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M Barnet

Publications and source records attributed to M Barnet.

At least 19 recordsLinked to original sources
PubMed2005

Are platelets activated after a rapid, one-step density gradient centrifugation? Evidence from flow cytometric analysis.

This procedure describes the preparation of platelets from whole blood of healthy donors and pregnancy-induced hypertensive (PIH) patients by a rapid, one-step density gradient centrifugation, and the direct immunofluorescence staining of obtained platelets (CD63). Platelets are relatively fragile structures. Consequently, for the investigation of their biochemical properties it is recommended to isolate them by a simple method that does not damage their functional parameters and induce their activation. During platelet activation, several changes occur at the platelet surface. CD63 is the receptor for a lysosomal glycoprotein expressed in activated platelets. Currently, flow cytometry (fluorescence-activated cell sorting) is the most sensitive method to detect increased surface exposure of activation antigens on the platelet surface. The present technical note describes that compared with other whole blood flow cytometric techniques, our one-step density-gradient centrifugation method using OptiPrep can also prevent artificial, sample manipulation-related platelet activation.

Blood Platelets↗
PubMed2001

IgG subclass switch capacity is low in switched and in IgM-only, but high in IgD+IgM+, post-germinal center (CD27+) human B cells.

Recent studies have shown that in humans the germinal center reactions produce three types of V(D)J mutated B cells in similar proportions, i.e. Ig-switched, IgD-IgM+ (IgM-only) and IgD+IgM+ cells, and that together they form the CD27+ compartment of recirculating B cells. We investigated the Ig isotype switch capacity of these cells. Peripheral blood B subsets were sorted and IgG subclass secretion in presence or absence of IL-4 was compared in B cell assays which lead to Ig secretion in all (coculture with EL-4 thymoma cells) or only in CD27+ (CD40L stimulation) B cells. Already switched IgG+ B cells showed no significant sequential switch and IgM-only cells also had a low switch capacity, but IgD+CD27+ switched as much as IgD+CD27- B cells to all IgG subclasses. Thus, in switched B cells some alterations compromising further switch options occur frequently; IgM-only cells may result from aborted switch. However, IgD+CD27+ human B cells, extensively V(D)J mutated and "naive" regarding switch, build up a repertoire of B cells combining (1) novel cross-reactive specificities, (2) increased differentiation capacity (including after T-independent stimulation by Staphylococcus aureus Cowan I) and (3) the capacity to produce appropriate isotypes when they respond to novel pathogens.

B-Lymphocyte Subsets↗
PubMed1998

Quiescent memory B cells in human peripheral blood co-express bcl-2 and bcl-x(L) anti-apoptotic proteins at high levels.

The anti-apoptotic proteins bcl-2 and bcl-xL seem to exhibit strictly opposite expression patterns in normal lymphoid cell differentiation stages, with bcl-2 low and bxl-xL high in immature and mature proliferating cells, the reverse being the case in recirculating quiescent cells. However, it is in fact not known whether recirculating memory cells are bcl-xL low or high. We analyzed memory (immunoglobulin isotype-switched) B cells in human peripheral blood, which were small lymphocytes in the G0 phase of the cell cycle, but proliferated better than naive B cells in response to Staphylococcus aureus Cowan I. Ex vivo these cells co-expressed bcl-2 together with bcl-xL mRNA and protein at high levels. The mcl-1 mRNA level was low. The bcl-xL mRNA level decreased during culture in medium containing fetal calf serum, which implies that it is maintained in vivo by continuous or frequent, non-mitogenic signal(s). The high bcl-xL expression of memory B cells may be relevant with regard to their longevity and/or their capacity to undergo an accelerated secondary type immune response.

Apoptosis↗
PubMed1993

High IgE secretion capacity of human plasma cells.

