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Biomedical subjects

D Blanchard

Publications and source records attributed to D Blanchard.

At least 109 records · Page 6Linked to original sources
PubMed1995

CD40-activated human naive surface IgD+ B cells produce IgG2 in response to activated T-cell supernatant.

Human IgG2 is an isotype associated with immune responses to carbohydrates. While interleukin-10 (IL-10) induced CD40-activated naive surface (s)IgD+ human B cells to secrete IgG1 and IgG3, none of 20 recombinant cytokines tested alone, or in combination with IL-10, was able to induce these cells to produce IgG2. This was not due to a specific inability of these sIgD+ B cells, as they could be induced to secrete microgram amounts of IgG2, as well as the three other IgG subclasses, when cultured with an anti-CD3-activated CD4+ T-cell clone. The supernatant of this activated CD4+ T-cell clone contained a soluble factor(s) able to induce the secretion of IgG2 by CD40-activated sIgD+ B cells. Following activation, blood T cells also produced a factor(s) inducing CD40-activated naive B cells to secrete IgG2. This CD4+ T-cell clone will thus permit us ultimately to define the presently uncharacterized cytokine(s) inducing naive B cells to secrete IgG2. This will provide a new insight for the study of immunodeficiencies involving a selective defect in IgG2.

B-Lymphocytes↗
PubMed1994

Protection of rat endothelial cells from primate complement-mediated lysis by expression of human CD59 and/or decay-accelerating factor.

The present study analyzed the ability of human decay-accelerating factor (DAF) and CD59 to protect rat endothelial cell (EC) clones from human and primate complement-mediated lysis. By flow cytometry and Scatchard analysis, we show that human DAF and/or CD59 cDNAs under the transcriptional control of elongation factor 1-alpha promoter were expressed at levels similar to or higher than that of a human EC line. Human DAF and CD59 were released from the surface of transfected rat cells by phosphatidylinositol phospholipase C, demonstrating that the two molecules were linked to the cell membrane by means of a glycolipid anchor. The functional activity of the two human C regulatory proteins expressed on rat EC lines was studied using an in vitro assay of C-dependent cytotoxic in which rat EC were incubated with human or nonhuman primate sera as sources of xenogeneic natural antibodies and C. We demonstrate that human and monkey xenogeneic natural antibodies bind to rat cells and induce lysis by a C-dependent mechanism involving mainly the C direct activation pathway. Our data indicate that human DAF and CD59, expressed either alone or in combination, abrogated all EC cytotoxicity, even in the presence of 50% human serum. This protective phenotype was correlated with decreased membrane attack complex (CD59 and/or DAF transfectants) and C3 deposition (DAF transfectants) on EC surface. Antibodies against the transfected molecules abolished the protection against C-mediated lysis.

Animals↗
PubMed1994

Variation in fetal hemoglobin parameters and predicted hemoglobin S polymerization in sickle cell children in the first two years of life: Parisian Prospective Study on Sickle Cell Disease.

Intracellular hemoglobin S (HbS) polymerization is most likely to be the primary determinant of the clinical and biologic manifestations of sickle cell disease (SCD). Fetal hemoglobin (HbF) does not enter the HbS polymer and its intracellular expression in sickle erythrocytes inhibits polymerization. HbF levels, high at birth but decreasing thereafter, protect the newborn from the clinical manifestations of this hemoglobinopathy. We have measured the sequential changes in HbF, F reticulocytes, and F cells in the first 2 years of life in 25 children with SCD and compared the results with those obtained in 30 normal children (AA). We have also calculated HbF per F cell (F/F cell), the preferential survival of F cells versus non-F cells, as measured by the ratio F cells versus F reticulocytes (FC/FR) and polymer tendency at 40% and 70% oxygen saturation. HbF levels decreased from about 80.4% +/- 4.0% at birth to 9.2% +/- 2.9% at 24 months. During this time, we observed a regular decrease of the F reticulocytes and the F cells. The kinetics of the decline of F/F cell was comparable with the decline of HbF, rapid from birth (mean, 27.0 +/- 3.6 pg) to 12 months of age (mean, 8.5 +/- 1.5 pg) and then slower from 12 to 24 months of age (mean, 6.2 +/- 1.0 pg) in the SCD children. In the AA children, the decrease in HbF, due to changes in both numbers of F cells and F/F cell, was more precipitous, reaching steady-state levels by 10 months of age. Calculated values for mean polymer tendency in the F-cell population showed that polymerization should begin to occur at 40% oxygen saturation at about 3 months and increase progressively with age, whereas polymerization at 70% oxygen saturation would not occur until about 24 months. These values correspond to HbF levels of 50.8% +/- 10.8% and 9.2% +/- 2.9%, respectively, and F/F cell levels of 15.6 +/- 4.5 pg and 6.2 +/- 1.0 pg, respectively. In the non--F-cell population, polymerization was expected at birth at both oxygen saturation values. Three individuals had significantly greater predicted polymerization tendency than the remainder of the group because of early decreases in HbF. These individuals in particular, the remainder of the cohort, as well as other recruited newborns, will be studied prospectively to ascertain the relationship among hematologic parameters, which determine polymerization tendency and the various clinical manifestations of SCD.

