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I Lefkovits

Publications and source records attributed to I Lefkovits.

At least 19 recordsLinked to original sources
PubMed1992

Putative invasion-specific proteins in mouse T-cell hybridomas that differ in invasive and metastatic potential.

Fusion of invasive, activated T-lymphocytes with non-invasive BW5147 T-lymphoma cells mainly yields highly invasive (HI), highly metastatic T-cell hybridomas. In addition, several non-invasive (NI), non-metastatic hybrids have been obtained, probably due to loss of involved gene(s) by chromosome segregation. Here we have compared a panel of HI and NI hybrids in a search for proteins specifically expressed by either cell type. MAbs were raised against HI hybrids, but out of more than 1,000 none bound exclusively to HI cells. Furthermore, polyclonal rat, rabbit and chicken antisera did not immunoprecipitate specific proteins from total lysates, and the expression of 18 (T-cell) surface markers did not correlate with invasiveness. These results indicated that the number of differences between HI and NI hybridomas was surprisingly small. This notion was confirmed by 2-dimensional gel electrophoresis. Among 1,000 detectable spots, we found only 2 clear-cut differences between HI and NI T-cell hybridomas, whereas multiple differences were found between individual hybrids. One protein (p130) was expressed at much higher levels by HI than by NI hybrids in this panel, whereas the other (p15) was only seen in NI hybrids. These proteins are primary candidates for a role in invasion.

Animals↗
PubMed1991

Light chain heterogeneity in the amphibian Xenopus.

Three subpopulations of light chains in Xenopus can be distinguished by monoclonal antibodies as well as by electrophoretic mobility on SDS-PAGE, peptide map and cell surface distribution. Analysis of these proteins from LPS-stimulated lymphocytes culture supernatants by two-dimensional gel electrophoresis showed a heterogeneity comparable to that observed for mouse kappa light chains. However, evidence from the selective expression of light chain subpopulations, as well as highly restricted light chain representation in anti-DNP antibodies, supports earlier findings that an antibody response in Xenopus is greatly limited in heterogeneity.

Animals↗
PubMed1990

A strategy for founding a global lymphocyte proteinpaedia and gene catalogue.

In this article, Ivan Lefkovits, John Kettman and Christopher Coleclough describe a strategy for a comprehensive analysis of the genes active in lymphocytes. It allows direct access to the coding sequence of any gene, provided that the two-dimensional gel spot coordinate position is known. The authors have constructed representative lymphocyte cDNA libraries and partitioned them into many sectors, each containing a unique set of cDNA clones. Data from the analysis of natural lymphocyte proteins is compiled into a 'proteinpaedia', which is matched to a gene catalogue comprising the partitioned recombinant phage collection.

Animals↗
PubMed1990

Molecular analysis of genetically determined target organ abnormalities in spontaneous autoimmune thyroiditis.

We have shown in earlier studies, that the development of spontaneous autoimmune thyroiditis (SAT) in chickens of the Obese strain (OS) depends on the presence of both, two dominant genes coding for an altered immune regulation and one recessive gene responsible for the susceptibility of the target organ for the autoimmune attack. The product(s) of the latter is (are) still not known. The present study was aimed at identifying possible candidates of cellular components of the thyroid gland of OS chicken and its SAT susceptible parental Cornell C-strain (CS) by high resolution 2-dimensional (2D) gel electrophoresis. For this purpose organ cultures of the thyroid, bursa, thymus and liver were established and the synthesized polypeptides were labelled by 35S-methionine. OS and CS organs were compared with those of healthy normal White Leghorn (NWL) controls. The autoradiographs of the 2D-gels obtained from individual samples after various labelling periods were subjected to comparative analysis. We have found both quantitative and qualitative differences of polypeptide spots between OS/CS and NWL organ samples, some of them specific for the thyroid gland. Although one has to be aware that in this multidimensional analytical approach numerous, still elusive pattern differences are revealed, the thyroid specific phenomena will be further scrutinized.

Animals↗
PubMed1990

Regulation of mRNA abundance in activated T lymphocytes: identification of mRNA species affected by the inhibition of protein synthesis.