In accordance with results obtained in another culture system, it has previously been shown that human B cells frequently switch to immunoglobulin E (IgE) when they are co-cultured with irradiated mutant EL4 thymoma cells (which provide a CD40 ligand-mediated B cell activation signal), T cell supernatant and recombinant interleukin (IL)-4. However, because of the potentially severe side effects of IgE, such as anaphylaxis, B cells could have a limited capacity to produce this isotype. The IgE secretion rate of plasma cells is not known. In the present study, we compared the secretion rates for different Ig classes by means of limiting dilution analysis of plasmocytic cells that were harvested after 8 to 9 days from primary EL4/B cell cultures and titrated into secondary cultures in the presence of a cell proliferation-blocking concentration of hydroxyurea. These cells secreted Ig at constant rates for periods of up to 2 weeks; IgE secretion was IL-4 independent. The mean cellular secretion rates were similarly high for IgE (150 pg/cell/24 h) and other isotypes (IgM 273 pg, IgG 112 pg, IgA 136 pg/cell/24 h). In terms of molecules per min this represents 3.3 x 10(5) for IgE versus 1.2 x 10(5) for IgM, 3.1 x 10(5) for IgG and 3.6 x 10(5) for IgA. The relative frequency of IgE-secreting cells was only 0.3% of the total number of Ig-secreting cells, suggesting a small size of IgE-producing clones in this in vitro system. Whether this is relevant regarding an in vivo response is not known. Clearly, the Ig secretion capacity of plasma cells would not limit an IgE response in the absence of extrinsic control.

Animals↗
PubMed1989

Hydatid disease: analysis of parasite antigens in circulating immune complexes and in preformed hydatid antigen-antibody complexes.

Fifty-three sera from 29 patients with hydatid disease, all but one positive for specific anti-parasite antibodies and all negative for specific circulating antigens, were studied for the presence of circulating immune complexes (CIC) by conglutinin binding-assay (KgBA). Fourteen serum samples (26%) from eight patients (27%) were positive. These positive sera were pooled for each patient and the eight samples were PEG-precipitated and analysed for the presence of specific Echinococcus granulosus antigens in the CIC using a human anti-human-hydatid cyst fluid antiserum capable of recognizing the major antigenic systems of the parasite namely, antigens 4 and 5. The assays utilized for detecting antigen in CIC were: (a) blotting on nitrocellulose paper after sodium dodecil sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and specific immunological detection; (b) ultracentrifugation in acid buffer and subsequent detection of antigens by a sandwich-radioimmuno assay (RIA); (c) protein separation by isoelectric focusing (IEF) and specific immunological recognition. In addition, all positive sera were analysed for the presence of antigen in the CIC by a modified KgBA and by polyethylenglicol (PEG)-precipitation in acid buffer followed by immunological recognition of antigen. All tests gave negative results with the patients' samples, but were positive with preformed in vitro complexes between parasite antigens and corresponding antibodies. Failure to detect antigen in the CIC could be due to: 1) insufficient sensitivity of the assays used to detect hydatid antigens in CIC; 2) rapid clearance of antigen or CIC from the circulation; 3) presence of parasite antigen not recognized by the antiserum employed; 4) production of CIC as a result of polyclonal B-cell activation. This last hypothesis is supported by the demonstration of IgM-rheumatoid factor (RF) and anti-F(ab')2 antibodies respectively in 11 (44%) and 13 (52%) out of 25 patients.

Animals↗
PubMed1985

Beta-endorphin in normozoospermic and pathologic human semen.

beta-Endorphin was estimated in normozoospermic, oligozoospermic and azoospermic human semen. The mean amount in normozoospermic specimens was 278.6 +/- 43.6 (SE) pg/ml while in the others only 191.1 +/- 25 pg/ml. Both values are significantly higher than those present in the blood.

Adult↗
PubMed1985

Characterization of parasite antigens from human hydatid cyst fluid by SDS-PAGE and IEF.