Anemia, Sickle Cell↗
PubMed1994

Human natural antibodies to porcine platelets.

The specificity of human natural antibodies directed against blood cells from pigs was investigated by ELISA and immunoblotting. Both IgG and IgM were identified as xenoantibodies reacting with pig platelets adsorbed to microplates. The antibodies could be absorbed on platelets as well as on RBC, suggesting that the corresponding antigens are expressed on the surface of a variety of cells. Galactose (20 mM) and melibiose (10 mM) partially inhibited (approximately 50%) the binding of antibodies to platelets, whereas lactose and cellobiose (300 mM) did not. On immunoblots, platelet glycoproteins of 115, 125, 135, 180, and 210 kDa were specifically revealed with human sera diluted 1/20. In contrast with the results obtained by ELISA, xenoantibodies reactive with blotted glycoproteins were of the IgM class and the binding was not significantly inhibited by galactose or melibiose. "Anti-Gal" antibodies, purified from human sera by affinity chromatography on a melibiose-Sepharose immunoabsorbent, represented only a minor portion of the antibodies reactive with porcine platelets. Purified anti-Gal antibodies bound to the 115- and 135-kDa components, whereas the antibodies in the nonretained fraction revealed the 125-kDa molecule. As deduced from these data, human serum contains natural antibodies of both IgG and IgM classes directed to several porcine antigens. Gal-reactive structures were identified on the 115- and 135-kDa platelet glycoproteins, which might be homologous to their counterpart on endothelial cells. Also, the present work suggests that a majority of the natural antibodies reacted with other unidentified structures.

Animals↗
PubMed1994

Activated CD4+ T cells induce CD40-dependent proliferation of human B cell precursors.

Anti-CD3-activated human CD4+ T cell clones were found to induce proliferation of CD10+, CD19+, surface(s) Ig- B cell precursors (BCP) isolated from human fetal bone marrow. The great majority of the B lineage cells recovered in cocultures of BCP and activated T cells displayed a BCP phenotype (Ig- or cytoplasmic mu+ and kappa/lambda-), including most of the cycling cells, indicating that the cultures do not favor a transition to mature B cells. Supernatants of activated T cells were ineffective in inducing BCP proliferation, indicating the necessity of close association with stimulator cells. In line with this finding, the CD40 molecule was found to represent an important component of the cocultures, as BCP proliferation was strongly inhibited by soluble anti-CD40 antibody. In addition, CD4+ T cell clones from a hyper-IgM patient expressing a truncated CD40 ligand (CD40-L) failed to induce BCP proliferation. Finally, a combination of cytokines (IL-2, IL-3, IL-7, and IL-10) enhanced the observed T cell-dependent BCP proliferation, but could not substitute for the deficient CD40-L. Taken together, our data demonstrate that CD4+ T cells exert a stimulatory effect on in vitro B human lymphopoiesis via the CD40 pathway. The present results suggest that T cells may play an important role in regulating B cell ontogeny in the bone marrow.

Antigens, CD↗
PubMed1994

Role of CD40 antigen and interleukin-2 in T cell-dependent human B lymphocyte growth.

