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Biomedical subjects

H Avraham

Publications and source records attributed to H Avraham.

At least 73 records · Page 4Linked to original sources
PubMed1993

Characterization of adhesive interactions between human endothelial cells and megakaryocytes.

Cell-cell adhesion is essential for many immunological functions and is believed to be important in the regulation of hematopoiesis. Adhesive interactions between human endothelial cells and megakaryocytes were characterized in vitro using the CMK megakaryocytic cell line as well as marrow megakaryocytes. Although there was no adhesion between unactivated human umbilical vein endothelial cells (HUVEC) and megakaryocytes, treatment of HUVEC with inflammatory cytokines such as IL-1 beta, tumor necrosis factor alpha, INF-gamma, or the phorbol ester phorbol myristate acetate (PMA) resulted in a time- and dose-dependent increase in adhesion. Stimulation of marrow megakaryocytes or CMK cells with the cytokines IL-1 beta, GM-CSF, IL-6, IL-3, or PMA augmented their adhesion to endothelium. Monoclonal antibodies against the LFA-1 subunit of the leukocyte adherence complex CD18 inhibited the binding of marrow megakaryocytes or CMK cells to HUVEC. Adhesion blocking experiments also demonstrated that the VLA-4/VCAM-1 pathway was important for megakaryocyte attachment to HUVEC. Adhesion promoted maturation of megakaryocytic cells as measured by increased expression of glycoproteins GpIb and GpIIb/IIIa and by increased DNA content. These observations suggest that alterations in megakaryocyte adhesion may occur during inflammatory conditions, mediated by certain cytokines, resulting in augmented megakaryocyte maturation.

Bone Marrow↗
PubMed1992

Interaction of human bone marrow fibroblasts with megakaryocytes: role of the c-kit ligand.

Human kit ligand (KL), also known as stem cell factor (SCF), steel factor, or mast cell growth factor, is a recently identified hematopoietic growth factor whose receptor is the product of the c-kit proto-oncogene. Alternative splicing of the pre-mRNA of KL/SCF results in secreted and membrane-bound forms of the protein. We and others have recently shown that the c-kit gene product is expressed on human megakaryocytes and that soluble KL/SCF in combination with granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3), or IL-6 increased megakaryocyte progenitor colony formation (CFU-MEG) and stimulated mature megakaryocytes. Here we show that adhesion of human megakaryocytes to bone marrow stromal fibroblasts, which express the membrane-bound form of KL/SCF (mKL/SCF), is mediated in part by the interaction between mKL/SCF and the c-kit protein. This interaction also results in marrow fibroblast-stimulated proliferation but not an increase in ploidy of megakaryocytes; when the two cell types were separated by a transoluble membrane, proliferation did not occur. Adhesion and proliferation of human megakaryocytes to an immortalized murine stromal cell line SI/SI lacking the KL/SCF gene was impaired, whereas transfection of SI/SI cells with human mKL/SCF significantly increased both adhesion and proliferation. Marrow stromal fibroblast mKL/SCF may serve both as an adhesion structure and as a growth-potentiating factor for megakaryocytes in the bone marrow.

Animals↗
PubMed1992

Effects of the stem cell factor, c-kit ligand, on human megakaryocytic cells.

The kit ligand (KL), also termed stem cell factor (SCF), is a recently discovered hematopoietic growth factor that augments response of early progenitor cells to other growth factors and supports proliferation of continuous mast cell lines. Histological studies suggest that the receptor for SCF/KL, the c-kit proto-oncogene product, is present in bone marrow megakaryocytes. We studied the effects of SCF/KL on immortalized human megakaryocytic cell lines (CMK, CMK6, and CMK11-5) and on isolated human marrow megakaryocytes. Human SCF/KL alone or in combination with the hematopoietic growth factors, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6, stimulated proliferation of these megakaryocytic cell lines. SCF/KL treatment did not alter expression of gpIb, gpIIb/IIIa, LFA-1, ICAM-1, or GMP-140 in CMK cells. No effect on ploidy was observed. Furthermore, human SCF/KL induced expression of IL-1 alpha, IL-1 beta, IL-2, and IL-6 in CMK cells. In a fibrin clot system, SCF/KL modestly potentiated megakaryocyte colony formation when added alone to cultures containing CD34+, DR+ bone marrow cells. Addition of SCF/KL with IL-3 or GM-CSF to these cultures resulted in a more marked marrow megakaryocytic cells. SCF/KL may directly affect megakaryocytopoiesis, as well as secondarily modulate hematopoiesis through induction of cytokines in target cells.