Combining high resolution power of SDS-PAGE and IEF with the specific immunological recognition of a human antiserum directed against Echinococcus granulosus antigens, we could identify, in 4 hydatid cystic fluids of human origin, 4 antigens with a molecular weight in the range 32-13 KD, and an antigen of 200 KD which, in reducing conditions, gave 2 bands of 67 and 52 KD. In addition, mainly in one of the cystic fluids, there were at least another 4 specific non-reducible bands with a molecular weight ranging from 80 to 40 KD. Specific parasite antigens, which constitute not more than 3% of total protein content of the cystic fluid, migrate, in isoelectric focusing, from a pH of less than 5 to more than 8.

Antigens, Helminth↗
PubMed1985

Deoxyribonuclease activity in human sperm.

A previous failure to detect DNase activity in human sperm sonicates was ascribed to an inadequate amount of sperm used. In the present work aliquots contained more sperm (1.5-2.5/10(6)), permitting the detection of enzyme activity. The latter was significantly higher in spermatozoa of oligozoospermic origin compared with activity in normospermic specimens.

Deoxyribonucleases↗
PubMed1983

ATP contents of human semen, seminal plasma and isolated sperm at time intervals after ejaculation.

1. ATP was estimated in 105 samples of human semen, seminal plasma and sperm of normozoospermic and oligozoospermic origins at time intervals after ejaculation. 2. In semen with sperm counts up to 40 millions per ml and in seminal plasma, ATP levels were lower than in specimens with higher sperm counts. 3. In isolated sperm, the ATP content decreased with the increase in sperm density. 4. The decrease in ATP after 24 hr was the highest in sperm, lower in semen and the lowest in seminal plasma, being maximal in specimens with high sperm counts.

Adenosine Triphosphate↗
PubMed1983

Sialic acid content of human spermatozoa and seminal plasma in relation to sperm counts.

Sialic acid levels were determined in spermatozoa and seminal plasma of 47 semen samples divided into two groups according to sperm counts. Group 1, up to 40 x 10(6) sperm/ml seminal fluid and group 2, above 40 x 10(6) sperm/ml seminal fluid. The content of sialic acid in spermatozoa ranged from 4.4-28.1 micrograms/10(8) spermatozoa and from 70-95 mg/100 ml seminal plasma. Sialic acid was significantly higher in sperm and lower in seminal plasma of group 1 as compared to group 2. The lower content of sialic acid in seminal plasma might have a deteriorating effect on structural integrity of sperm.

Humans↗
PubMed1983

Hyaluronidase activity in untreated human semen and following laboratory manipulations.

Hyaluronidase activity was examined in 104 oligospermic and normospermic human semen specimens promptly after liquefaction, after repeated cycles of washings with a solution containing 1.3 M sucrose and 0.15 M NaCl, and after freezing and thawing. We assessed enzyme activity by measuring areas of the digestion of substrate (hyaluronate). After washing the oligospermic and normospermic semen specimens twice 34%-43% enzyme activity was still present in the sedimented sperm as compared to the initial values. Following additional washings no enzyme activity could be detected. Freezing and thawing of semen increased enzyme activity by 40%-50%, respectively, after the second cycle whereas further treatment did not increase the enzyme activity rate. It is suggested that two types of hyaluronidase are present in human spermatozoa, differing in membrane-binding properties which would be connected with their localization in the acrosome.

Acrosome↗
PubMed1983

Identification of components of IC purified from human sera. I. Immune complexes purified from sera of patients with SLE.

Immune complexes (IC) were purified by affinity chromatography on conglutinin columns from human sera (five SLE, one AML and one leishmaniasis) and compared with IC formed in vitro in the presence of normal serum (NHS). First, analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated a common qualitative pattern, but with marked quantitative differences, in IC obtained from five patients' sera (four SLE, one leishmaniasis) and for in vitro formed IC. In two other patients (one SLE, one AML), the pattern of IC components was very different, with a major band in the 26 kD region. Secondly, after electrophoretic transfer of the SDS-PAGE bands to nitrocellulose membranes, the nature of IC components was studied by defining the reactivity of the bands with antisera against human serum antigens. Several serum proteins were identified in the purified IC:IgG, IgA, IgM, C1q, C1r, C1s, C3bi and Bb. A few bands did not correspond with any normal serum protein. One of them, at 26 kD was shown to react with anti-C reactive protein (CRP) antiserum. From all the constituents observed in the SDS-PAGE analysis of purified IC, only two bands in one SLE patient might be corresponding to unidentified antigens.

