralization for cognitive processes should be extended to T cell immune recognition. The phenomenon is present at a population level.

Multicenter evaluation of nine commercial kits for the quantitation of anticardiolipin antibodies. The Working Group on Methodologies in Haemostasis from the GEHT (Groupe d'Etudes sur l'Hémostase et la Thrombose).

The performances of nine commercial kits and an in-house method (HM) for the quantitation of anticardiolipin antibodies (ACA) have been evaluated in a multicenter study. Ninety control and patient samples and six standards from Louisville University were run with kits and with the HM. Marked differences in positivity rate between kits were observed, ranging from 31 to 60% for IgG and 6 to 50% for IgM. Concordance between kits occurred in 59 and 51% of samples for IgG and IgM respectively. Concordance coefficients (kappa) ranged from 0.13 to 0.92. Slopes of regression lines between the declared units of Louisville standards and the units measured from the calibrators of the kits showed great diversity and ranged from 0.159 to 0.931 for IgG and from 0.236 to 0.836 for IgM. The beta 2-glycoprotein I (beta 2-GPI) content of the dilution buffers and the wells supplied with the kits revealed noticeable differences. However samples containing anti-beta 2-GPI antibodies were classified similarly by all but one kit. In contrast the ability to measure samples devoid of anti-beta 2-GPI antibodies differed markedly between the kits. This study shows that differences in positivity rates between the commercial kits may contribute to the differences in ACA prevalence rate found in the literature. The choice of cut-off levels may partly explain the moderate concordance between the kits. In addition some samples behave very differently depending on the kits. In spite of the expression of results in PL units, standardization of ACA assays has not been achieved.

Lipid and protein components of the syncytiotrophoblast plasma membrane inhibit lymphocyte proliferation by two distinct pathways.

The ability of syncytiotrophoblast plasma membrane lipid and protein fractions (STPM lipids, STPM proteins), tested under a reconstituted form, to inhibit lymphocyte proliferation induced by PHA was investigated. The cytostatic activity of STPM proteins appeared greater than that of the STPM lipids. Furthermore, IL-2 production and IL-2 receptor expression by activated lymphocytes were markedly decreased in the presence of STPM proteins compared to the native membrane but remained unaffected in the presence of STPM lipids. Finally, the inhibition of lymphoproliferation could be maintained after removal of the protein fraction from lymphocytes prior to stimulation by PHA. The biological and immunological significance of these results is discussed.

Correlation of plasma hormone levels and peripheral circulating lymphocyte subpopulations during human pregnancy.

Using monoclonal antibodies (OKT3, OKT4, OKT8) peripheral blood lymphocyte subsets were determined in 40 normal primiparous pregnant women and compared with those of 31 nonpregnant controls. In pregnant women plasma concentrations of estradiol, progesterone, human placental lactogen (HPL), beta subunit of human chorionic gonadotropin (beta HCG), and alpha-fetoprotein were measured by means of radioimmunoassay. We studied if correlations between peripheral lymphocyte subsets and plasma hormone levels might exist. We observed in pregnant women from 10 to 40 wk of amenorrhea a decrease in the percentage of OKT3 and OKT8 cells, and during the course of pregnancy an increase in the percentage of OKT4 cells. This increase inversely correlated with plasma beta HCG levels and directly correlated with plasma HPL levels.

Is anti-topoisomerase I a serum marker of pulmonary involvement in systemic sclerosis?

STUDY OBJECTIVE: To determine the value of the level of anti-topoisomerase I (anti-topo I) to evaluate lung involvement defined by abnormal high-resolution computed tomography (HRCT) score and pulmonary function tests (PFTs) in systemic sclerosis (SS). PATIENTS: Forty-eight patients with SS, 20 with lung involvement and 28 with no lung involvement. DESIGN: PFT measurement, HRCT scoring of lung involvement, and anti-topo I assay by enzyme-linked immunosorbent assay. Normal anti-topo I level was defined as < 30. RESULTS: There was a significant association between cutaneous extent and anti-topo I level (6.5% of patients with limited cutaneous scleroderma had abnormal anti-topo I levels vs 70.6% of patients with diffuse cutaneous scleroderma, p = 0.0001). In patients with diffuse cutaneous scleroderma, pulmonary involvement was associated with a higher percentage of abnormal anti-topo I level: 91.7% vs 20% (p = 0.010). In patients with diffuse cutaneous scleroderma, a significant association was found between the class of anti-topoII level and total lung capacity (median, 69 in patients with abnormal anti-topo I level vs 87 in patients with normal anti-topo I level, p = 0.010), between the class of anti-topo I level and HRCT score (median, 12 in patients with abnormal anti-topo I level vs 5 in patients with normal anti-topo I level, p = 0.05). CONCLUSION: Anti-topo I can be considered as a marker of lung involvement in patients with diffuse cutaneous scleroderma.