In the present study, we examined the participation of CD40 ligand (L)-CD40 interaction in T cell-dependent B cell responses. To this end, purified B lymphocytes were cultured over irradiated CD4+ cloned T cells activated with immobilized anti-CD3 antibody. The anti-CD40 mAb 89 strongly blocked, in a specific fashion, both proliferation and Ig secretion of tonsil B cells. Interestingly, proliferation of surface (s)IgD+ B cell was significantly less inhibited by anti-CD40 than that of sIgD- cells. Preactivated T cells induced B cells to grow and secrete immunoglobulins preferentially in response to IL-2. This contrasts with the CD40 system where B cells are essentially responsive to IL-4 and IL-10 but not to IL-2 alone. Collectively, these data indicate that CD40L-CD40 interaction plays an important role in IL-2 mediated T cell-dependent B cell responses. However, the activation of a subset of sIgD+ cells may be independent of this interaction.

Antigens, CD↗
PubMed1994

B cells regulate expression of CD40 ligand on activated T cells by lowering the mRNA level and through the release of soluble CD40.

The expression of CD40 ligand (CD40L) on activated T cells (CD4+ T cell clone MT9) is diminished when the T cells are cultured in the presence of B cells. This effect, observed both with normal tonsil B cells and with the B cell line JY, was detected after 6 h and sustained at least until 18 h of co-culture. Analysis of mRNA showed that CD40L mRNA levels were not modified after 6 h, but were significantly down-regulated after 18 h of co-culture with B cells. Although CD40L expression could not be detected by a CD40-Fc chimera, the molecule was still expressed at the membrane as shown with a polyclonal antiserum against CD40L (anti-TRAP). In addition, T cells activated in the presence of B cells were stained by a polyclonal antiserum against CD40, without the appearance of CD40 mRNA. These results indicated that a soluble form of CD40 (sCD40) bound to the expressed CD40L on T cells. The existence of sCD40 was confirmed by detection of sCD40 in B cell supernatants using a specific enzyme-linked immunosorbent assay. Collectively, these data show that B cells can regulate the expression of CD40L on activated T cells at least by two different mechanisms.

Antigens, CD↗
PubMed1994

Influence of the size of the polar head of non-ionic detergents on membrane proteins immunoaffinity purification.

Nonionic polyoxyethylene type detergents (CxEy) are widely used to solubilize and purify membrane proteins. The detergent hydrophobic moiety (Cx) replaces phospholipids at exposed hydrophobic regions of the membrane proteins. During chromatography on an immobilized anti-Kell antibody to purify Kell protein (an integral erythrocyte protein), it was observed that the size of the polar head of an non ionic detergent added to the mobile phase appeared to influence the interaction of the detergent-protein complex with the immobilized antibody. Further studies were performed using another erythrocyte membrane protein, Glycophorin C and three anti-GPC monoclonal antibodies directed against three epitopes of the extracytoplasmic domain of the protein. The interaction of GPC with the three Protein A-coupled monoclonal antibodies was studied in the presence of three detergents C12E<9>, C13E<15> and C12E<23>. It was observed in batch mode and in column chromatography experiments that the adsorption of GPC to the immunoaffinity supports decreased as the size of the detergent polar head increased. Thus, the polyoxyethylene chain of a detergent might prevent the interaction of the detergent-protein complex with the immobilized antibody.

Animals↗
PubMed1994

Serological analysis by A60 antigen ELISA and BCG immunoblotting in domestic carnivores experimentally vaccinated with mycobacterium bovis BCG.

The serological response to Mycobacterium bovis BCG vaccination was studied in 3 cats, 3 dogs and 3 puppies. The animals received two doses of 0.1 mg of BCG and were studied over a period of 8 months simultaneously by ELISA with antigen A60 (Anda Biologicals, Strasbourg, France) and immunoblotting with BCG. The two methods detected an increase of antibodies at 3 or 5 weeks. In the cats, specific antibody titer remained high and stable for more than one year, in the dogs they diminished quickly within 23 weeks. Carnivores elicit a serological response against specific protein antigens in the bands corresponding to 18 and 25kDa, 30 and 45kDa, and especially 35 and 42kDa bands, and to minor bands at higher molecular weight. Non-specific bands at 50-55 kDa and 70 kDa, assigned to heat shock proteins, were enhanced by BCG vaccination. Cat immunosera recognized on A60 immunoblots the specific homologous bands at 20-25 kDa and 32 kDa, but also two other dominant bands: one was partially specific (65 kDa) and the other was absent from the M. bovis and M. paratuberculosis profiles. The suitability of A60 ELISA for detection of mycobacterial infection in carnivores may be highlighted by immunoblot analysis.