Antigen-Antibody Complex↗
PubMed1982

Vitamin A and beta-carotene content of seminal fluid and sperm of normospermic and oligozoospermic men.

Beta-carotene and vitamin A were estimated in 125 specimens of human seminal fluid and spermatozoa. In 72 of the specimens triiodothyronine, tetraiodothyronine, and T4-binding globulin were also determined. Vitamin A in seminal fluid increased from 10.4+/-l.8 microgram (SE) in severe oligozoospermia up to 19.1+/-2.5 microgram/100ml in specimens with high sperm densities. In spermatozoa, however, there was a trend for a decrease in vitamin content from 2.1+/-0.8 to 0.2 +/-0.07 microgram/108. In the majority of cases beta-carotene was undetectable and, when present, the levels were much lower than those of vitamin A. The amounts of triiodothyronine and tetraiodothyronine in seminal fluid were 0.19+/-0.03 ng/ml and 1.1+/-0.18 microgram/100ml, respectively. No correlation was found between levels of both hormones and quality of seminal fluid. T4-binding globulin was either very low or undetectable.

Alpha-Globulins↗
PubMed1982

Inhibition and enhancement of macrophage migration in the presence of lymphocytes and seminal fluid of men with fertility disorders.

Macrophage migration inhibition factor (MIF) towards autologous seminal fluid was studied in 29 infertile men, 16 of whom were also examined towards normal seminal fluid. In 10 patients, inhibition of macrophage migration was observed in the presence of 50 microgram of protein of autologous semen sonicates. Nine patients had enhanced macrophage migration in the presence of 5 microgram protein. Macrophage enhancement was more antigen dependent than the migration inhibition. In all patients seminal abnormalities were observed, being milder in the patients whose lymphocytes enhanced macrophage migration.

Humans↗
PubMed1982

Estimation of hyaluronidase activity of human semen and its relationship with sperm density by means of a simplified method.

Hyaluronidase activity was examined in 109 specimens of human semen of various sperm densities, seminal plasma, and spermatozoa sedimented by centrifugation. We used a modification of a method originally devised for estimation of hyaluronidase activity in Clostridia and which was bases on measurements of the area of hyaluronate digestion in agar plates. Enzyme activities in both semen and seminal plasma increased with increase in sperm density. The activity in seminal plasma, which appeared promptly after liquefaction and represented enzyme released from spermatozoa, ranged from 31% to 61% of the activity in semen. Activities of spermatozoa in sediments exhibited lower values than those calculated for sperm of whole semen, possibly due to leakage. Both activities, calculated per million sperm cells, gradually increased with decrease in sperm density. The possibility that with severe oligozoospermia the acrosome may become less prone to enzyme release or that the initial activity per cell may increase is discussed. These phenomena would represent additional characteristics of oligozoospermia.

Humans↗
PubMed1982

Ferritin levels and circulating immune complexes in patients with solid tumors.

The level of ferritin and circulating immune complexes (IC) were measured in 53 patients with solid tumors and metastasis using C1q binding assay and the conglutinin binding assay. The level of circulating IC was increased in patients (p less than 0.001) as compared to the controls. Ferritin level was increased in both male and female patients (p less than 0.001), even when the ferritin value was corrected by the aspartic transaminase value, to take for hepatic damage in account. Ferritin was not found to be an important component of immune complexes as IC ferritin did not exceed 2 per cent of total serum ferritin in the 10 patients that were investigated. These results suggest that ferritin levels and circulating IC were increased in patients with solid tumors and metastasis, but that these two parameters are not correlated.

Aged↗