Syncytiotrophoblast plasma membrane inhibits membrane expression of activation markers on PHA-stimulated human lymphocytes.

Studies have been carried out on the effects of full-term human syncytiotrophoblast plasma membrane (STPM) preparations on the membrane expression of the lymphocyte activation markers HLA-DR, IL-2R, TfR and CD69 during PHA-induced lymphoproliferation. STPM considerably decreases the expression of both late (HLA-DR) and early (IL-2R, TfR, CD69) activation markers at doses which inhibit PHA-induced lymphoproliferation. These results favour the hypothesis that STPM inhibits a very early phase of PHA-induced lymphocyte activation.

In vitro inhibitory effect of human syncytiotrophoblast plasma membranes on the cytolytic activities of CTL and NK cells.

We have previously described the purification procedure for syncytiotrophoblast plasma membranes (STPM) and have now studied their immunomodulatory properties on the in vitro cytotoxic assays of generated cytotoxic T-lymphocytes (CTL) and natural killer cells (NK). STPM inhibited the cytolytic activities of CTL and NK against their target cells, whereas RBC ghosts, even at the highest protein concentration used, were ineffective. This inhibitory effect was dose-dependent upon the STPM-protein concentration and appeared to be particularly distinct at low effector/target ratios. It is hypothesized that the inhibitory activity of STPM may be exerted by blocking the effector cells or by masking their targets. Regardless of the mode of action, since cytotoxic cell activities are known to play an important role in the allograft rejection process, this suppressive inhibitory effect of STPM might be a crucial mechanism in the tolerance of the semiallogenic fetal graft.

Effect of human syncytiotrophoblast extract on in vitro proliferative responses.

The authors studied the effects of soluble syncytiotrophoblast extract (STE) on the proliferative responses of lymphocytes to different lectins (PHA and Con A) and to allogeneic cells in one-way mixed lymphocyte cultures. STE suppressed lymphocyte reactivity to lectins and to allogeneic cell cultures. The inhibitory effect was dose dependent. Increased concentrations of lectins failed to overcome the inhibitory effect of STE. Lymphocytes preincubated with STE for 18 hr, then washed and exposed to lectins still exhibited an inhibition of cellular proliferation. STE added to lymphocyte cultures at various times in the presence of both mitogens or of allogeneic cells continued to inhibit lymphoproliferative responses even when it was added after 43 hr of cell culture. Furthermore, STE was able to reduce the spontaneous proliferation of tumor cell lines K562 and LHN13 maintained in vitro culture prior to testing. In all cases, the inhibition observed was not due to lymphocytotoxicity or to tumor cell mortality. The inhibitory effect of STE on the proliferative responses of lymphocytes to stimulants may be due to a cytostatic effect that may represent a contributing factor in the nonrejection of the fetus by a competent immune system.

Sodium diethyldithiocarbamate influences the early immunological changes evoked by an acute non-antigenic inflammation process.

Calcium pyrophosphate (CaPp) produces an acute local inflammatory process when administered intra-pleurally. It is the simplest model of an inflammation uncomplicated by pharmacological properties of the agent or the presence of an antigen. Spleen or lymph-node T- or B-cell numbers and activities, including NK activity, are modified within 2 h under these conditions. A treatment with sodium diethyldithiocarbamate (lmuthiol), already known as a T-cell augmenting agent, restores towards normal values both the inflammatory reaction and the modified immune parameters induced in mice by CaPp pleurisy. The use of lmuthiol as a non-toxic, non-steroidal antiinflammatory agent is therefore suggested.

