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Biomedical subjects

E Sahar

Publications and source records attributed to E Sahar.

At least 19 recordsLinked to original sources
PubMed2006

Antigen-chip technology for accessing global information about the state of the body.

Traditionally, immunologic diagnosis has been based on an attempt to correlate each disease with a specific immune reactivity, such as an antibody or a T-cell response to a single antigen specific for the disease entity. The state of the body, however, appears to be encoded by the immune system in collectives of reactivities and not by single reactivities. Here we describe our use of microarray technology and informatics to develop an antigen chip capable of detecting global patterns of antibodies binding to hundreds of antigens simultaneously. The patterns fashion diagnostic signatures.

Animals↗
PubMed1994

Flow cytometric analysis of entire microbial colonies.

Much could be gained if the scope of flow cytometry could be broadened to the study of entire cell colonies, rather than to populations of single, separate cells. This can be achieved by encapsulating single microbial cells in small spheres in a way that allows each cell to multiply and form a colony within its respective microbead, which is then amenable to analysis by flow cytometry. Methods for performing the encapsulation within beads of appropriate size and homogeneity have been developed (Nir et al., Appl. Environ. Microbiol. 56:2870-2875, 1990). We describe here how a variety of properties of the entrapped colonies, such as mass, growth rate, enzymatic activity, and expression of specific antigens, can be measured, and we discuss how these constructs can be utilized to select microbial mutants.

Animals↗
PubMed1991

Effect of sex hormones on human T cell activation by concanavalin A.

The effect of sex hormones on concanavalin A (Con A)-activated human T cells was studied. We show that neither 17 beta-estradiol (E2) nor progesterone, in concentrations of up to 10(-6) M, alters the proliferative response of peripheral-blood mononuclear cells (PBMC) of healthy postmenopausal women. Furthermore, the hormones had no effect on the composition of T cell populations and on the expression of activation markers. We extended our study to a unique T cell population that is characterized by the ability to form rosettes with human erythrocytes, following Con A activation (designated autorosette-forming cells; ARFC) and known to manifest suppressive activity. Indeed, the in vitro addition of E2 (neither progesterone nor testosterone) to Con A-stimulated PBMC brought an about 2- to 4-fold increase in the frequency of ARFC. Tamoxifen, an antiestrogen drug, reduced the frequency of estrogen-stimulated ARFC to the original low level. Furthermore, the inhibitory effect of growth medium from ARFC cultures originally stimulated with Con A + E2 was found to be higher than that of ARFC cultures originally stimulated with Con A alone.

Concanavalin A↗
PubMed1990

Flow cytometry sorting of viable bacteria and yeasts according to beta-galactosidase activity.

We describe a novel method for quantitative measurement of beta-galactosidase (beta-gal) levels in bacteria and yeasts by using flow cytometry, a method which allows viable microbial cells to be sorted on the basis of the expressed activity and to be recultivated. The method is based on encapsulating single cells in agarose microbeads 20 to 30 microns in diameter and analyzing the beta-gal activity of the colonies that develop (containing several hundred cells) by using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG). Three strains of Escherichia coli, containing different levels of beta-gal, served as a model system. A high degree of correlation was found between the average fluorescence measured per bead and the level of the enzyme in extracts of the respective strain. Although the use of FDG necessitates cell permeabilization, conditions were found under which a small part of each colony remained viable, yet most of the enzyme was exposed to the substrate. This allowed sorting of microcolonies and plating with close to 100% efficiency. The potential of the technique was demonstrated by selecting beta-gal-positive cells from an artificial mixture of beta-gal-positive and beta-gal-negative E. coli strains.

Candida↗
PubMed1990

Single-cell entrapment and microcolony development within uniform microspheres amenable to flow cytometry.

A method is presented for encapsulating single microbial cells in small spheres suitable for analysis and sorting by flow cytometry. The entrapped cells are able to multiply and form colonies contained within their respective microspheres. The system is based on ejecting the cells suspended in a gellable liquid through an orifice vibrating at ultrasonic frequencies, thus shearing the cell-containing jet into uniform droplets. When low-melting-temperature agarose was used, the droplets could be gelled into solid spheres during flight by appropriately directed colling air streams. This gelling was accompanied by significant dehydration, resulting in a twofold decrease in bead diameter and a corresponding increase in agarose concentration. Nevertheless, the microbeads obtained were highly uniform and had diameters which could be precisely controlled in the range of 10 to 40 microns. A variety of bacterial and yeast species were entrapped in agarose beads by using this system. In all cases the cells were able to develop into microcolonies containing as many as several hundred cells. This system enables one to apply the powerful method of flow cytometry to the analysis and sorting of whole microbial colonies. Potential applications of this technology in various areas of microbiology are considered.

