Selection of down-regulated sequences along the monocytic differentiation of leukemic HL60 cells.

In order to dissect the molecular mechanisms of monocytic differentiation we have developed a subtractive hybridisation method based on a simplified 'representational difference analysis'. We have selected 16 sequences and confirmed their down-regulation along the TPA-induced monocytic differentiation of HL60 cells. Among these sequences we have identified the alpha-tubulin, the TaxREB protein and two ribosomal protein sequences which had not been previously described as differentially expressed. These results add to our knowledge about the molecules implicated along the monocytic differentiation and growth arrest of leukemic cells and provide a first step in the study of their respective roles.

Granulocyte activation and adhesion molecules during hemodialysis with cuprophane and a high-flux biocompatible membrane.

Hemodialysis with complement-activating membranes, such as cuprophane, induces neutropenia and expression of the granulocyte adhesion receptor Mac-1 (CD11b/CD18), while hemodialysis with noncomplement-activating membranes does not. Increased expression of CD11b by neutrophils may mediate cuprophane-induced leukopenia. However, the rebound granulocytosis that follows leukopenia is not fully understood. Ten patients on regular hemodialysis were included in a cross-over study. Hemodialysis was performed for 2 weeks with cuprophane and 2 weeks with polyamide, a high-flux noncomplement-activating membrane. At the end of each period, the following parameters were determined during a hemodialysis session: C5a concentration by enzyme immunoassay and the neutrophil expression of CD11b, LFA-1 (CD11a/CD18), and the antigen recognized by MoF11 (MoF11 Ag), a monoclonal antibody that recognizes activated neutrophils, by immunofluorescence flow cytometry. Hemodialysis with cuprophane induced an increase in C5a concentration and in the expression of CD11b and MoF11 Ag, which were maximal after 15 minutes of hemodialysis, at the nadir of neutropenia. CD11b expression was maintained throughout hemodialysis, despite the reversal of neutropenia. Conversely, after peak expression, C5a and MoF11 Ag decreased as the neutrophil count increased to baseline values. Polyamide hemodialysis did not induce variations in C5a concentration, nor in CD11b and MoF11 Ag expression. CD11a/CD18 expression remained stable during hemodialysis with both membrane types. Neutrophil activation, as determined by MoF11 Ag expression, was correlated with the evolution of neutrophil count and C5a concentration during cuprophane hemodialysis, while CD11b expression was not correlated with neutrophil count throughout dialysis. A decrease in neutrophil activation could explain in part the detachment of neutrophils previously bound to endothelium and, therefore, the reversal of neutropenia.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration that anti-phospholipid auto-antibodies react with both anionic and zwitterionic phospholipids.

The specificity of anti-phospholipid auto-antibodies present in the serum of systemic lupus erythematosus patients towards the phospholipids has been studied by two different methods. The antibodies have been characterized either after affinity purification or by inhibition experiments. Our results clearly demonstrate that anti-phospholipid auto-antibodies recognize all phospholipids, whatever their polar head.

Effect of PGE2 on thymocyte proliferation induced by Con A or IL-4 + PMA.

Prostaglandin E2 (PGE2) is known to inhibit peripheral T-lymphocyte and thymocyte proliferation activated by antigens, mitogens or anti-CD3 antibodies. In this study, we have investigated, the effect of PGE2 on thymocyte proliferation induced by the combination of IL-4 plus PMA. PGE2 inhibits the proliferation of thymocytes activated by ConA, whatever the culture period; in contrast PGE2 shifts the kinetics of thymocyte proliferation after stimulation by IL-4 plus PMA, but does not sustain the proliferation beyond day 3. This effect depends upon cell density, IL-4 concentration and on the time that PGE2 is added to the culture. By use of the cAMP inducer, forskolin, or a cAMP analog, db-cAMP, we observed the same results, PGE2 increases the proliferation of CD8+ corticoresistant thymocytes (CRT) activated by IL-4 plus PMA, but inhibits that of CD4+ CRT. These results suggest that PGE2 can regulate thymocyte proliferation differently according to the activation pathway and the thymic subpopulations.

Flow cytometry analysis of adhesion molecules on human Langerhans cells.