Antigens, Surface↗
PubMed1992

Negative and positive cis-acting elements in the promoter of the mouse gene that encodes the serine/glycine-rich peptide core of secretory granule proteoglycans.

The gene that encodes a proteoglycan peptide core rich in serine and glycine (SG-PG) is selectively expressed by hematopoietic cells that store in their cytoplasmic granules negatively charged proteoglycans bound ionically to numerous positively charged proteins. With deletion analysis, a negative transcription regulatory element was located between residues -250 and -190 of the 5'-flanking region of the mouse SG-PG gene, and a positive regulatory element was located between residues -118 and -81. The negative regulatory element was dominantly active in fibroblasts that do not express the SG-PG gene whereas the positive regulatory element was dominantly active in hematopoietic cells that do express the SG-PG gene. Site-directed mutagenesis was used to demonstrate that the proximal element within the gene's atypical promoter resided between residues -40 and -20. As assessed by gel mobility shift analyses, the nuclei of rat basophilic leukemia-1 cells and rat-1 fibroblasts contain a number of trans-acting factors that interact with the positive and negative cis-acting regulatory elements of the SG-PG gene. Furthermore, some of these trans-acting factors appear to be different for the two cell types. These studies on cell types that do and do not express the SG-PG gene indicate that transcription of this proteoglycan peptide core gene is regulated constitutively by both positive and negative cis-acting elements located 5' of an atypical promoter.

3T3 Cells↗
PubMed1992

Cytokine gene expression and synthesis by human megakaryocytic cells.

Cytokine expression and production by human megakaryocytic cells were studied using the CMK cell line as a model for cytokine gene expression by cell line as a model for cytokine gene expression by polymerase chain reaction (PCR) and for cytokine protein synthesis by specific radioimmunoassays. CMK cells at all stages of maturation were found to constitutively express moderate mRNA levels for tumor necrosis factor (TNF-alpha), transforming growth factor beta (TGF-beta), interleukin (IL) 1 beta, and endothelial cell growth factor (ECGF) transcripts. After 6-h treatment with the phorbol ester PMA, gene expression for IL-1 alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and the IL-6 receptor were increased. After 24 h of exposure to PMA, levels for most cytokines declined to baseline, except for IL-6 which appeared as a new transcript. PMA-stimulated CMK lines synthesized low levels of TNF-alpha and IL-6, and higher levels of GM-CSF, IL-1 beta, and IL-1 alpha protein. These observations suggest that cells of megakaryocytic lineage are capable of producing a repertoire of cytokines which could mediate an autocrine role as well as modulate the replication and function of other hematopoietic cells.

Base Sequence↗
PubMed1992

Effects of transforming growth factor beta on megakaryocytic cell fusion and endomitosis.

Transforming growth factor beta (TGF-beta) is a cytokine secreted by megakaryocytes and platelets which inhibits the proliferation of megakaryocyte progenitors in vitro. We have studied the effects of TGF-beta on more mature megakaryocytes, using cell lines CMK, DAMI, and CHRF as well as isolated human marrow megakaryocytes as models. Proliferation of these megakaryocytic cell lines was inhibited by TGF-beta 1 at concentrations of 10-100 ng/ml. Although these cell lines secreted small amounts of TGF-beta (up to 100 pg/ml), it was predominantly in an inactive form. Upon induction with 12-phorbol 13-myristate acetate (PMA), they secreted greater amounts of TGF-beta (500-1250 pg/ml), most of which was still inactive in a bioassay. Addition of exogenous active TGF-beta 1 had no effect on ploidy of unstimulated megakaryocytic cell lines or on [3H]thymidine incorporation of isolated human marrow megakaryocytes. Following PMA induction, exogenous TGF-beta 1 had a significant inhibitory effect on ploidy in DAMI cells but not CMK or CHRF cells, suggesting that the phenomenon is restricted to DAMI cells. Because certain cell lines may fuse in vitro, and phorbol esters can promote this phenomenon, we investigated the possibility that fusion was contributing to the increase in ploidy in megakaryocytic cells. Unstimulated megakaryocytic cells did not show spontaneous fusion; when PMA was added to the cultures, fusion was markedly increased, particularly in DAMI cells. These results demonstrate that in vitro fusion accounts for a proportion of the apparent increase in DNA content of megakaryocytic cell lines upon induction with PMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