Bacteria↗
PubMed1989

Characterization of lympho-myeloid-erythroid-megakaryocytic stem cells in peripheral blood of hairy cell leukemia patients.

Circulating stem cells with lympho-myeloid-erythroid differentiative capacity have been described in the peripheral blood of hairy cell leukemia (HCL) patients (Michalevicz R. & Revel M. (1987) Interferons regulate the in vitro differentiation of multilineage lympho-myeloid stem cells in Hairy Cell Leukemia. Proc. natn. Acad. Sci. U.S.A. 84, 2307.) The aim of the present work was to enrich the progenitors and characterize their antigenic and growth properties. Peripheral blood (PB) from HCL patients was stained with antibodies (BI3C5 (CD34) and/or My10 as well as RFB7 (CD20) and RFT12 (CD7) and sorted using flow cytometry (FCM) into positive and negative fractions. Peripheral blood cells from several patients showed 4-16% cells positive for the BI3C5/My10. The positive fraction contained all the colony-forming cells and LGEM/LG/LGM colonies were enriched approximately ten-fold as compared to the unsorted population. No colony was found in the negative fraction. All colony forming cells were in the RFB7 and RFT12-negative fractions. Thus, circulating stem cells with lymphoid/myeloid potential can be isolated from PB of HCL patients.

Antibodies, Monoclonal↗
PubMed1989

Production and characterization of monoclonal antibodies identifying breast tumor-associated antigens.

We have generated a mouse monoclonal antibody (H23) against the retrovirus-like particles (human mammary tumor virus) released in vitro by the human breast adenocarcinoma cell line T47D. This antibody reacts specifically with a glycoprotein with an apparent molecular mass of 68 kDa (gp68) that is detected in the growth medium of T47D cells as well as in pleural effusion fluids from breast adenocarcinoma patients. No detectable levels of this antigen could be observed in pleural effusions of patients with cancers other than of breast origin. The H23-related antigen was localized in the cytoplasm of breast tumor cells as well as on the cell surface of both T47D cells and metastatic cells from breast cancer patients. A survey of tissue from 812 patients was performed by using H23 in an indirect immunoperoxidase assay. The results showed that the antigen was detectable in 91% of all breast tumors tested. No cytoplasmic staining was observed in either normal tissues or nonbreast carcinomas. Only one of the benign breast tissues tested (out of a total of 56 samples of tissue) was positive for this antigen. Given the ability of this antibody to specifically detect breast tumor cells, H23 may be of importance in diagnosis and in clinical follow-up of patients for the detection of metastatic lesions by imaging and for therapy.

Adenocarcinoma↗
PubMed1989

Cell-density-dependent lysis and sporulation of Myxococcus xanthus in agarose microbeads.

Vegetative cells of Myxococcus xanthus were immobilized in 25-microns-diameter agarose microbeads and incubated in either growth medium or sporulation buffer. In growth medium, the cells multiplied, glided to the periphery, and then filled the beads. In sporulation buffer, up to 90% of the cells lysed and ca. 50% of the surviving cells formed resistant spores. A strong correlation between sporulation and cell lysis was observed; both phenomena were cell density dependent. Sporulation proficiency was a function of the average number of cells within the bead at the time that sporulation conditions were imposed. A minimum of ca. 4 cells per microbead was necessary for efficient lysis and sporulation to proceed. Increasing this number accelerated the lysis and sporulation process. No lysis occurred when an average of 0.4 cell was entrapped per bead. Entrapping an average of 1.7 cells per bead resulted in 46% lysis and 3% sporulation of survivors, whereas entrapping an average of 4.2 cells per bead yielded 82% lysis and 44% sporulation of the surviving cells. Sporulation and lysis also depended upon the cell density in the culture as a whole. The existence of these two independent cell density parameters (cells per bead and cells per milliliter) suggests that at least two separate cell density signals play a role in controlling sporulation in M. xanthus.

Cell Survival↗
PubMed1989

Rapid flow cytometric bacterial detection and determination of susceptibility to amikacin in body fluids and exudates.

