Search PubMed⌕ Search

Biomedical subjects

K Shortman

Publications and source records attributed to K Shortman.

At least 163 records · Page 9Linked to original sources
PubMed1979

Cytotoxic and fluorescent assays for thymocyte subpopulations differing in surface thy-1 level.

A number of analytical techniques for distinguishing and separating the "high theta" "low theta" subpopulations of mouse thymocytes have been compared. A differential cytotoxic assay was compared to a quantitative immunofluorescent assay on individual cells using flow cytofluorometry and cell sorting. Conventional anti-Thy-1 antisera were compared with a monoclonal IgM anti-Thy-1. The monoclonal reagent greatly improved both types of assay, eliminated a number of artifacts and allowed either procedure to be used to give a clear distinction, based on Thy-1 level, between the two subpopulations. The distribution of Thy-1 on thymocytes is bimodal, rather than continuous. These separate "high theta" and "low theta" categories each includes a population a population of dividing cells.

Animals↗
PubMed1979

The differentiation of T lymphocytes. V. Evidence for intrathymic death of most thymocytes.

The DNA of CBA mouse thymocytes was simultaneously labelled with the efficiently reutilized precursor [3H]TdR and the poorly reutilized precursor [125I]UdR, and the subsequent rate of loss of the two thymidine analogues was used to compute the extent of local thymidine reutilization. This should provide a minimum estimate of thymocyte death within the thymus. In agreement with published data, reutilization of thymidine in the "steady state' adult mouse thymus was about 61%. Adrenalectomy was used to show that this turnover was not due to steroid mediated stress effects; nor could it be attributed to direct effects of [125I]UdR itself. Most of the initial uptake and the subsequent turnover was within the major "high theta' thymus subpopulation. A similar study with the growing thymus of 7-day-old mice, when the demand for T cells in the periphery should be high, also revealed a high level (63%) of thymidine reutilization. The results support the view that a minimum of 60% of newly formed thymocytes die within the tyhmus, and that this is a constant and normal aspect of thymus function. This would be compatible with some form of negative selection for the appropriate developing T cells.

Animals↗
PubMed1978

Some requirements for a linear cell dose response in vitro assay for the T-cell progenitors of cytotoxic lymphocytes.

To develop a precise assay for the T-cells progenitors of cytotoxic lymphocytes (CL-progenitors), lymphoid were cultured under optimal conditions in Marbrook vessels with mitomycin-treated allogeneic stimulator cells, and the total level of CL produced 5 days later estimated by a modified 51Cr release assay. Conditions were adjusted so an arithmetically linear cell dose response relationship was obtained. Three aspects of the cell dose response curve required attention. (1) At low responding cell inputs a macrophage-like cell became limiting (despite the presence of allogeneic macrophages in the stimulating cell population), leading to a lag in the response. This limitation was overcome by adding a low level of irradiated syngeneic macrophages, or by using irradiated syngeneic spleen 'filler' cells. (2) The slope of the resultant linear dose response region could be reduced if desired by changing from cellophane dialysis membranes to 0.1 mu pore size nuclepore membranes, suggesting a stimulatory role for some higher molecular weight soluble factor produced in the cultures. (3) At higher responding cell inputs a marked and extensive plateau was obtained. CL developing early in the response appeared to be destroying the allogeneic stimulator cells causing the response to be self-limiting. This problem was overcome by using a responding cell concentration lower than commonly employed. Assays using mixed leukocyte cultures in the lag or plateau regions could give misleading vlaues for CL-progenitor activity. It is suggested that some examples of apparent synergism in CL generation may have resulted from these effects, rather than T-cell helper T-cell progenitor interactions.

Animals↗
PubMed1978

The differentiation of T lymphocytes. IV. Net increases in low theta cells, maintenance of biological activity and death of high theta cells during short-term culture of mouse thymocytes.

Dissociated mouse thymocytes were cultured under optimized conditions in Marbrook vessels in order to follow some aspects of T-cell differentiation. Under particular conditions it was possible to obtain a 50% net increase in the total number of the peripheral T-cell like, minor low theta thymocyte subpopulation over the first day of culture, although this increase was lost with further incubation. The increase arose from proliferation of preexistent low theta cells,and not from transformation of the major, high theta population. Under all conditions the high theta cells died rapidly in culture. These results are in accordance with the view that the high theta and low theta thymocytes represent separate streams of T-cell development. There was a striking increase in responsiveness to phytohemagglutinin and concanavalin A after culture of thymocytes under these conditions. However, this was ascribed, not to the development of new immunocompetent T cells, but to selective cell death in the cultures eliminating some inhibitory elements. No increase in progenitors of cytotoxic lymphocytes was obtained. The results demonstrate some potential artifacts in assessing the immunocompetence of cultured thymocytes.

Adrenal Glands↗
PubMed1978

Antigen-initiated B lymphocyte differentiation. XIV. Nonspecific effects of antigen stimulation cause proliferation in the "pre-progenitor" subset of primary B cells.