Animals↗
PubMed1994

Epidural anesthesia impairs both central and peripheral thermoregulatory control during general anesthesia.

BACKGROUND: The authors tested the hypotheses that: (1) the vasoconstriction threshold during combined epidural/general anesthesia is less than that during general anesthesia alone; and (2) after vasoconstriction, core cooling rates during combined epidural/general anesthesia are greater than those during general anesthesia alone. Vasoconstriction thresholds and heat balance were evaluated under controlled circumstances in volunteers, whereas the clinical importance of intraoperative thermoregulatory vasoconstriction was evaluated in patients. METHODS: Five volunteers were each evaluated twice. On one of the randomly ordered days, epidural anesthesia (approximately T9 dermatomal level) was induced and maintained with 2-chloroprocaine. On both study days, general anesthesia was induced and maintained with isoflurane (0.7% end-tidal concentration), and core hypothermia was induced by surface cooling and continued for at least 1 h after fingertip vasoconstriction was observed. Patients undergoing colorectal surgery were randomly assigned to combined epidural/enflurane anesthesia (n = 13) or enflurane alone (n = 13). In appropriate patients, epidural anesthesia was maintained by an infusion of bupivacaine. The core temperature that triggered fingertip vasoconstriction identified the threshold. RESULTS: In the volunteers, the vasoconstriction threshold was 36.0 +/- 0.2 degrees C during isoflurane anesthesia alone, but significantly less, 35.1 +/- 0.7 degrees C, during combined epidural/isoflurane anesthesia. Cutaneous heat loss and the rates of core cooling were similar 30 min before vasoconstriction with and without epidural anesthesia. In the 30 min after vasoconstriction, heat loss decreased 33 +/- 13 W when the volunteers were given isoflurane alone, but only 8 +/- 16 W during combined epidural/isoflurane anesthesia. Similarly, the core cooling rates in the 30 min after vasoconstriction were significantly greater during combined epidural/isoflurane anesthesia (0.8 +/- 0.2 degrees C/h) than during isoflurane alone (0.2 +/- 0.1 degrees C/h). In the patients, end-tidal enflurane concentrations were slightly, but significantly, less in the patients given combined epidural/enflurane anesthesia (0.6 +/- 0.2% vs. 0.8 +/- 0.2%). Nonetheless, the vasoconstriction threshold was 34.5 +/- 0.6 degrees C in the epidural/enflurane group, which was significantly less than that in the other patients, 35.6 +/- 0.8 degrees C. When the study ended after 3 h of anesthesia, patients given combined epidural/enflurane anesthesia were 1.2 degrees C more hypothermic than those given general anesthesia alone. The rate of core cooling during the last hour of the study was 0.4 +/- 0.2 degrees C/h during combined epidural/enflurane anesthesia, but only 0.1 +/- 0.3 degrees C/h during enflurane alone. CONCLUSIONS: These data indicate that epidural anesthesia reduces the vasoconstriction threshold during general anesthesia. Furthermore, the markedly reduced rate of core cooling during general anesthesia alone illustrates the importance of leg vasoconstriction in maintaining core temperature.

Adolescent↗
PubMed1994

Characterization of new murine monoclonal antibodies directed against glycophorins C and D.

Six new murine monoclonal antibodies (mAbs) directed to the erythrocyte membrane glycophorins C (GPC) and D (GPD) were obtained from splenocytes of different BALB/c mice immunized with human red blood cells, and fully characterized. The mAbs were selected by agglutination tests with control and Gerbich-negative cells, and by immunoblotting analysis. They showed specificity for the N-terminal domain(s) of GPC (and GPD) and were classified into three categories by competitive analysis using 125I-labelled antibodies and real-time biospecific interaction. The first group (NaM10-7G11, NaM70-1G4 and NaM77-7B6) compete for epitope(s) located at the N-terminal portion of GPC. Agglutination-inhibition tests revealed that the 7G11 epitope involves the amino group of Met1 and sialic acid residue(s) whereas the 1G4 and 7B6 epitopes contain O-glycans. NaM89-2G11 belongs to a second group; its epitope is located in a region including Glu17, Asp19 and (an) O-glycan(s). The third group comprises mAbs NaM19-3C4 and NaM98-3C1 which bind to both GPC and GPD in proximity of the binding site of human anti-Ge:3 antibodies. In addition, mAb 3C4 (anti-GPC/GPD) was found to bind to approximately 125,000 sites per red cell. Considering that the ratio of the GPC to GPD is about 3-4 to 1, the number of GPC and GPD molecules was estimated as 95,000 and 35,000, respectively.