A flow cytometry-based method for rapid and quantitative detection of bacteria in various clinical specimens and for rapid determination of antibiotic effect is described. Achieving such a measurement with high sensitivity required discrimination between bacteria and other particles which were often present in clinical samples in high concentrations. This discrimination was facilitated by detecting the bacterial characteristic light scatter and fluorescence signals following staining, e.g., with the fluorescent nucleic acid-binding dye ethidium bromide, as well as by measuring bacterial proliferation during short time intervals. Antibiotic susceptibility was measured by observing the inhibition of such proliferation. The method was applied to 43 clinical specimens from various sources, such as wound exudates, bile, serous cavity fluids, and bronchial lavage. Bacterial detection, achieved in less than 2 h, agreed with results of conventional methods with a sensitivity of 74% and a specificity of 88%. Susceptibility to amikacin was detected in 1 h in 92% of 13 positive specimens.

Amikacin↗
PubMed1988

Flow cytometric screening and isolation of Escherichia coli clones which express surface antigens of the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1.

Flow cytometry (FCM) in conjunction with immunocytochemical-labeling was used to analyze and screen a population of Escherichia coli clones containing a genomic library from the oil-degrading microorganism Acinetobacter calcoaceticus RAG-1 surface antigens. Reconstruction experiments using mixed populations indicated that RAG-1 cells could be clearly distinguished at a ratio of one RAG-1 cell to 500 Escherichia coli cells. Using this technique two clones, WM143 and WM191, were isolated and shown by restriction endonuclease cleavage and Southern hybridization to contain plasmids carrying inserts of RAG-1 DNA of 9.4 and 9.8 kb respectively.

Acinetobacter↗
PubMed1988

Comparison of NK activity in mouse spleen and peripheral blood lymphocytes.

Natural killer (NK) cells originating in mouse peripheral blood were studied with regard to their lytic activity against YAC-1 target cells and to their expression of asialo-GM1 marker on their surface. In Balb/c, CBA/LAK and A/J mice, PBL were found to be approximately twice as effective as splenocytes. Splenic and peripheral NK cells were shown by flow cytometry to have similar lytic potential per cell; the difference in NK activity found in the spleen and in PBL was solely due to the differences in the size of the NK cell population found in the two sites. Strain distribution of NK activity in PBL followed the same pattern observed in splenocytes. The difference in NK activity between CBA and Balb/c mice was shown to be due to the fact that the lytic potential per NK cell was approximately twice as high in the former.

Animals↗
PubMed1988

Late immune and haemopoietic functions in plasmacytoma-bearing mice cured by melphalan.

Alkylating agents can cause latent and permanent damage to the bone marrow. We compared the long term effects of melphalan on a number of immune and haemopoietic functions of plasmacytoma bearing BALB/c mice with that of normal mice treated with a similar dose of melphalan. The drug administered orally at a dose of 250 micrograms and 400 micrograms on day 14 and 24 following i.m. inoculation of MOPC-315 plasmacytoma cells resulted in cure of the mice. Their spleen cells showed a permanent impairment of MLR activity, T-cell number and IL-2 production as well as a mild suppression of NK activity for one year after cessation of melphalan therapy. The number of B cells was elevated. In contrast, plasmacytoma-free mice treated with melphalan retained long term normal immune functions, although shortly after melphalan therapy a temporary suppression was noted. On the other hand, melphalan was responsible for bone marrow myeloid stem cell damage since the number of myeloid progenitor cell (CFU-GM) colonies was reduced in both melphalan-treated groups compared to untreated normal controls. Plasmacytoma bearing mice had a shorter survival. These results demonstrate that some late sequelae of alkylating agents are not due to the drug alone; shorter survival and T-cell deficiency are related to the previous presence of the tumour.

Animals↗
PubMed1988

Carcinogen-mediated co-activation of two independent genes in Chinese hamster cells.

Following carcinogen treatment, elevated expression levels of dihydrofolate reductase (dhfr) were measured by labeling cells with fluorescent methotrexate which binds to this enzyme. Fractionation of carcinogen-treated, Simian virus 40 (SV40)-transformed Chinese hamster embryo cells (C060) into subpopulations differing in their levels of dhfr expression revealed co-expression, at enhanced levels, of dhfr and SV40 T antigen in the same cells. The increased expression of dhfr was amplification independent, while the T antigen coding sequences were amplified. The co-expression of dhfr and the bacterial chloramphenicol acetyltransferase gene linked to the early SV40 regulatory region was measured in CHO cells stably transformed by pSV2CAT-SVgpt (CC24). Both these sequences were expressed at higher levels in treated cells and the elevated expression levels were observed in the same subpopulation of cells, although no increase in their gene copy number was detected. The concomitant activation of enhanced expression of two independent genes in the same cells suggests that cellular factors governing gene expression are activated in the carcinogen-treated cells. The implications of these findings to cellular control mechanisms and to the carcinogenic process are discussed.

Animals↗