Decreased proliferative activity associated with activation markers in patients with alcoholic liver cirrhosis.

Peripheral blood mononuclear cells from 15 patients with alcoholic cirrhosis (AC) and 15 matched healthy controls were tested for their proliferative response to mitogens such as PHA and con A, tumour promoter PMA and OKT3 monoclonal antibody, for their capacity to produce IL-2 and to respond to recombinant IL-2 (rIL-2). The expression of IL-2 receptor (Tac) together with two other activation markers, the receptor for transferrin (T9) and Ia antigen have also been assessed. A profound decrease of proliferative response was observed after stimulation by lectins and PMA. IL-2 production was significantly decreased (0.39 +/- 0.07 versus 0.82 +/- 0.009 units; P less than 0.001) in alcoholic cirrhosis. Resting PBMC of these patients disclosed spontaneous responsiveness to rIL-2 without requiring prestimulation with PHA as observed in healthy subjects. This abnormal response was associated with significantly increased percentages of Tac (19.4 +/- 3.1 versus 5.1 +/- 1.1%; P less than 0.001), T9 (13.6 +/- 3.3 versus 6.6 +/- 1.1%; P = 0.04) and Ia positive cells (21.4 +/- 5.4 versus 11.5 +/- 1.4; P less than 0.05) in alcoholic cirrhosis. Defective proliferative activity and impaired IL-2 production, combined with an increase of lymphocytes bearing T cell activation markers and of rIL-2 responsiveness could represent in vitro correlates for mechanisms leading to immunoregulatory disturbances in cirrhotic patients.

Internalization of human T lymphocyte receptors.

Monoclonal antibodies specific to human T lymphocyte receptors are currently being used to define the biochemical structure of these proteins as well as of functionally distinct cell subsets. Since one of the antibodies (OKT3) recognizing the T3 (CD3) receptor mimics vital physiological processes involved in the activation of the immune system and has been successfully used as a therapeutical agent, we investigated one of the mechanisms underlying this antibody-receptor interaction. Our results show that after binding of OKT3, the complex (OKT3-T3) disappears rapidly from the cell surface. Using electron microscopy, we found that this down-regulation is due to the internalization of the complex. Parallel experiments performed on the T11 (CD2) and T4 (CD4)/AIDS retrovirus receptor indicate that the same mechanism applies for the down-regulation of those molecules. These data suggest that the T3, T11 and T4 receptors have a behavior comparable to other well characterized, hormonal and viral receptors; they provide information on the metabolization pathway of surface receptors and on the possible intracellular penetration of ligands like the HTLV-III/LAV agent in human T lymphocytes.

Chorionic biopsy in management of severe Kell alloimmunization.

Determination of fetal red blood cell Kell antigen is possible on fetal red blood cells obtained at 10 weeks of gestation by chorionic biopsy and may contribute to the management of severe Kell-hemolytic disease.

An immunogold silver staining method for the light microscopic analysis of blood lymphocyte subsets with monoclonal antibodies.

This paper introduces an immunogold silver staining procedure for the identification of human lymphocyte subpopulations in optical microscopy. The described procedure is performed on cells in suspension (peripheral mononuclear cells or blood buffy-coat cells) and uses conventional antilymphocytic monoclonal antibodies (OKT3, OKT4, OKT8, OKIa1) followed by gold-labeled secondary antibodies (GAM G30 or G40) and silver sensitization. It ends with the preparation of permanent records (smears or cytocentrifuge preparations) which are counterstained with standard panoptic Wright or May-Grünwald Giemsa stain. In all cases, the surface antigens appear as numerous black dots on the lymphocytes, with a strong labeling reaction that allows one to clearly distinguish between negative and positive cells. The comparison with immunofluorescence microscopy in normal individuals and in patients indicates that this new immunostaining technic is specific and more sensitive. It has the advantages of using small amounts of blood (1 mL per test), of being rapid (three hours), and of allowing the simultaneous immunophenotypic and morphologic evaluation on smears. This makes it suitable for the analysis of other biologic specimens not already accessible with immunofluorescence. The labeled preparations apparently can be stored indefinitely, and are thus useful for longitudinal studies in the same patient. Finally, the method does not require sophisticated equipment and may be subjected to automated analysis.

Immunogold staining: an alternative method for lymphocyte subset enumeration. Comparison with immunofluorescence microscopy and flow cytometry.

An immunogold staining procedure for light microscopic enumeration of peripheral blood lymphocyte subsets defined by monoclonal antibodies (OKT3, OKT4, OKT8, OKIa1, Leu 1, Leu 4, Leu 2a, Leu 3a, Leu 10, Leu 12, B1) is described. It uses colloidal gold-labeled goat anti-mouse Ig (GAM G40 and GAM G30) as second layer and a methyl-green pyronin counterstain. Performed on small volumes of blood without sophisticated laboratory equipment, this method allows accurate cell type recognition and permanent records, essential for longitudinal observations. By enumerating the gold particles on positively labeled cells, it was shown that the staining reactivity depended on the monoclonal antibody used. Lymphocytes reacting with OKT8 or OKIa1 or B1 exhibited the strongest labeling whereas OKT4+ cells were weakly labeled. When compared with flow cytometry analysis in healthy subjects, the accuracy, precision and sensitivity of both methods were very similar. Similarly, a close correlation (97%) was found between immunogold staining and immunofluorescence microscopy in 35 patients with various diseases suggesting that immunogold staining may be useful in a clinical context.

Active T rosettes, human autologous T rosettes, OKT8 and OKT4 cells, con A-induced suppressive activity, and autoantibodies: clinical correlations.

Correlations between various T-cell subsets: OKT4, OKT8, TEh (human autologous T rosettes), and TEa (active T rosettes) cells and the concanavalin A-induced suppressor function assessed on Con A proliferation have been analyzed in 46 patients with various secondary immunological disorders. The different T subsets and the suppressive activity were also compared in patients with and without autoantibodies. Significant positive correlations were found between T-cell markers TEa-OKT8 and TEh-OKT4. Significant inverse correlations were also found between Con A-induced suppressive activity-OKT4/OKT8 ratios, and TEa-OKT4/OKT8 ratios suggesting that the subpopulations identified by the active T rosettes are probably involved in T immunoregulatory mechanisms. There was no association between the Con A-induced suppressive function or the T markers TEa, OKT4, and OKT8, and the presence of autoantibodies. Only subjects with autoantibodies had a lower percentage of T lymphocytes forming autologous rosettes. These observations emphasize the fact that lack of correlation may exist between markers or function and immune status in some patients.