Deleterious effect of anti-insulin antibodies on diabetes control.

We studied the effect of anti-insulin antibodies (AIA) on glycemia control in patients with insulin-dependent diabetes (IDD) by following the AIA titer changes with time. Although expected to remain constant, AIA levels were found to either increase or decrease in most patients. These AIA titer changes correlated significantly with changes in glycohemoglobin but not with changes in insulin dose. The results suggest that AIA may have a deleterious effect on glycemia control in IDD.

Adolescent↗
PubMed1989

A rapid procedure for cell colony hybridization using DNA probes.

A rapid, direct method for screening single cell-derived colonies or foci is described. The method allows the screening of a large number of colonies or foci by nitrocellulose filter hybridization using DNA probes. This technique simplifies current screening procedures and is a reliable, rapid, and sensitive method for the selection of cell clones containing a desired transfected gene.

Animals↗
PubMed1989

Characterization and expression of the human rhoH12 gene product.

The rho genes constitute an evolutionarily conserved family having significant homology to the ras oncogene family. These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rat, and human; their 21,000-dalton products show strong conservation of structure. In humans, three classes of rho cDNA clones have been identified which differ by virtue of the presence of variable C-terminal domains: rhoH12, rhoH6, and rhoH9. The predicted 193 amino acids of human rhoH12 protein show 88% similarity with those of the human rhoH6 clone, 96.8% similarity with those of the Aplysia rho product, and 81.8% similarity with those of the yeast RHO1 protein. Rat-1 and NIH 3T3 mouse fibroblasts were transfected with clones containing the normal human rhoH12 allele as well as the variants encoding valine in place of the glycine and leucine in place of the glutamine normally found at residues 14 and 64, respectively. These replacements mirror the changes responsible for oncogenic activation of the related ras-encoded p21 proteins. These mutant rhoH12 clone alleles did not cause focus formation in monolayers or growth in soft agar. However, amplification of normal rhoH12 via cotransfection with a dihydrofolate reductase gene resulted in colonies that displayed reduced dependence on serum for growth, grew to higher saturation densities, and were tumorigenic when inoculated into nude mice. Normal p21rho protein was detected in the transfected cell lines as well as in normal cell lines by Western immunoblot and immunoprecipitation analysis with rabbit antibodies raised against the peptide corresponding to amino acids 122 to 135.

Amino Acid Sequence↗
PubMed1986

RAB-1: a new monoclonal antibody to leukemic hairy cells.

RAB-1, a new monoclonal antibody (McAb) to human leukemic hairy cell (HC) was produced. Using indirect immunofluorescence methods and microscopic or flow cytometric analysis, it was found that the RAB-1 antigen was expressed on few resting B cells and not on resting T lymphocytes, platelets, monocytes, erythroid and myeloid cells. RAB-1 expression on malignant cells was as follows: strongly positive in 15/15 hairy cell leukemia (HCL), negative with non-T and T-acute lymphoblastic leukemia and T-chronic lymphocytic leukemia (CLL); weakly expressed on myeloma and Waldenstrom cells; moderately on 10-25% of the cells in 4/10 B-CLL and 6/10 B lymphomas and in 7/7 B-prolymphocytic leukemia (PLL). Amongst human cell lines that were tested, RAB-1 reacted strongly with one HC line, moderately with the EBV-lymphoblastoid Daudi and Raji Burkitt's lines and was not expressed on Ramos, ALL, myeloid and erythroid cell lines. Normal B cells activated with PWM or anti-mu beads, and malignant B cells activated with anti-mu and TPA did not show an increase of expression of RAB-1 antigen. Interestingly, 30-40% of T4-Class II antigen positive cloned cells and T cells activated with PHA and Con.A expressed RAB-1, suggesting that this McAb recognizes surface molecule, newly induced during T-cell activation and constitutively expressed on HC and some B-cell malignancies.