The Langerhans cell (LC) migrates between the epidermis and the regional lymph nodes to present antigens. This migration pattern requires the expression of a changing repertoire of cell-surface molecules. In this work, we have investigated the expression of the adhesion molecules CD 11/CD 18 and CD 58 on LCs. Human epidermal cell suspensions were enriched in LCs (mean enrichment 75%) using a two-step technique including a Ficoll-Hypaque gradient followed by Fc receptor panning with IgG-coated sheep erythrocytes. The number of cells obtained per experiment was 750,000 (extremes 280,000-1,800,000), and the following antibodies were tested on fresh suspensions and/or after 48 hours in culture: BB3 (antithyroglobulin negative control IgG2a), OKT6 (anti CD1a, Ortho), anti HLA-DR (Becton-Dickinson), MHM 24 (anti CD 11a, leukocyte typing workshop n(0)3), MO1 and 44 (anti CD 11b, leukocyte typing workshop n(0)3), anti CD 11c (Immunotech), 60.3 and MHM 23 (anti CD 18, leukocyte typing workshop n(0)2), TS2/9.1.1 (anti CD 58, leukocyte typing workshop n(0)3). We found that amongst CD 11 subunits, only CD 11c was expressed in fresh suspensions, but was weaker than CD 18, and disappeared with culture. CD 58 was not detected in fresh suspensions but appeared after 2 days of culture, confirming earlier work. Thus the LC exhibits cell surface characteristics similar to tissue macrophages (CD 18 and CD 11c) prior to culture. The expression of CD 58 after culture is in accordance with the interaction of LC with CD2 bearing T-lymphocytes during antigen presentation in peripheral lymph-nodes.

HILDA/LIF urinary excretion during acute kidney rejection.

Recently, a new lymphokine called HILDA (human interleukin for DA cells) has been described and cloned. This cytokine, initially described to be produced by alloreactive T lymphocyte clones grown from a rejected human kidney allograft, is identical to other factors termed D-factor, differentiation-inducing factor, differentiation inhibitory activity, hepatocyte-stimulating factor III, and leukemia inhibitory factor. HILDA/LIF induces various effects on neural, hemopoietic, embryonic cells as well as on bone remodeling and acute phase protein synthesis in hepatocyte. In this study we demonstrate the presence of HILDA/LIF in the urine but not in the serum of kidney graft recipients during acute rejection episodes, whereas this lymphokine was detectable neither in the serum nor in the urine of kidney transplanted patients with stable renal function. These data reinforce the notion of a possible role for this lymphokine in the inflammatory and/or the immune response.

Production of epidermal sheets in a serum free culture system: a further appraisal of the role of extracellular calcium.

Rhenwald and Green's technique is currently the standard method for growing stratifying epidermal cell cultures. The serum free system developed in Ham's laboratory (MCDB 153) was designed to grow keratinocyte monolayers in clonogenic conditions. Our aim was to optimize conditions in serum-free MCDB 153 for culturing epidermal sheets from adult normal skin, and to assess the effect of extracellular calcium and temperature on proliferation and differentiation of cultured keratinocytes. Sixteen strains derived from plastic surgery specimens (mean age of donors 37 years; range 5-89) were used. Primary cultures were seeded at an optimal density of 8 x 10(4) cells/cm2 in primary cultures and 10(4) cells/cm2 in secondary cultures in complete medium including EGF, insulin, hydrocortisone and bovine pituitary extract, supplemented with isoleucine, tyrosine, methionine, phenylalanine, tryptophane and histidine. Amino acid (AA) supplementation allows a 5.8-fold increase in cell counts at confluency and monolayers with densely packed cells are obtained. In AA supplemented cultures, confluency is obtained in 16 +/- 3 days in primary cultures and in 13 +/- 0.5 days at first passages. Switches to 1.1 mM calcium at first or second passages resulted in a significant increase in cell counts (P less than 0.001), when compared with AA supplemented low calcium cultures. Low temperature/low calcium cultures resulted in a 50% decrease in cell counts. Low temperature/high calcium cultures gave similar cell counts as the 37 degrees C controls. AA and calcium supplemented cultures were evaluated for differentiation markers: involucrin expression was increased, keratins 5, 6, 14, 17 were expressed, and the sheets were 6-10 layers thick by electron microscopy, with keratohyalin granules and cornified envelopes appearing at layers 3-6 (from basal layer). Dispase treatment allowed an easy detachment of these sheets. These results show that the culture medium MCDB 153 can be adapted without serum supplementation to batch culture of human adult keratinocytes to produce epidermal sheets suitable for grafting. They also indicate that extracellular calcium in physiological range of concentration is not a sufficient signal for growth arrest when other growth conditions are optimized.

