Negative selection of human germinal center B cells by prolonged BCR cross-linking.

The antigen receptors on T and B lymphocytes can transduce both agonist and antagonist signals leading either to activation/survival or anergy/death. The outcome of B lymphocyte antigen receptor (BCR) triggering depends upon multiple parameters which include (a) antigen concentration and valency, (b) duration of BCR occupancy, (c) receptor affinity, and (d) B cell differentiation stages. Herein, using anti-immunoglobulin kappa and lambda light chain antibodies, we analyzed the response of human naive, germinal center (GC) or memory B cells to BCR cross-linking regardless of heavy chain Ig isotype or intrinsic BCR specificity. We show that after CD40-activation, anti-BCR (kappa + gamma) can elicit an intracellular calcium flux on both GC and non-GC cells. However, prolonged BCR cross-linking induces death of CD40-activated GC B cells but enhances proliferation of naive or memory cells. Anti-kappa antibody only kills kappa + GC B cells without affecting surrounding gamma + GC B cells, thus demonstrating that BCR-mediated killing of GC B lymphocytes is a direct effect that does not involve a paracrine mechanism. BCR-mediated killing of CD40-activated GC B cells could be partially antagonized by the addition of IL-4. Moreover, in the presence of IL-4, prestimulation through CD40 could prevent subsequent anti-Ig-mediated cell death, suggesting a specific role of this combination in selection of GC B cells. This report provides evidence that in human, susceptibility to BCR killing is regulated along peripheral B cell differentiation pathway.

[Comparison of different methods for serum folate assay]. / Comparaison de différentes méthodes de dosage des folates sériques.

Folates were determined in 148 patient sera, using four different methods: a microbiological assay (Reference Method), two radioassays (Magic B12 FOL [NB] and SimulTRAC SNB no boil kits) and a non isotopic competition method (Magic Lite kit). Folate mean varied according to the methods 10.0 nmol.l-1 (reference method), 12.2 nmol.l-1 (Magic Lite), 8.7 nmol.l-1 (Magic B12 FOL [NB]) and 10.8 nmol.l-1 (SimulTRAC SNB NO boil). Poor correlations were also noted (0.83 < r < 0.95, figures 1 and 2). Differences between methods were explained according to the literature.

[Determination of tumor marker CA 125 in the cancer of the ovary: comparison of immunoenzyme and immunoradiometry methods]. / Dosage du marqueur tumoral CA 125 dans les cancers de l'ovaire: comparaison des méthodes immunoenzymatique et immunoradiométrique.

CA 125, tumor marker newly appeared, is used in ovarian carcinomas. Its determination in serum is an important progress, not only in diagnosis but above all at the time of monitoring of clinical course during the treatment. The determination is possible with immunoradiometric assay (IRA) or enzyme immunoassay (EIA). The comparative study show a good correlation between these two methods. Both give comparable results. The choice for either method depends on equipment of laboratory.

Influence of metabolic disturbances of diabetes mellitus on serum CA 19-9 tumor marker.

CA 19-9 is a monoclonal antibody-defined tumor marker expressed by exocrine pancreas. It has been shown that exocrine atrophy was associated with deficiency. Hyperamylasemia has been described during ketoacidosis. Our study aimed at investigating the relationships between CA 19-9 and metabolic control of diabetes. Study was performed on 51 adult consecutive diabetic patients (21 type 1 and 30 type 2), with ketoacidosis or hyperosmolarity (group A, n = 15), poor glycaemic control (group B, n = 19), or good control (group C, n = 17). Serum CA 19-9 and metabolic parameters were evaluated on day 1 and day 30. Analysis of variance showed a very significant global difference between groups for CA 19-9 (p less than 0.0001); group A (66.1 +/- 11.4 u/ml) significantly differed from group B (36.4 +/- 4 u/ml) (p less than 0.01) and group C (22.4 +/- 2.8 u/ml) (p less than 0.001). Simple regression showed a significant correlation between CA 19-9 and fasting blood glucose (r = 0.6, p less than 0.001), plasma creatinine level (r = 0.37, p = 0.01), bicarbonate (r = 0.47, p = 0.001) and HbA 1c (r = 0.33, p = 0.032). The Ca 19-9 decrease on day 30 paralleled the improvement of glycaemic control. We conclude that CA 19-9 in diabetic patients is raised in acute metabolic situations and correlated very well with blood glucose concentration. A careful interpretation of this tumor marker assay is required when screening for pancreatic carcinoma among diabetic patients. CA 19-9 could be a useful and sensitive marker for the severity of exocrine damage and functional cellular disorders following metabolic disturbances in diabetes.

Comparison of three commercial kits and a microbiological assay for the determination of vitamin B12 in serum.

