Changes in the subunit distribution of prosomes (MCP-proteasomes) during the differentiation of human leukemic cells.

The subunit composition of cell-internal and surface prosomes during phorbol myristate acetate (PMA)-induced differentiation of human leukemic T lymphocytes (CCRF-CEM cell line) was studied in relation to clusters of differentiation (CD) markers. PMA inhibited cell growth and decreased the amounts of CD1a and CD4 while CD3, CD8, CD25, CD45, CD57 and MHCI increased it; the p53 anti-oncogene increased while actin levels remained constant. Cells incubated with the inducer PMA for 3 days and placed in fresh inhibitor-free medium resumed growth at a low rate, while the CD values slowly reverted to those of the initial phenotype.

Proteasome (prosome) subunit variations during the differentiation of myeloid U937 cells.

20S proteasomes (prosomes/multicatalytic proteinase) are protein particles built of 28 subunits in variable composition. We studied the changes in proteasome subunit composition during the differentiation of U937 cells induced by phorbol-myristate-acetate or retinoic acid plus 1,25-dihydroxy-cholecalciferol by western blot, flow cytometry and immuno-fluoresence. p25K (C3), p27K (IOTA) and p30/33K (C2) subunits were detected in both the nucleus and cytoplasm of undifferentiated cells.

The oxidative burst triggered by Salmonella typhimurium in differentiated U937 cells requires complement and a complete bacterial lipopolysaccharide.

The bacterial and serum factors involved in the oxidative response triggered by Salmonella typhimurium in differentiated U937 cells were investigated. Complement activation was shown to be required, using sera deficient in complement factors. An original dot-blot technique was developed to study the activation of complement by either bacteria or purified lipopolysaccharide (LPS).

Dimethyl sulfoxide-induced apoptosis in human leukemic U937 cells.

Dimethyl sulfoxide (DMSO), which induces differentiation of myeloid cells, was found to cause apoptosis in human leukemic U937 cells. Apoptosis was assessed by DNA electrophoresis and flow cytometry. The time needed to induce apoptosis varied from a few hours to 2-3 days, depending on the concentration of DMSO used.

Differentiated U937 cells and human monocytes exhibit a differential production of extracellular oxygen species: O2.- excretion versus H2O2 diffusion.

The nature and the localization of the oxidative response triggered by different stimuli in either differentiated U937 cells and peripheral blood-derived human monocytes was investigated using luminometric and cytofluorometric techniques. Differentiated U937 cells essentially produced extracellular superoxide anion (O2.-), whatever the stimulus used.

Infection of differentiated U937 cells by Salmonella typhimurium: absence of correlation between oxidative burst and antimicrobial defence.

The human histiocytic lymphoma cell line U937 can be induced to differentiate along the monocyte/macrophage pathway by either phorbol myristate acetate (PMA) or by the combination of retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (VD). U937 cells treated with either PMA or RA/VD were able to phagocytose Salmonella typhimurium in the presence of non-immune human serum. However, only cells differentiated by RA/VD were capable of developing an oxidative metabolic burst in response to infection.

Rapid fluorometric measurement of the intra-cellular concentration of ciprofloxacin in mouse peritoneal macrophages.

A simple fluorometric assay requiring only a single sample of cells to determine the number of cells, from the DNA linked to the fluorochrome 4',6-diamidino-2-phenylindol (DAPI), and the uptake of ciprofloxacin, a natural fluorescent quinolone is described. Mouse peritoneal macrophages were found to concentrate ciprofloxacin up to 12.7 (+/- 1.

[Use of flow cytometry in the study of a non-specific immunodeficiency in children. A case].

Using flow cytometry, we explored a case of nonspecific immunodeficiency in a seven month old girl with repeated infections. This method showed evidence of granulocyte phagocytosis and oxidative metabolism abnormalities suggesting the diagnosis of a variant form of chronic granulomatous disease (CGD). Findings also showed that flow cytometry can be useful to study phagocytic cells during the neonatal period as it allows rapid multiparametric analysis with a very small amount of blood.

Suspended mouse peritoneal macrophages. Preparation and properties.

Since macrophages (MPH) are able to adhere firmly to solid surfaces, the recovery of viable and functional MPH has proven to be extremely difficult. We have developed a simple method using agarose coating for preparing MPH and culturing the cells in suspension. Their properties were tested over 72 h.

Cellular responses to "brucelline INRA" during the experimental brucellosis of the mouse.

"Brucelline INRA" is an aqueous extract of a rough strain of Brucella melitensis that is used to test cutaneous delayed hypersensitivity in man and animals. The cellular response of mice (either normal, or sensitized by a previous brucella infection) was investigated with local implanted "cell-traps" and by immunological testing in vitro. Brucelline markedly reduced the number of polymorphonuclear cells in the local infiltrate of sensitized animals during the early, nonspecific phase of the response.