Antibodies, Monoclonal↗
PubMed1986

A human monoclonal antibody to insulin.

To elucidate the immune aspects of insulin-dependent diabetes mellitus (IDDM), we attempted to generate human monoclonal anti-insulin antibodies by fusing peripheral blood lymphocytes obtained from 10 insulin-treated IDDM patients with cells from a human lymphoblastoid cell line. Hybridomas that secreted immunoglobulins appeared in 9 of 400 wells. One of these hybridomas secreted anti-insulin antibody of the IgM class. The lymphocytic partner of this hybridoma was obtained from an IDDM patient who had undetectable levels of antibodies to insulin in his serum. Thus, by employing the hybridoma technique, it was possible to reveal the presence of insulin-sensitized B-lymphocytes in a patient who was serologically negative for anti-insulin antibodies. The monoclonal antibody recognized intact human insulin and insulins of other species, but not isolated A- and B-chains. This indicates that the antibody was functionally an autoantibody directed to an epitope formed by the native conformation of a highly conserved portion of the insulin molecule. This is the first report of a human hybridoma antibody to insulin.

Animals↗
PubMed1983

Leishmania tropica and Leishmania donovani: solid phase radioimmunoassay using leishmanial excreted factor.

A radioimmunoassay for the quantitative determination of anti-leishmanial excreted factor (EF) antibody in rabbit sera was developed. The assay, using Leishmania tropica and Leishmania donovani promastigotes EF, purified by either extraction with phenol followed by fractionation on a Sephadex G-100 column or by the dissociation of EF antibody complexes, was shown to be sensitive and reproducible. Using monospecific anti-EF antibodies, levels of as low as 0.06-0.12 micrograms/ml of anti-EF IgG could be detected. The specificity of the assay was assessed by inhibition with homologous and heterologous EF. Only minor cross-reactivity with heterologous EF was observed, and as little as 2.5 micrograms/ml of EF could be detected. Sera from kala-azar patients showed only 1.8-3.1 times more anti-EF activity, as compared with uninfected controls. No specificity was observed with sera from kala-azar patients with regard to the type of EF used. Almost the same activity was obtained with both EF from L. tropica and L. donovani. No anti-EF antibodies were detected in sera from patients with cutaneous leishmaniasis.

Animals↗
PubMed1983

Preliminary field trial of a radioimmunoassay for the diagnosis of malaria.

A radioimmunoassay (RIA) has been developed for the detection of Plasmodium falciparum in infected blood. The assay is based on the ability of solubilized, infected red blood cells (RBC) (P. falciparum "antigen") to combine with anti-P. falciparum antibodies and thus prevent the subsequent interaction of the latter with "antigen"-coated microtiter plates. A preliminary trial was carried out in Thailand to determine the usefulness of the RIA for the immunodiagnosis of malaria. Blood samples from malarious and non-malarious patients were examined both by standard microscopy and by RIA. Efficient solubilization of the parasites proved to be a major requirement for the successful performance of the RIA. Sonication or freezing and thawing, which were perfectly satisfactory for the solubilization of cultured, infected RBC, were found to be totally inadequate when applied to RBC taken from patients. However, parasites in RBC from patients could be solubilized efficiently by treatment with detergents (e.g., NP40, Triton X-100, etc.). Of the 108 blood samples tested, 23 were found positive for falciparum parasitemia by microscopy and 39 by RIA. One sample from a patient with patent falciparum parasitemia and three with patent vivax parasitemia were negative by RIA. Ten of the samples positive only by RIA belonged to patients with recent malarial infection, as shown by microscopy. Thus, the RIA detected almost all of the patients with microscopic evidence of falciparum malaria. The proportion of false positives in the RIA test was low.

Detergents↗
PubMed1983

The inhibitory effect of human immune sera upon the in vitro development of Plasmodium falciparum.

