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Biomedical subjects

I Lefkovits

Publications and source records attributed to I Lefkovits.

At least 73 records · Page 4Linked to original sources
PubMed1984

Changes in the protein pattern of murine B cells after mitogenic stimulation.

The technique of two-dimensional gel electrophoresis has been applied to study the changes in protein patterns occurring in murine B lymphocytes after activation with the synergistic B cell mitogens lipopolysaccharide and dextran sulfate. Whole cell lysates from [35S]methionine-labeled mitogen-activated B cells were analyzed and radiofluorographs of the gels compared. We found that a large number of proteins appear and disappear during the 5-day activation period (74/206 spots of a nonimmunoglobulin region of the gel). The B cell pattern (in the nonimmunoglobulin region) was also compared with that of mitogen-activated T cells. We identified 19 T cell-specific and 29 B cell-specific proteins, the remaining 1000 or so proteins being common.

Animals↗
PubMed1984

Optimization of the culture medium composition for the antibody response of mouse spleen cells.

The Mishell-Dutton culture system for in vitro primary antibody response of mouse spleen cells was used to optimize the amino acid composition of RPMI 1640 media. Each of the 20 amino acids was tested over a broad range of concentrations always leaving the remaining 19 amino acids unaltered (i.e. at the formula recommended concentration). In several instances, higher plaque-forming cell responses were obtained with an amino acid concentration that was either higher or lower than that recommended: (a) the optimum concentration for valine, glutamine, and lysine lies considerably above the recommended one, (b) the optimum concentration for leucine as well as for several other amino acids lies below the recommended concentration, and (c) the optimum concentration for arginine corresponds exactly to the recommended concentration. The second round of optimization, i.e. combining of two conditions that individually yielded an improved response often caused a decrease of response. The possibility is discussed that for an optimal response a ratio of two or several amino acids rather than the absolute concentration of any one amino acid is of importance.

Amino Acids↗
PubMed1984

An approach to molecular analysis of T-cell clones by two-dimensional gel electrophoresis: is there interclonal and intraclonal heterogeneity?

To understand the regulation of immunity one must understand the processes and the consequences of the activation of the lymphoid cell, its clonal proliferation, and the ultimate differentiation of the progeny into the effector elements that constitute the immune system. By use of polyclonal mitogens, individual T-cell precursors can be activated for study of their clonal growth and generation of T effector cells. We used two-dimensional gel electrophoresis to study the gene products expressed by such T-cell clones. On comparing the radiofluorograms of such gels for a large set of T-cell clones we find that many gene products are expressed in common, as expected, but that another large set of products varies from one clone to the other and might be designated as markers of T-cell subsets or stages of differentiation. By subdividing the growing clone and analyzing the family of daughter clones, we find considerable variation in gene expression during clonal expansion.

Animals↗
PubMed1984

A metrological study of autoradiographs from two-dimensional gel electrophoresis.

Samples prepared from a single batch of labeled cells were subjected to two-dimensional gel electrophoresis and autoradiography. Three factors were varied: total quantity of protein, quantity of labeled protein, and exposure time. The mean background absorbance of the film remained identical (about 0.5 A) for all the treated series, whatever the exposure time and whether or not there were unlabeled proteins in the sample. Hence any spot with a peak A of the same order of magnitude can be seen. The standard deviation was about 0.05 A. Thus, the measurement precision is 2.5% of full scale for digitalization over 0 to 2 A. We derived experimental calibration curves, which are neither linear nor logarithmic because of the film response and which can be used on randomly chosen spots.

Animals↗
PubMed1984

Two-dimensional gel pattern of proteins synthesized de novo in lymphoid organs of the mouse.

Mice were injected intravenously with 1 mCi of [35S]methionine, and serum and tissue were sampled 4 h later. Serum and lymphoid and non-lymphoid tissues had all incorporated enough labeled methionine to allow radiofluorographic detection on two-dimensional gels of proteins synthesized de novo and, by comparing radiofluorographs, we could distinguish spots peculiar to a given tissue from others more ubiquitous. We selected a protein of 17-kDa apparent molecular mass and pl about 4 to demonstrate tissue specificity. All patterns obtained for thymus samples yielded this spot; in all other immunologically relevant sites it was missing or weak. It also was not detected on gels previously obtained from lymphocyte subpopulations biosynthetically labeled in vitro. The labeling method described here will be especially helpful for characterizing cell populations that cannot be radiolabeled under cell-culture conditions. In contrast to detection methods that detect all proteins of a sample, in vivo labeling allows specific recognition of de novo synthesized proteins. Therefore it will facilitate comparison and detection of proteins produced by an animal in response to a given treatment.