Animals↗
PubMed1994

Production of a new murine monoclonal antibody with Fy6 specificity and characterization of the immunopurified N-glycosylated Duffy-active molecule.

A murine monoclonal antibody (mAb i3A; IgG1, kappa light chain) was obtained using human red blood cells as immunogen. The antibody showed Fy6 specificity since it agglutinated all but Fy(a-b-)-untreated red cells and failed to agglutinate chymotrypsin-treated cells. An erythrocyte membrane protein of 42-46 kD was revealed as the major component recognized by the antibody on immunoblots. The antibody also bound to 92- to 95- and 200-kD proteins, tentatively identified as oligomers of the 42- to 46-kD monomeric form. The affinity-purified Fy6-active protein was converted to a sharp band of 35 kD after N-glycanase treatment. The molecule appeared as a slightly broadly band after neuraminidase treatment but was not further altered by O-glycanase. The i3A mAb bound to 6,000 +/- 1,000 receptor sites on either Fy(a-b+), Fy (a+b+) and Fy(a+b-) red cells with an affinity constant in the range of 3-6 x 10(8) M-1. No binding was observed to other blood cells nor to several cells (B, T, myelomonocytic and erythro-leukemia cell lines). Also, the bulk of i3A-Fy6 immune complexes could be dissociated from the red cell membrane with as low as 0.2% Triton X-100, showing that the Fy6-active glycoprotein is not tightly associated with the membrane skeleton. Our data obtained with a new monoclonal antibody directed to the Fy6 antigen demonstrate that the blood group Duffy-active component is a red cell-specific glycoprotein carrying one or more N-linked oligosaccharides.

Animals↗
PubMed1994

The CD40 antigen and its ligand.

CD40 is an integral membrane protein found on the surface of B lymphocytes, dendritic cells, follicular dendritic cells, hematopoietic progenitor cells, epithelial cells, and carcinomas. It is a 45-50 kDa glycoprotein of 277 aa, which is a member of the tumor necrosis factor receptor superfamily. The CD40 gene maps to human chromosome 20q11-2-q13-2. CD40 binds to a ligand (CD40-L) which is an approximately 35 kDa glycoprotein of 261 aa, a member of the tumor necrosis factor superfamily. The CD40-L gene maps to human chromosome Xq24. This CD40-L is expressed on activated T cells, mostly CD4+ but also some CD8+ as well as basophils/mast cells. The CD40-L is defective in the X-linked hyper-IgM syndrome. Cross-linking of CD40 with immobilized anti-CD40 or cells expressing CD40-L induces B cells to proliferate strongly, and addition of IL-4 or IL-13 allows the generation of factor-dependent long-term normal human B cell lines and the secretion of IgE following isotype switching. Addition of IL-10 results in very high immunoglobulin production with limited cell proliferation. IL-10 induces naive B cells to produce IgG3, IgG1, and IgA1, and further addition of TGF beta permits the secretion of IgA2. Several evidences suggest that CD40-dependent activation of B cells is important for the generation of memory B cells within the germinal centers: (i) CD40 activated germinal center B cells cultured in the presence of IL-4 acquire a memory B cell phenotype, (ii) CD40 activated B cells can undergo isotype switching, (iii) the deficit of CD40-L results in the hyper-IgM syndrome characterized by lack of germinal centers in secondary lymphoid organ follicles and lack of IgG, IgA, and IgE, and (iv) CD40-L positive T cells are present in secondary follicles. Thymic epithelial cells, activated monocytes, and dendritic cells express CD40 antigen which may be involved in an enhanced cytokine production by these cells, allowing an amplification of T cell proliferation. Finally, as other members of the tumor necrosis factor receptor family have been shown to bind several ligands, it is possible that CD40 may bind other ligands that may trigger CD40 on different cell types such as hematopoietic cells or epithelial cells.