Inhibition of protein synthesis has often been observed to increase the concentration of mRNAs that encode proteins associated with the regulation of cell division. As two-dimensional gel electrophoresis permits the simultaneous monitoring of individual elements in large populations of gene products, we have used this technique to assess the effect of cycloheximide treatment on the mRNA complement of activated mouse T cells in an objective fashion. Two-dimensional gels of proteins generated by cell-free translation of mRNA from T-cell blasts display about 400 spots; only 5 of these are reproducibly enhanced by cycloheximide treatment and about 4 are diminished. The cDNA cloning vector lambda jac allows analysis of large arrays of molecular clones by cell-free expression, and we have used it in a sibling selection scheme to isolate a clone of one of the prominently induced mRNA species, which we refer to as chx1. chx1 mRNA concentration is increased by cycloheximide treatment of activated B cells, as well as T cells, and it is rapidly and transiently induced, in a cycloheximide-enhanced manner, upon serum stimulation of resting 3T3 fibroblastoid cells. The chx1 protein is hydrophilic, is slightly basic, and has patches of homology with the Jun-D gene product. The chx1 gene is remarkable in its lack of detectable introns and of strong bias against CpG dinucleotides.

Amino Acid Sequence↗
PubMed1990

Polyoma-induced thymic epithelial tumors: analysis by 2D gel electrophoresis of tumors upon labeling the entire tumor bearing host.

Injection of neonatal C3H/Bi mice with polyoma virus (PTA-5) results after 4 to 20 weeks in the development of thymic epithelial tumors in 2/3 of the mice. These tumors appear to be the result of a neoplastic proliferation or transformation of thymic epithelial cells. The tumors contain immature lymphoid infiltrates characteristic of normal tissue and express the same function as normal tissue by providing the microenvironment for T cell maturation. To establish more comprehensively the relation of neoplastic to normal thymic epithelial cells, tumor-bearing mice and their normal counterparts were labeled in vivo by injection of S35-L-methionine. The stroma and lymphoid cells were separated and analyzed by two dimensional gel electrophoresis followed by radiofluorography. It was found that the epithelial portion of the tumor differed in several structural polypeptide components from normal tissue while the polypeptide composition of the thymocyte population remained unaltered. It was concluded that the neoplasia is confined to the epithelium compartment and the function of the neoplastic organ is maintained.

Animals↗
PubMed1989

Immunoglobulin diversification in bursal duct-ligated chickens.

The role of external antigen contact on immunoglobulin (Ig) diversification occurring in chicken bursal cells was evaluated. The entry of environmental antigens into the lumen of the bursa of Fabricius was prevented by ligating the bursal duct prior to hatching (BDL: bursal duct ligation). We used two-dimensional gel electrophoresis to compare the heterogeneity of Ig molecules from bursa cells of normal and BDL chickens. We have found that Ig diversity obtained from BDL chickens' bursae in two-dimensional gel analysis was similar to that of control birds. Furthermore, by using two monoclonal anti-idiotype antibodies to study intrabursal Ig diversification we have shown that frequencies of the Cld-1 and Cld-2 idiotypes were also unaltered following bursal ligation. We conclude that primary B cell diversification in the bursa is independent of the external antigen flow from the bursal lumen.

Age Factors↗
PubMed1989

Consecutive radiofluorography and silver staining of two-dimensional gel electrophoretograms: application in determining the biosynthesis of serum and tissue proteins.

The methodology for conventional radiofluorography of two-dimensional gels, followed by rehydration of the gel and subsequent silver staining, is described. The image obtained by radiofluorography is referred to as biosynthetic image, and the image obtained by silver staining as constitutive image. Since the two images are already in close register (the same gel), reliable identification of polypeptides by the two different assays is possible, and the comparison provides valuable information on the catabolism of each entity. The utility of this procedure is illustrated in experiments involving a labeling with L-[35S]methionine of an entire mouse. Both serum and tissue samples were analyzed by two-dimensional gel electrophoresis with the aim of determining several categories of polypeptides in terms of their biosynthetic rates and their composition.

Animals↗
PubMed1988

Prospective partition analysis of independently assorted sets. Accessory elements supporting clonal lymphoid activation by mitogens.