Different methods are available for cobalamin determination in serum. Microbiological and radio ligand binding assays are the most commonly used. Kits involving non-isotopic competitive-binding assay have been recently commercialized. In the present work, cobalamins were determined in 146 patient sera, using four methods: a microbiological method, two no boil radio ligand binding assay kits (Magic B12 FOL (NB) from Ciba-Corning and SimulTRAC SNB No Boil from Becton Dickinson) and a non-isotopic kit with acridinium ester labelled cobalamin (Magic Lite from Ciba-Corning). Median (range) cobalamin concentrations in pmol l-1 were 317 (15-1291) using the microbiological method, 355 (25-3469) using the Magic Lite kit, 355 (35-2312) using the Magic B12 FOL (NB) kit and 380 (37-2021) using the SimulTRAC SNB No Boil kit. The ANOVA test indicated that differences between methods were statistically significant (p < 0.01). Competitive-binding methods gave higher results than the microbiological method. Although correlation coefficients were not excellent (0.88 < r < 0.96), the results obtained with the different methods were generally similar and confirmed that competitive methods are useful for detecting low serum concentration of vitamin B12.

Antibodies to HLA class I alpha1 domain trigger apoptosis of CD40-activated human B lymphocytes.

We analyzed herein whether antibodies to HLA class I alpha1 domain, which trigger apoptosis of activated T cells, may also control the growth/survival of human B lymphocytes. Addition of monoclonal antibody (MoAb) 90 (mouse IgG1) or YTH862 (rat IgG2b) was found to strongly inhibit the proliferation of CD40-activated total tonsil B cells as well as that of purified naive, germinal center, and memory B-cell subsets. This inhibitory effect was not prevented by addition of B-cell tropic factors, such as interleukin-2 (IL-2), IL-4, and IL-10, and was a result of induced B-cell apoptosis as shown by using a TUNEL assay and DNA electrophoresis. In contrast, engagement of another epitope of the alpha1 domain, as well as that of the alpha2 and alpha3 domains by specific anti-HLA class I MoAbs, failed to inhibit DNA synthesis and to induce apoptosis of CD40-activated B cells. As recently reported for acquisition of sensitivity to Fas (APO-1/CD95) -dependent apoptosis, susceptibility to MoAb90-and YTH862-induced death was restricted to CD40-activated B cells, because resting and anti-IgM-activated B cells did not undergo apoptosis after HLA class I engagement. Moreover, ligation of the B-cell receptor protected CD40-activated B cells from both HLA class I- and Fas-mediated growth inhibition and apoptosis. Taken together, these results show that engagement of the alpha1 domain of HLA class I induces apoptotic cell death of CD40-activated, but not of antigen-activated B cells, and would, therefore, suggest a possible role for HLA class I molecules in the control of B-cell homeostasis.

Induction of somatic mutation in a human B cell line in vitro.

Both the B cell-surface trigger(s) and the intracellular molecular mechanism(s) of somatic hypermutation in immunoglobulin (Ig) variable region genes remain unknown, partly because of the lack of a simple and reproducible in vitro model. Here, we show that upon surface immunoglobulin cross-linking followed by co-culture with activated cloned T cells, the Burkitt's lymphoma cell line BL2 is induced to mutate its IgV(H) gene. Repeated activation of BL2 cells increased the frequency of mutation. The in vitro-induced mutations, which do not affect the IgM constant region, are point mutations distributed over the entire V(H)DJ(H) gene segment and do not show evidence of antigen-driven selection.

Fas-independent apoptosis of activated T cells induced by antibodies to the HLA class I alpha1 domain.

In addition to their major function in antigen presentation and natural killer cell activity regulation, HLA class I molecules may modulate T-cell activation and proliferation. Monoclonal antibodies (MoAbs) that recognize distinct epitopes of HLA class I molecules were reported to interfere with T-cell proliferation. We show here that two MoAbs (mouse MoAb90 and rat YTH862) that bind to an epitope of the alpha1 domain of HLA class I heavy chain induce apoptotic cell death of activated, but not resting, peripheral T lymphocytes. Other reference anti-HLA class I antibodies specific for distinct epitopes of the alpha1 (B9.12.1), alpha2 (W6/32), or alpha3 (TP25.99) domains of the heavy chain decreased T-cell proliferation but had little or no apoptotic effect. Apoptosis shown by DNA fragmentation, phosphatidylserine externalization, and decrease of mitochondrial transmembrane potential was observed whatever the type of T-cell activator. Apoptosis did not result from Fas/Fas-L interaction and distinct though partly overlapping populations of activated T cells were susceptible to Fas- and HLA class I-mediated apoptosis, respectively. Induction of apoptosis did not require HLA class I cross-linking inasmuch as it could be observed with monovalent Fab' fragments. The data indicate that MoAb90 and YTH862 directed against the alpha1 domain of HLA class I trigger apoptosis of activated T lymphocytes by a pathway which does not involve Fas-ligand.