Fatty acids rendered immunogenic.

A method allowing the covalent binding of fatty acids to a carrier was developed. The absence of absorbed fatty acids was controlled by HPTLC. Rabbits were immunized with these immunogens. The antibodies obtained were studied by ELISA and were shown to be directed against the fatty acyl chain, whatever its length.

In vitro culture of hybridoma cells in agarose beads producing antibody secretion for two weeks.

A new process for embedding cells in agarose is described. Beads were obtained by extruding an ultralow gelling temperature agarose solution in a capillary containing a hydrophobic medium flowthrough. The toxicity of the procedure has been evaluated by monitoring the energy status of agarose-embedded C(6) glioma cells with (31)P nuclear magnetic resonance (NMR). Suspension and microbead cultures of hybridoma cell line were compared. In suspension culture the number of cells and the antibody concentrations increased for 5 days before the stationary phase began, when the cultures were stopped. In agarose bead cultures, the gel provided an enormous support surface area (50 m(2)/ mL of gel). It was possible to seed 20-fold more cells. The gel pressure modified the proliferative process and antibody pattern secretion. In particular, the antibodies could be harvested for two weeks.

Effect of LTB4 on the inhibition of natural cytotoxic activity by PGE2.

NK activity is regulated by arachidonic acid metabolites. More precisely PGE2 and LTB4 decreases and increases respectively non-MHC-restricted cytotoxicity in humans. We have observed similar data in mice since NK activity was inhibited by PGE2 (10(-6) to 10(-8) M) and enhanced by LTB4 (10(-8) to 10(-12) M). On the other hand when PGE2 and LTB4 were combined during the same assay the lysis percentage was smaller than the one which was induced by PGE2 alone. Because PGE2 increases intracellular cyclic AMP and that LTB4 augments cyclic GMP we used a cAMP inducer (forskolin) and a cGMP analogue (8 Br-cGMP) instead of eicosanoids and we observed similar data (i.e., a decrease of natural killing) as when PGE2 was combined with LTB4. When splenocytes are cultured for 1-4 days alone, cytotoxic activity decreases unless they are cultured in the presence of indomethacin. Cytotoxic activity of spleen cells cultured in the presence of PGE2 or LTB4 is respectively decreased or increased. However, splenocytes that were cultured alone for at least 24 hr were no longer sensitive to inhibition by PGE2 but were still PGE2-sensitive when cultured in the presence of LTB4.

[Seroepidemiologic study of sexually transmitted diseases caused by Chlamydia trachomatis in Casablanca (Morocco)]. / Enquête séro-épidémiologique sur les MST a Chlamydia trachomatis a Casablanca (Maroc).

Screening 579 sera specimens obtained in Casablanca (Morocco) we have performed a retrospective seroepidemiological survey with regard to the prevalence of Chlamydia trachomatis involved in genital tract infections. The surveyed populations are divided into 3 groups: 177 patients affected with sexually transmitted diseases (STD), 319 reference patients, 83 maghrebian patients affected with STD but living in France. The study was performed by the indirect microimmunofluorescence technique. The prevalence of Chlamydia trachomatis ranging from 50% in the moroccan reference groupe to about 70% among patients with STD both in Morocco and Bordeaux indicates that Chlamydia trachomatis takes a significant part in STD observed in moroccan populations.

In vitro study of platelets and circulating mononuclear cells of subjects presenting an intolerance to aspirin.

Aspirine-sensitive asthma (ASA) is a disease defined only by clinical criteria. It is an intrinsic asthma related to a hypersensitivity to aspirin. The illness is linked to abnormalities in platelet and macrophage arachidonic acid metabolism. We assessed in vitro the platelet chemoluminescence (CL) and lymphocyte proliferative response of ASA patients. We observed that platelets from patients and control do not generate any CL in the presence of aspirin. Concerning the proliferative response of lymphocytes, the in vitro effect of aspirin depends upon the origin of the lymphocytes tested. Thus aspirin clearly enhances the proliferative response of lymphocytes from normal subjects but diminishes the thymidine uptake by lymphocytes from ASA patients. This discrepancy in the in vitro response of lymphocytes from normal subjects and patients might be useful for in vitro diagnosis of ASA.

Synthesis of a monoclonal antibody-indium-111-porphyrin conjugate.