Accessory cells for mitogen-induced clonal proliferation of lymphocytes can be found after in vitro culture of murine spleen cells. Mitogenic activation of T cells (concanavalin A) depends on such accessory cells. When the cultures in which the accessory cells developed are partitioned, assortment of the accessory cells in cultures is found. When small numbers (1-10) of responder cells, T cells, are dispensed into the above cultures, clonal growth of the T cells is achieved. Neither the dose-response profile of accessory cells nor that of responding cells shows single hit kinetics. Re-categorizing the data set reveals that only cultures with over 20 adherent cells were likely to promote T cell growth and single hit kinetics for the responding cell clone and the accessory cell clone were obtained.

Animals↗
PubMed1988

Immunoglobulin diversification in embryonic chicken bursae and in individual bursal follicles.

Previous studies have shown that the same immunoglobulin (Ig) V lambda gene (V lambda 1) is rearranged in all chicken B cells, and that extensive sequence diversification of this gene occurs during B cell development in the bursa of Fabricius. We used two-dimensional gel electrophoresis to compare the heterogeneity of Ig lambda light chains produced by B cells at different stages of bursal development. Somatically diversified light chains were observed in Ig molecules produced by bursal cells as early as 15 days of embryonic incubation. The two principal species of light chain observed probably represent glycosylated and nonglycosylated forms of lambda chain encoded by alleles of a single lambda gene. Extensive diversification was observed during late embryogenesis. We also studied lambda light chain diversity in cyclophosphamide-treated birds repopulated with normal bursal cells. In these birds, individual bursal follicles are repopulated by single B cell precursors. Follicular cells derived from single B cell precursors were able to produce a spectrum of light chains almost as diverse as that of the total bursal cell population. We used two monoclonal anti-idiotype antibodies to study idiotype expression in individual normal or reconstituted follicles. About 30% of follicles contained 0.1% to 5% of lymphocytes which reacted with one or both of the antibodies. The results indicate that within individual bursal follicles bursa stem cells undergo Ig hyperdiversification.

Animals↗
PubMed1988

Toward an objective classification of cells in the immune system.

The relative abundance of individual proteins shared among clones of lymphocytes provides a meaningful basis for cellular classification. Twelve clones of T cells (obtained by limiting dilution) were analyzed by two-dimensional gel electrophoresis for polypeptide content and then evaluated by the computational technique known as principal component analysis. As a result, relatedness of the clones was established and expressed in terms of taxonomic distances. The data show that a comprehensive and objective classification of the cells involved in the immune system can be approached.

Animals↗
PubMed1988

Increased helper cell activity of NZB mice against H-2-identical allogeneic cells.

The T cells of NZB mice become hyperreactive after stimulation with minor histocompatibility (MIH) antigens. This hyperreactivity has previously been demonstrated only for cytotoxic T cells of NZB, although there was some evidence for an increase of their T-helper cell activity facilitating the response. Here we report a quantitative analysis of T-cell help and help of T-cell subpopulations against autologous, MIH, and H-2 antigens in a limiting dilution assay. After stimulation of NZB T cells with autologous and H-2 antigens, the T-helper cell frequencies did not differ from that of normal mice. After stimulation with MIH antigens however, Lyt 1+2+ T-cells of NZB showed a higher response than those of BALB/c origin. The same difference was seen after prestimulation with ConA or the specific antigen. This demonstrates that NZB-helper cells are more easily activated by weak antigenic differences, and it is possible that this contributes to the prevalence of autoimmune disease in this strain.

Animals↗
PubMed1987

In vivo priming of helper and suppressor T cells by alloantigens. Frequency analysis with the use of an in vitro limiting dilution assay.

Earlier studies have demonstrated that T cells activated in mixed lymphocyte reactions can exert positive as well as negative allogeneic effects on B cells expressing the appropriate alloantigens on their surface. We investigated the effect of in vivo priming of T cells with alloantigens on their capacity to help or suppress allogeneic B cell cultures against sheep erythrocytes. We used immunization protocols that have been shown to be optimal for induction of alloantigen-specific delayed-type hypersensitivity (DTH) and alloantigen-specific suppressor T (Ts) cells for DTH. The results show that in vivo stimulation with alloantigens, depending on the immunization route and the lymphoid organ studied, can be as effective as in vitro stimulation in increasing the frequency of alloantigen-specific helper T (Th) cells and Ts cells. Subcutaneous immunization induced a 10-fold frequency raise of Th cells as well as of Ts cells in the lymph nodes. In the spleen the Th cell population was hardly affected by s.c. immunization, whereas the Ts cell population increased by at least a factor 20. Intravenous immunization, on the other hand, selectively expanded the Th cell population in the spleen, whereas the splenic Ts cell population and the Th and Ts cells in the lymph nodes were not affected. Comparison of these results with our previous data concerning characteristics and the requirements of in vivo activation of alloantigen-specific DTH reactive T cells and of alloantigen-specific Ts cells suggest that different Ts cell populations are involved in suppression of alloantigen-specific DTH in vivo and of allogeneic suppression of in vitro induced sheep erythrocytes specific antibody formation.