Kinetics of the uptake of rifampicin and tetracycline into mouse macrophages. In vitro study of the early stages.

The in vitro uptake by mouse peritoneal macrophages, of chlortetracycline (by fluorescence microscopy) and of tetracycline and rifampicin (by scintillation spectrometry of radioactive antibiotics) has been studied over a six hours period, using various concentrations of the antibiotics, close to the therapeutic concentrations. The incidence of the conditions of the assays, especially that of the use of heterologous serum for the cultivation of cells, has been investigated; a medium supplemented with homologous serum at low concentration has been devised with the technique. The uptake of these antibiotics was a three-phases process suggesting the superposition to a passive diffusion of either an active incorporation, or a restriction of the outflow (perhaps associated).

Ultrastructural localization of NADPH-oxidase activity in murine peritoneal macrophages during phagocytosis of Brucella. Correlation with the production of superoxide anions.

The localization of the enzyme NADPH oxidase in mouse peritoneal macrophages unstimulated or after phagocytosis of Brucella suis was investigated by electron microscopy in normal mice and mice immunized against B. suis. The enzyme was clearly visualized on mitochondrial cristae, smooth endoplasmic reticulum, and the plasma membrane; its activity correlated mainly with the state of the endoplasmic reticulum which itself reflected macrophage activation.

Modifying factors on phagocytosis of polymorphonuclear allergic subjects.

Supernatants from pollen-activated cultures of mononuclear (MN) cells obtained from two subjects allergic to gramineae pollen were tested for their effect on phagocytic activity of human polymorphonuclear leucocytes (PMN). An enhancement of phagocytosis was observed. Similarly prepared supernatants from MN cells of non-allergic control subjects did not affect PMN phagocytic activity.

In vitro immunosuppressive activity of gram-positive (Streptococcus pyogenes) peptidoglycan on mouse lymphocytes.

A peptidoglycan fraction prepared from group-A streptococcus was assayed for in vitro mitogenicity on mouse lymphocytes. This fraction reduced considerably the uptake of radioactive thymidine both on unstimulated cell suspensions and on suspensions stimulated by T(PHA)- or B(LPS)-mitogens. The immunosuppression was induced by relatively moderate doses of the fraction, and was dose-dependent.

[Attempts of ultrastructural and biochemical characterisation of cell wall deficient "Brucella" (L forms) (author's transl)].

In previous studies the in vivo conversion of Brucella suis to L-form state was put in evidence. The L forms isolated from mouse spleen had original structural aspects in common: the absence of the cell wall layer and the extracellular multilayer "membranous" structures. The biological characterization of these L forms and the preliminary identification of specific chemical markers of the bacterial envelope is reported in the present study, performed with the stable L forms well-growing in the liquid media.

Isolation of L-forms from the spleens of Brucella suis-infected, penicillin-treated mice.

Previous attempts to obtain in vitro wall-deficient stable L-forms of various strains of Brucella have failed because the obtained spheroplasts revert quickly to bacterial form. Here, we report the isolation of L-forms from mice infected with a B. suis strain type 1 and treated with penicillin.

Cellular responses of the mouse to the peptidoglycan of a gram-positive bacterium (Streptococcus pyogenes).

The cellular responses and the stimulation of the reticulo-macrophagic system induced in the mouse by a purified bacterial peptidoglycan (PGL) as previously described, were studied by the changes in the peritoneal cytology, the macrophage-migration-inhibition test and the clearance of colloidal carbon. PGL was submitted to chemical and immunochemical characterization and was shown to be substantially free of contamination by polysaccharides, phospholipids, teichoic acid and nucleic acids, but to contain a detectable amount of peptide contaminants; N-acetylglucosamine and the tetrapeptide (with terminal D-alanine) were shown to be the main antigenic determinants. This substance had no action on polymorphonuclear leucocytes but induced an inhibition of the migration of macrophages.

Response of the lymphoid organs of the mouse to the peptidoglycan of a gram-positive bacterium (Streptococcus pyogenes).

The response of the lymphoid organs of the mouse to the injection of a Gram-positive (Streptococcus pyogenes) bacterial peptidoglycan (PGL) has been investigated by several complementary techniques. Special cares were brought to: a) the extraction mode and the characterization of the bacterial fraction, b) the sanitary and microbiological status of the mice, and c) the lack of biologically active contaminants, even at a trace level. In these conditions, the local reaction at the site of inoculation was very moderate, transient and not very distinctive.

Loss of plasmid-mediated resistance after conversion of a group B streptococcus strain to a stable cell wall deficient variant.

A group B streptococcus strain carrying plasmid DNA determining resistance to several drugs was converted by penicillin to cell wall (CW) defective and then to CW deficient variants (L-forms). The stable CW deficient variants became susceptible to antibiotics in study. Dye-buoyant density analysis of the DNA of CW deficient variants showed that the loss of antibiotic resistance was associated with the loss of extrachromosomal DNA.