Amino Acid Sequence↗
PubMed1994

Long-term of patients receiving an aorto-bi-femoral prosthesis for atherosclerotic occlusive disease of the aortic bifurcation.

Over a 14-year period (1975-1989), 547 patients with a mean age of 57 years received an aorto-bi-femoral prosthesis for atherosclerotic occlusive disease (AOD) and underwent regular follow-up over a 13-year period. Immediate post-operative mortality was 2.5% with few deaths due to cardiovascular causes (0.5%). The long-term mortality was significantly elevated at 5 years (10% higher than controls) and at 10 years (16%). These deaths were due to the AOD and its surgical treatment (10%), other cardiovascular disease (30%) and cancer (41%). Approximately 5% of patients required an immediate post-operative, or delayed amputation, compared to 33% which had a threatened limb before the operation. Thrombosis of the prosthesis was relatively rare. At 5 years, 81% of the survivors remained largely improved; at 10 years, 67% and at 13 years, 62%. The benefit of this intervention is therefore unquestionable with regards to the functional state and survival. Also, in avoiding immobility due to the disabling intermittent claudication or amputation, it considerably improves the quality of life which largely compensates for the low mortality rates and subsequent complications of the prosthesis.

Aged↗
PubMed1993

[Paroxysmal nocturnal hemoglobinuria. Diagnosis aided by a monoclonal antibody directed against the decay accelerating factor glycoprotein].

The laboratory diagnosis of paroxysmal nocturnal haemoglobinuria (also called Marchiafava-Micheli disease) is based on the sensitivity of the patient's red cells to complement-induced lysis. In view of the clonal expression of the disease, haemolysis tests are difficult to interpret when the abnormal red cell population is small. The sensitivity of abnormal red cells to haemolysis is due to the absence of proteins attached to the cell membrane by a phosphatidyl-inositol link, which intervene in the regulation of the complement-induced lysis mechanism. Using a monoclonal antibody directed against one of these proteins, the decay accelerating factor (DAF, protein CD 55), makes it possible to diagnose paroxysmal nocturnal haemoglobinuria. DAF expression on patients' blood cells was measured by quantitative agglutination and by indirect flow cytometry. The agglutination test using polybren is a fast detection method, but it may be uninterpretable, notably in cases with positive antiglobulin (Coombs') test. In contrast, DAF expression measured by indirect flow cytometry correlates perfectly with measurement of red cell sensitivity by haemolysis tests. Using the monoclonal antibody by indirect flow cytometry is the method of choice to confirm the diagnosis of paroxysmal nocturnal haemoglobinuria and to measure the proportions of normal and abnormal red cells in case of haematological disorder.

Adolescent↗
PubMed1993

[Bilateral aorto-iliac prosthesis. Long-term results].

Over a 12-year period (1975-1987), 565 patients (male: 94.5 percent; female: 5.5 percent) underwent aorto-iliac bilateral reconstruction. The operations were performed by the same surgical team and all patients were followed up for 13 years. The patients' mean age was 59.4 years; 16.6 percent were over 70; 16 percent had a non-fissured aneurysm. The postoperative mortality rate was 2.8 percent, with relatively few deaths of cardiovascular origin (0.5 percent). The late mortality rate was significantly higher than that of an age-matched control population. Arteritis and its surgical treatment accounted for a low proportion of late deaths: 10.7 percent as opposed to other cardiovascular diseases (33 percent) and chiefly to cancer (39 percent)--a figure not found in other reports. Five percent of the patients had to be amputated soon after the operation or later; this is a low figure compared with the 33 percent of patients who had one limb threatened before reconstructive surgery. After the aorto-iliac reconstruction 81 percent of the patients remained considerably improved at 5 years and 67 percent at 10 years. Long-term arterial patency was satisfactory (95 percent at 5 years, 90 percent at 10 years). This type of surgery, therefore, benefits the patients' functions and their life: it avoids rupture of aneurysms and sedentarity due to a disabling intermittent claudication, and it considerably increases the duration of life.

Aged↗