Animals↗
PubMed1983

Effect of whole-body irradiation of mice on the number of background plaque-forming cells.

Mice were exposed in whole-body fashion to several doses of radiation and killed at various times thereafter for a determination of the number of background plaque-forming cells (PFCs) as assayed on either sheep erythrocytes or bromelain-treated autologous mouse erythrocytes. Increased numbers of both types of PFC were found in the irradiated groups. These increases were dependent on radiation dose and time after exposure. They did not appear to be caused by a disruption of normal lymphocyte traffic or a switch in immunoglobulin isotype. An increased number of PFCs on bromelain-treated mouse RBCs but not on sheep RBCs were found in irradiated congenitally athymic nude mice. On the basis of this and related observations, background PFCs on bromelain-treated mouse RBCs and on sheep RBCs appear to fall under different forms of homeostatic control.

Animals↗
PubMed1981

Helper T-cell-replacing factors and T-cell-growth factors produced by concanavalin A-stimulated and pokeweed mitogen-stimulated mouse spleen cells.

Variables affecting helper factor induction by two T-cell mitogens, concanavalin A (Con A) and pokeweed mitogen (PWM), were examined. It was found that both factors are capable of reconstituting T-cell-dependent antibody response as well as enhancing mitogen-stimulated thymocyte proliferation. Detectable factor production is obtained within 6-12 h of culture; Con A factor production reaches a peak usually around 24 h, while no factor activity is detectable after 72 h of culture period. PWM factor production persists throughout 72 h of culture. Con A factor production can be rescued at culture periods beyond 24 or 48 h by using supraoptimal Con A doses. Con A factor, if incubated at 37 degrees C under cell-free conditions in a serum-free medium, shows a rapid rate of decay; in the presence of cells the decay is considerably reduced.

Animals↗
PubMed1980

Radiation-induced augmentation of the immune response.

Radiation-induced augmentation of the immune response has been shown to occur both in vivo and in vitro. Evidence is presented to implicate injury to an extremely radiosensitive T cell in the expression of this phenomenon. Experiments are outlined which could be employed to support or reflect this hypothesis.

Animals↗
PubMed1980

Limiting dilution analysis of the suppressive effect mediated by alloantigen-primed cells.

T cells primed in mixed lymphocyte culture exert both positive and negative allogeneic effects on B cells expressing the appropriate alloantigens. The positive and negative effects can be separated by limiting dilution analysis: positive effects, measured by production of anti-sheep erythrocyte antibody, are revealed when low numbers of primed T cells are added to cultures of B cells and sheep erythrocytes, while suppression of the response occurs at higher T-cell inputs. In the present report, these negative allogeneic effects have been analysed in detail. Suppression was qualitatively and quantitatively similar when helper T cell activity was provided from any of several sources. Helper T cells in the alloantigen-primed population gave rise to active T-cell replacing factors even under conditions in which all microcultures were suppressed and suppressor cells were present at a high multiplicity in every well. The degree of suppression was influenced by the multiplicity of B cells in culture; as the number of B cells increased, more suppressor cells were required to inactivate a microculture. Taken together, these data indicate that the targets of the suppressor cells are B cells and not helper T cells or T-cell replacing factors. Although suppressor cells can prevent the activation of B cells by the more frequent helper cells in the primed T-cell population, detailed analysis of the stoichiometry of the suppression demonstrated that a single suppressor cell is capable of inactivating only a limited number of B cells, suggesting that a 'ratio-dominance' model of suppression is operative in this system.

Animals↗
PubMed1980

Effects of irradiation on the in vitro immune response.

Primed and unprimed murine spleen cells and suspensions enriched for T or B cells were exposed to various doses of radiation in vitro and evaluated with respect to their capacity to respond to sheep red blood cells (SRBC) in 1-ml or 10-microliter cultures. The dose-response data show three components: (1) a low dose (less than 50 rad) phase associated with an augmented anti-SRBC response; (2) a radiosensitive phase associated with precipitous loss of anti-SRBC activity; (3) a relative radioresistant phase with a loss of anti-SRBC activity which occurs over a broad range of radiation doses. Data obtained utilizing irradiated subpopulations enriched for T or B cells combined with excess numbers of nonirradiated lymphocytes of the corresponding cell type suggest that low dose augmentation is the result of radiation-induced injury to T cells or subcomponent thereof. With respect to the second component of the dose-response curve, similar experiments show that B cells are more radiosensitive than T cells (D37 of approximately 80 vs. approximately 220 rad, respectively) in this response. The third component of the curve appears to involve the interaction of radio-resistant subpopulations of T and B cells.

Animals↗
PubMed1979

T cell activation of primed rabbit blood lymphocytes by antigen.

