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.

[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.

Adhesive properties of choriocarcinoma cells toward lymphocytes activated or not by interleukin-2.

Choriocarcinoma cells (CC) in vitro are resistant to NK lysis but sensitive to lysis by blood or decidual effectors activated by interleukin-2 (IL-2). Because lytic activity requires a step of adhesion, the adhesive properties of the choriocarcinoma cells BeWo, JEG-3, and JAR were examined functionally toward peripheral blood lymphocytes. The adhesion of lymphocytes to choriocarcinoma cells was very low and did not increase after stimulating lymphocytes with IL-2. As demonstrated by cytofluorimetry analysis, choriocarcinoma cells and cytotrophoblast cells prepared from term placenta expressed intercellular adhesion molecule-1 (ICAM-1), whereas only CC expressed CD56. Tumor necrosis factor-alpha or interferon-gamma increased the expression of ICAM-1 on choriocarcinoma cells without modifying the adhesion of lymphocytes to choriocarcinoma cells. These results suggest that resistance of choriocarcinoma cells to lysis by cytotoxic effectors could partially be attributed to the low level of lymphocyte adhesion to these cells.

[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.

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.

Acute measles encephalitis of the delayed type: neuroradiological and immunological findings.

A case of delayed acute measles encephalitis in an immunosuppressed child is reported. Detailed immunological studies have shown defective humoral immunity (defective IgA, IgG2 and IgG3) and decreased natural killer activity. Neuroradiological examination by magnetic resonance imaging revealed several high signal lesions on T2-weighted images in the gray matter without clinical or pathological correlation. The implications of these findings are discussed.

Inhibitory effect of human syncytiotrophoblast plasma membrane vesicles on Jurkat cells activated by phorbol ester and calcium ionophore.

The effects of syncytiotrophoblast plasma membrane vesicles (STPM) on stimulated Jurkat leukemic T cells have been investigated. STPM inhibited IL-2 production and the expression of protein P55 of the IL-2 receptor (IL-2R P55), when Jurkat cells were stimulated by a combination of calcium ionophore A23187 (CaI) + phorbol 12-myristate 13-acetate (PMA). STPM also inhibited IL-2R P55 when cells were stimulated by PMA alone, a situation in which IL-2 production is negligible. On the other hand, STPM had no effect on the sustained mobilization of intracellular Ca2+ induced by CaI nor on the PKC-dependent CD3 down regulation induced by PMA. Finally STPM had no effect on intracellular cAMP levels. These results show that (i) the inhibitory effect of STPM on IL-2R P55 expression is independent of the inhibition of IL-2 production, and (ii) the inhibitory effects of STPM are at least partially independent of phosphatidylinositol 4,5-bisphosphate hydrolysis. They suggest that STPM affect a signaling pathway activated by PMA but possibly PKC independent.

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 plasma membrane-soluble extracts on in vitro mitogen-induced lymphocyte proliferation. A possible inhibition mechanism involving the transferrin receptor.

Transferrin (Tf) binding to lymphocytes and to syncytiotrophoblast plasma membranes (STPM) and their soluble extracts (STPM-SE) was examined, as well as the effect of the latter on mitogen-induced lymphoproliferation. Lymphocytes only express Tf receptors (Rtf) after mitogen (phytohemagglutinin or concanavalin A) stimulation, and the percentage of Tf bound by stimulated lymphocytes increased as a function of contact time with the mitogen, reaching a maximum at 72 hr. Studies of Tf binding to STPM-SE showed that the percentage of bound Tf increased proportionally to the protein concentration, but was additionally enhanced by a factor of three when STPM were pretreated with 3 M KCl. Scatchard analysis of Tf binding to lymphocytes cultured for 72 hr in the presence of mitogen, as well as to STPM and STPM-SE, revealed that this binding was specific and occurs via a single category of identical and independent receptors the numbers and affinity constants (Ka) of which have been determined. The results obtained by using STPM indicate that the Ka does not vary significantly from one preparation to another, but that the number of sites per milligram of protein increases by a factor of 10 when the STPM are pretreated with 3 M KCl (KCl-STPM). Finally, STPM-SE inhibited the mitogen-induced lymphoproliferative response whether or not they were treated with 3 M KCl. This inhibition was not due to lymphocytotoxicity, was dose dependent regardless of the preparation used, but was maximized with the KCl-STPM-SE fraction. The correlation between the inhibitory capacities of the soluble STPM extracts and the numbers of RTf sites present on their membranes leads to the hypothesis that the observed inhibition could involve the RTf. This effect may help in protecting the fetus from the maternal immune system at the time of the semi-allogenic 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.

Involvement of brain neocortex and liver in the regulation of T cells: the mode of action of sodium diethyldithiocarbamate (imuthiol).

Sodium diethyldithiocarbamate ( imuthiol ), a non-antigenic and non-carcinogenic compound, devoid of toxic effects at immunostimulant doses, shows distinctive properties in recruitment and activation of T cells. Studies on its mode of action disclosed unsuspected links between the immune system, the endocrine liver and the central nervous system. Evidence was obtained indicating that the brain neocortex modulates T-cell mediated events, most likely via control of specific hormonal synthesis by the liver. The influence of imuthiol is determined at the level of the brain neocortex.

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.

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.

Neocortical lateralization of NK activity in mice.

The natural killer (NK) reactivity of mouse spleen cells is controlled by the left brain neocortex and not by the right symmetrical brain area. The finding strongly suggests direct relationships between the central nervous system and the immune system, both involved in biological adaptation for the maintenance of homeostasis and body integrity in relation to the external environment.