Sera from patients who had recovered from malaria were examined for their capacity to inhibit the growth of Plasmodium falciparum in vitro. The sera were dialysed to eliminate any residual drugs taken by the donor. IgG purified from inhibitory sera was found to be as effective as the original sera. The inhibitory activity of some of the immune serum was pronounced after the second day in culture. This may suggest that the mechanism of action of antibodies affects the merozoites which appear for a short period in the two day cycle of the parasite. There was no correlation between antibody titre measured by IFA and the inhibitory capacity of the sera: some samples with high IFA titres did not inhibit the growth while some with low IFA titres inhibited the parasite. Autoantibodies directed against the erythrocytes, which were found in some sera, had no antimalarial inhibitory role and the absorption of them did not alter the inhibitory effect. There were more inhibitory sera among Nigerian than amount Thai and Ethiopian sera. The experiments were performed with a Nigerian strain and therefore the results may be explained by differences between the various falciparum strains of malaria.

Antibodies↗
PubMed1982

A highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies.

A highly sensitive radioimmunoassay for detection of P. falciparum antibodies and antigens is described. A partially purified P. falciparum antigen preparation is obtained from in vitro cultured parasites enriched after gelatin sedimentation by sonicating the infected red blood cells and precipitating the proteins with 50% saturated ammonium sulfate. The precipitate is dissolved in buffer, ultracentrifuged and used to coat wells of microtiter plates. Anti-P. falciparum antibodies are detected by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated staphylococcal protein A. P. falciparum antigens are detected by their ability to inhibit binding of antibodies to the coated wells. Sera of individuals with a history of P. falciparum infection contain antibodies detectable at a dilution of 1:75,000. P. falciparum RBC infected in vitro can be detected at levels of parasitemia of the order of 1 parasite or less per 10(6) RBC.

Antibodies↗
PubMed1982

Inhibition of antibody-binding as a radioimmunoassay for Plasmodium berghei infection in rats.

Sonicated red blood cells of rats infected with Plasmodium berghei (Pb) were used to coat plastic tubes, which were subsequently tested for capacity to bind anti-P, berghei antibodies. Binding was detected using radioiodinated Staphylococcus Protein A. Two antigenic preparations were used to make the solid-phase adsorbent (and also as inhibitors in the inhibition tests): sonicated red blood cells (SIRC), and a supernate of frozen-thawed, infected red blood cells (FTE). Treatment of the antigen-coated tubes with sera of immunized rats or by rabbits resulted in binding of antibodies to the tubes. The specificity of the binding was confirmed by inhibition tests. Preparations derived from infected blood but not from normal blood inhibited the binding of the anti-Pb antibodies. Using the inhibition tests, an infection corresponding to 0.00005% parasitemia could be detected. The sensitivity was higher when SIRC rather than FTE was used as antigen. The sensitivity of the inhibition test for the detection of parasites in the blood greatly surpassed that of the standard microscopical procedure. Parasites were demonstrated in blood samples 2 days after infection, 5 days before a microscopic diagnosis was positive. Repeated assays conducted with the same batch of reagents showed that the test was remarkable reproducible.

Animals↗
PubMed1981

Plasmodium falciparum: assay of antigens and antibodies by means of a solid phase radioimmunoassay with radioiodinated staphylococcal protein A.

Human red blood cells (RBC) infected in vitro with Plasmodium falciparum were employed to prepare several types of antigens (sonicated, infected RBC and purified, sonicated merozoites and schizonts). These antigens, as well as control preparations derived from non-infected RBC, were used to coat plastic tubes, which were subsequently tested for capacity to bind anti-P. falciparum antibodies. Binding was detected by means of radio-iodinated staphylococcus protein A. Sera from patients with recent disease or patients who had a history of P. falciparum infection gave strong binding, while sera of normal individuals had only a low binding activity. Some of the antibodies in the positive sera were directed against RBC, since they could bind to tubes coated with normal RBC antigens and could be removed by absorption with RBC. The specificity of the P. falciparum antibodies was confirmed by inhibition tests: preparations derived from infected blood but not from normal blood inhibited the binding activity of the positive sera, to antigen coated tubes.

Animals↗