Activation of primed rabbit PBL by homologous antigen in the early proliferative phase (on days 3--5) mainly involves lymphocytes which neither secrete specific antibody nor contain immunoglobulin in their cell membrane. This stimulation is antigen-specific, and evidence is given that the major proportion of these cells are T lymphocytes. The B cells forming Av-CHO-specific PFC were studied by autoradiography on days 6 and 12 of culture. Since incorporation of radioactive thymidine was found in the majority of PFC, these cells are also in proliferation.

Animals↗
PubMed1979

The purification and quantitation of helper T cell-replacing factors secreted by murine spleen cells activated by concanavalin A.

A microculture assay with low numbers of athymic (nude) spleen cells has been used to quantitate the helper T cell-replacing activity secreted by concanavalin A-treated spleen cells. This quantitation allows an estimate of the recovery of biologic activity during concentration from culture supernatants by salt precipitation, and purification by gel filtration, ion exchange chromatography, and isoelectric focusing. The T cell-replacing activity is found in protein of 30 to 40,000 daltons size as estimated by gel filtration, but shows heterogeneity in electric charge. The activity is active at concentrations of less than 10(-9) M, and is strictly antigen dependent in its mode of action.

Ammonium Sulfate↗
PubMed1979

Induction of suppression and help during in vitro immunization of mouse spleen cells.

Both specific and non-specific suppressor cells are induced during the in vitro culture of mouse spleen cells in Mishell-Dutton-type cultures. The induction of T cells capable of providing helper activity does not require the presence of antigen during the culture period. In a mixture of helper and suppressor cells, as generated during the culture of spleen cells in the presence or absence of antigen, suppression is dominant if tested on normal spleen cells. This same cell mixture can reveal potent T-cell help if added in low numbers to T-cell depleted B cells in the presence of antigen. Partitioning of this cell mixture in a microculture system reveals potent T-cell help. Up to 1/600 recovered viable cultured mouse spleen cells can provide T-cell help for an anti-SRBC response. Evidence points to a coupled induction of non-specific help and suppression.

Animals↗
PubMed1979

Frequency of allogeneic helper T cells responding to whole H-2 differences and to an H-2K difference alone.

Limiting dilution analysis was used to determine the frequency of splenic T cells that are stimulated by alloantigen to give help in a primary antibody response to SRBC. Several haplotype combinations were tested. A semilogarithmic plot of the fraction of nonresponding culture as a function of the number of T cells added to excess B cells gave a straight line intercepting with the origin. Thus a single cell-type was limiting, which was required to help B cells respond to SRBC. The frequency of syngeneic precursors of T helper cells specific for SRBC ranged from 1/10,000 to 1/55,000 with a mean of about 1/20,000. Allohelpers generated by whole H-2 differences gave precursor frequencies that ranged from 1/1000 to 1/7000 with a mean of about 1/2500. Thus allohelpers to whole H-2 differences were approximately 8-fold more frequent than SRBC-specific helpers. When the stimulation was limited to the H-2K difference between the mutant B6.C-H-2ba and wild-type B6, frequencies of from 1/2600 to 1/7900 allohelpers were found with a mean of about 1/5000, approximately half the frequency of allohelpers to whole H-2 differences. Thus some, but probably not all, of the magnitude of allogeneic halp can be attributed to the high frequency of helper T cells that respond to a given alloantigen.

Animals↗
PubMed1979

In vitro evaluation of radiation-induced augmentation of the immune response.

Small doses (5--25 rads) of radiation augment the in vitro response of murine spleen cells to sheep red blood cells (SRBCs). Such augmentation appears to result from radiation-induced disruption of a homeostatic component of the response that exerts maximum effect soon after the introduction of antigen. Evidence is presented to support the concept that augmentation is due to injury of an exquisitely radiosensitive subpopulation of T cells with suppressor activity.

Animals↗
PubMed1978

Activation of primed rabbit blood lymphocytes by antigen: early phase of triggering and the specificity of the response.

The early phase of antigen-dependent triggering of rabbit blood lymphocytes (PBL) is described. Specific activation of PBL by streptococcal vaccines requires primed lymphocytes from high responder rabbits. B-cell memory is conferred by Ig receptors by both mu and gamma chains. Antigen-induced incorporation of leucine and thymidine reach peak values at day 5, with stimulation indices of 10 to greater than 100, closely followed by increases in the number of living cells. A second and less intensive phase of cell proliferation is seen on days 8--11. A large fraction of cells (between 3 and 33%) participates in proliferation. A rough calculation of the average doubling time of cells during the exponential growth phase gives values of 8--16 h.

Animals↗