Animals↗
PubMed1986

Mitogenic activation of B cells in vitro: the properties of adherent accessory cells as revealed by partition analysis.

The requirement of B cells activated by mitogen (dextran sulfate plus lipopolysaccharide) for accessory cells was studied by partition analysis. Small numbers of splenic B cells were activated to clonal growth, as determined by visual inspection, and to immunoglobulin (Ig) synthesis, as determined by release of Ig into the culture fluid. By placing irradiated adherent cells in the periphery of the microculture wells and forcing responding cells to different areas of the well (slant experiments), it was observed that no cell contact was necessary for B cell activation, and that "promoted" contact ("Rock and Roll" experiments) does not increase the efficiency of activation. Sequential microcultures suggest that only some irradiated adherent cells act as accessory cells, but they can perform this function to more than one B cell. Attempts to perform limiting dilution analysis by varying irradiated adherent cell input showed non-single-hit behavior. When the data were rearranged, taking into account the distribution of irradiated adherent cells, then single-hit behavior with about 1 to 5% of irradiated adherent cells acting as an accessory cells for B cell clonal activation was observed. The evidence suggests that an uncommon irradiated adherent cell releases a soluble factor necessary for B cell activation and/or clonal proliferation.

Animals↗
PubMed1986

Parameters of the labeling of mitogen-activated murine lymphocytes by [35S]methionine for two-dimensional gel electrophoresis. I. Effect of culture conditions.

Labeling with [35S]methionine at a high specific activity is essential to the facile preparation of 2-dimensional gel electrophoretograms with the analytical 2-dimensional charge-size separation procedure (Anderson's ISODALT system). Mitogen-activated T and B lymphocytes subjected to low methionine concentrations would not proceed through cell cycle. In the case of activated B lymphocytes, the use of fetal bovine serum (FBS), dialyzed to lower endogenous methionine concentrations, prevented B cell growth even in the presence of otherwise satisfactory levels of methionine. High concentrations of [35S]methionine (greater than 300 mCi/1) induced B cell death, apparently by radiation damage. Despite these problems, good radioautograms and radiofluorograms of 2D electrophoretograms could be prepared by labeling activated B or T cells in bulk (10(6) cells/ml) with high specific activity [35S]methionine. The polypeptides labeled may be a biased sample since lymphoid cells do not proceed through cell cycle under these conditions. Small numbers (10(3] of activated T cells also yielded satisfactory samples but labeling of small numbers of activated B cells was not possible.

Animals↗
PubMed1986

Analysis of the activation of lymphoid cells by mitogens in vitro with limiting dilution methods: adherent peritoneal cells suppress B-cell activation and synergize with WEHI-3 in the activation of T cells by Con A.

Adherent peritoneal cells (APC) have often been used as a pure and effective macrophage population. Using partition analysis and small numbers of lymphoid cells activated by mitogens (concanavalin A for T cells (in the presence of TCGF) and LPS + DxS for B cells) we found that APC were accessory cells for T cell activation and growth but were not effective for B cells. Although APC were effective in assisting T-cell mitogenesis, they were not especially efficient. However, when APC were mixed with irradiated WEHI-3 cells (a tissue culture line previously shown to exhibit accessory cell activity in vitro for mitogenic activation T and B cells), the APC and WEHI-3 showed apparent synergy. One reason for failure of APC to assist B-cell mitogenesis was traced to the presence of a suppressor cell population which overcame the accessory cell help given by irradiated WEHI-3 cells to LPS-DxS stimulated murine B cells. It is thus possible to find "helper" effects (synergy of APC and WEHI-3 assisting the mitogenesis of T cells), as well as suppressor effects within the range of cells found in adherent accessory cells.

Animals↗