Antibodies were labelled with indium-111 with a view to their use in the radio-immunodetection of cancers. The covalent coupling between indium-111 porphyrin and monoclonal antibodies (IgG and F(ab')2 fragment) was achieved using the ester activated method [N-hydroxy-succinimide/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide]. After purification, this provided conjugated with specific activities of 6 muCi/micrograms Mab (9.3 molecules per Mab) or 1 muCi/microgram (F(ab')2 fragment (1.5 molecule per F(ab')2). ELISA procedures suggested the full retention of immunoreactivity by the radiolabelled antibodies.

Improvement of leucocytic Na+ K+ pump activity in uremic patients on low protein diet.

Leucocytic Na+ K+ pump activity was assessed in 20 patients with advanced renal failure. Na+ K(+)-ATPase activity was reduced when compared with the values obtained from normal subjects (101.8 +/- 48.6 versus 165.13 +/- 8.9 microM of Pi hr-1.g-1; P less than 0.001) and the mean 86Rb uptake by U 937 cells was depressed by 38% after the addition of patients' sera. Subsequently, patients were put on a diet providing 0.3 g protein/kg body weight daily and supplemented with ketoacids. After three months of dietary treatment Na+ K(+)-ATPase activity increased to 142 +/- 48.3 (P less than 0.01) and reached normal values at the sixth month (162.8 +/- 54.70 microM of Pi hr-1.g-1; P less than 0.001) whereas 86Rb uptake increased by 23 percent when compared to initial values. These data suggest that among the different mechanisms which have been advanced to explain the defects in the Na+ pump observed in uremic patients, circulating inhibitors deriving from alimentary protein intake may affect cation transport.

Autoantibodies against all the phospholipids: a comparative systematic study with systemic lupus erythematosus and healthy sera.

The sera of systemic lupus erythematosus patients were tested by ELISA for the presence of autoantibodies against all the phospholipids: cardiolipin, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylserine. The quantity of phospholipid coated (in comparison with that initially deposited) on the microtitration plates was precisely evaluated in order to determine if the results (absorbance values) obtained for each phospholipid could be compared directly. The systemic lupus erythematosus sera tested gave positive results for all the phospholipids. The highest level of autoantibodies was observed with phosphatidic acid followed by phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine. The sera seemed to contain antibodies directed either against all the 8 phospholipids tested or more specifically against one or two phospholipids. The results were compared with those obtained with 17 healthy sera. Much lower values were obtained for the sera of healthy subjects, the majority of which showed a weak binding, similar for all the phospholipids. These results suggest that the anti-phospholipid autoantibodies present in systemic lupus erythematosus sera are significantly higher than those of healthy subjects. It is concluded that in the investigation of anti-phospholipid antibodies, tests should be carried out for all the phospholipids.

Treatment of leg ulcers with an allogeneic cultured-keratinocyte-collagen dressing.

A living cellular allogeneic dressing made up of cultured keratinocytes adhering to a collagen film was used to treat 20 leg ulcers of various aetiologies in 16 patients. A reduction in pain was noted in 80% of cases, and promotion of granulation tissue in the ulcer bed in 70% of cases. In 10 patients, epithelialization of 71 +/- 29% of the ulcer was noted at Day 30.

Biocompatibility of polyacrylamide microcapsules implanted in peritoneal cavity or spleen of the rat. Effect on various inflammatory reactions in vitro.

The biocompatibility of polyacrylamide microcapsules was investigated by implanting microcapsules in the peritoneal cavity or the spleen of rats. The capsules were retrieved every four weeks for twenty weeks. They remained isolated and free in the peritoneal cavity, but led to a slight inflammatory reaction in the spleen. These results were confirmed, in vitro, by determinations of chemiluminescence and interleukin I levels, and by evaluation of attachment and growth of human fibroblasts. The microcapsules did not enhance these inflammatory reactions, and they were a poor support for cellular proliferation.

Effect of a low-protein diet on chemiluminescence production by leukocytes from uremic patients.

Chemiluminescence (CL) emission from leukocytes was studied in 20 uremic patients after ingestion of opsonized zymosan. CL production was impaired when compared with control groups. Six months after a low-protein, low-phosphorus diet supplemented with essential amino acids and ketoanalogues (SD) was started, a significant improvement in CL production was observed. SD may be expected to decrease the susceptibility of uremic patients to bacterial infections.