[Exacerbation of an autoimmune thrombocytopenic purpura during treatment with interferon alpha in a woman with chronic viral hepatitis C]. / Exacerbation d'un purpura thrombopénique auto-immun au cours du traitement par l'interféron alpha chez une femme atteinte d'une hépatite chronique virale C.

We report the case of a 40-year-old woman with chronic viral hepatitis C and latent idiopathic thrombocytopenic purpura who developed severe thrombocytopenia during alpha interferon therapy. Platelet-associated IgG titers were elevated, and platelet antibodies were detected in the serum. Intravenous administration of high-dose polyvalent immunoglobulins was ineffective, but a normal platelet count was obtained after corticosteroid therapy. A history of idiopathic thrombocytopenic purpura could be considered a contraindication for alpha-interferon therapy in patients with chronic viral hepatitis.

[Gougerot-Sjögren syndrome. Central neurological involvement with recurrent development]. / Syndrome de Gougerot-Sjögren primitif. Atteinte neurologique centrale évoluant par poussées.

A 64-year-old woman was repeatedly hospitalized for various recurrent clinical signs of central nervous system involvement. The diagnosis of primary Sjögren's syndrome was established 3 years 6 months after the onset of the disease. Sicca symptoms, as well as inflammatory biological abnormalities were absent. Moreover, both lacrymal and salivary gland secretions were affected. A high level of antinuclear antibodies to SSA and SSB was associated with inflammatory lesions in minor salivary glands biopsy samples consistent with the diagnosis of Sjögren's syndrome.

[Beginning rheumatoid arthritis revealed during bronchiectasis surinfections. Value of cyclic antibiotherapy?]. / Polyarthrite rhumatoïde débutante révélée au décours de surinfections de bronchectasies. Place d'une antbiothérapie cyclique?

Incidence of symptomatic bronchiectasis (BR) occurs in around 2% in patients with late rheumatoid arthritis (RA). Its seems that the association BR-RA could be a worsening factor for outcome of RA patients. A 58-year-old woman without dry syndrome, suffering from bronchial purulence over one year was admitted to the Department of Pneumology for hemoptysis and arthritis (knees, ankles, and wrists). Three prior episodes of inflammatory articular pain had occurred after transient bronchial purulence or pneumonitis. CT-scan showed bilateral bronchiectasis. Diagnosis of early RA was proved after the third episode of bronchial purulence related to a strain of Haemophilus influenzae. A strain of Coxiella burnetii was probably responsible for one of the three bronchial surinfections. Latex and Waaler Rose tests were transiently positive during the first episode, and became positive after the third one. At that time, RA was relevant in view of ARA criteria. Cyclic prophylactic antibiotic regimens could be proposed to patients suffering from RA-BR association, in contrast to the cases of patients with isolated BR. This approach could prevent destabilization of RA and reinforce of anti-rheumatic therapy. Activation and release of cytokines (NFk-B, TNF-alpha), and/or bacterial epitopes seems to be directly responsible for the articular destabilization.

Abnormalities in CD4+ T lymphocyte subsets in patients with common variable immunodeficiency.

In order to investigate whether deficient immunoglobulin production in common variable immunodeficiency (CVI) patients was related to defective T cells functions, phenotype and proliferative responses to mitogen of peripheral blood mononuclear cells (PBMC) were investigated in 9 patients with CVI. The results were compared to those of 12 age- and sex-matched normal controls. The numbers of CD3+ and CD8+ T cells in the patients were not different from those in the control group, but the numbers of CD4 T cells were decreased (511 +/- 237 vs 844 +/- 247/mm3; P less than 0.01). The decrease in CD4 T cells was due to a dramatic deficiency in the CD4+ CD45RA+ subset, observed as an absolute value of blood lymphocytes (126 +/- 91 vs 384 +/- 142; P less than 0.001) and as a percentage (9.0 +/- 7.1 vs 18.8 +/- 5.0; P less than 0.01). In contrast, the CD4+ CD29+ T cell subset was not different in CVI from those in the control group. Moreover, there was a strong positive correlation between the number of percentages of CD4+ CD45RA+ blood T cells and the proliferative response of PBMC to PHA (respectively, P less than or equal to 0.02 and P = 0.05) and to Con A (P less than or equal to 0.02). The decrease of CD4+ CD45RA+ T cells could reflect an abnormality in the physiological status of T cells and could be of critical importance in the antibody deficiency.