The effect of specific and nonspecific stimuli on the cycle status of subsets of primary B lymphocytes was assessed by preinjecting donor CBA mice 1 to 2 days previously with various substances, and then incubating the isolated spleen cells with high specific activity 3H-TdR before assay. AFC-progenitor activity was assessed as a response to NIP-POL antigen, either by adoptive transfer to irradiated recipients or by cell culture. Previous studies showed these assays reflected the activity of different subsets of B cells, termed "pre-progenitors" (adoptive assay) and "direct progenitors" (culture assay). Most functional primary B cells, whether assayed in culture or by adoptive transfer, were not initially in rapid cell cycle in normal adult mice. However, nonspecific stimulation for 1 day caused NIP-specific adoptive transfer IgM AFC-progenitors to enter rapid cell cycle. This effect was independent of T cells and not related to the antigenicity of the stimulus: particulate peritoneal irritants were the most effective stimulants. In contrast to adoptive transfer results. AFC-progenitors assayed in cell culture were unaffected by nonspecific stimuli, but were activated into cell cycle by specific antigen.

Animals↗
PubMed1977

Some requirements for the response of separated T-cell sub-populations to the mitogens phytohaemagglutinin and concanavalin A.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low theta sub-population appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high theta thymocyte population.

Animals↗
PubMed1977

Antigen-initiated B lymphocyte differentiation. IX. Characterization of memory AFC progenitors by buoyant density and sedimentation velocity separation.

The characteristics of memory B cell antibody-forming cell (AFC) progenitors from long-term hapten-primed CBA mice were investigated by using sedimentation velocity and buoyant density separation to isolate physically distinct B cell sub-sets. The isolated fractions were assayed by the adoptive immune response to NIP-POL antigen, under conditions where neither T cells nor other accessory cells were limiting the IgM or IgG AFC responses. The results were compared to previous studies on the IgM AFC-progenitors of unprimed adult mice. Splenic IgM and IgG memory AFC-progenitor activity was largely found among the typical B cells of slow to medium sedimentation rate, in contrast to the fastre sedimenting IgM AFC-progenitor activity of unprimed animals. Splenic IgM and IgG memory AFC-progenitor activity was found among the medium to light density cells, and so resembled by this parameter the IgM AFC-progenitor activity in unprimed animals. Thoracic duct lymphocytes from hapten-primed mice also exhibited memory IgM and IgG AFC-progenitor activity in the slow-medium sedimentation range. However, in contrast to spleen, the IgM and IgG memory AFC-progenitor activity in lymph was found among very dense B cells. Two physically distinct sub-populations of memory B cells have thus been identified, namely: i) small, medium-light density, presumably tissue-resident B lymphocytes found in spleen; and ii) small, dense, presumably recirculating B lymphocytes found in lymph. Both physical forms include IgM and IgG progenitors. Both forms are distinct from the larger, medium-light density "virgin" AFC-progenitors in the spleen of unprimed adult mice.

Animals↗
PubMed1976

Antigen-initiated B-lymphocyte differentiation. VIII. Sedimentation velocity and buoyant density characterization of virgin antibody-forming cell progenitors in the adoptive immune response of unprimed CBA mice to 4-hydroxy-3-iodo-5-nitrophenylacetic acid-polymerized bacterial flagellin antigen.

The characteristics of antibody-forming cell (AFC) progenitors lacking previous contact with specific antigen (virgin AFC progenitors) has been studied using sedimentation velocity and buoyant density separation for the investigation of physically distinct B-cell subpopulations. Functional characterization of isolated subsets was made using a quantitative adoptive immune assay for the IgM AFC progenitors responding to the antigen 4-hydroxy-3-iodo-5-nitrophenylacetic acid conjugated polymerized bacterial flagellin. Extensive heterogeneity is present among B lymphocytes, only some subpopulations of which exhibit AFC progenitor function. In the spleen of adult conventional CBA mice, atypically fast sedimenting cells of low buoyant density are active, while typical small B lymphocytes do not appear to be progenitors of IgM AFC. Spleen of adult specific pathogen-free (SPF), germfree, and athymic nude mice give similar results, although a minor population of typical slowly sedimenting dense cells are active in the latter two sources. Adult conventional bone marrow cells are as physically and functionally heterogeneous as splenic B cells, and although a significant proportion of AFC progenitor activity is found among dense, slowly sedimenting cells, most of the activity is among low density, faster sedimenting cells. In contrast to this situation in adult animals, where most of the unprimed AFC progenitors are large, atypical B cells, the spleens of neonatal mice provide a site where virgin AFC progenitors with the physical properties of typical small B lymphocytes are found. While being present in conventional and SPF neonatal spleens, these virgin cells are predominant in 7-day-old germfree mouse spleen. These findings suggest that the newborn virgin B cell is a typical small lymphocyte. However, few cells of this type are found in the adult animal. The unprimed AFC-progenitor population in the adult consists of large, fast sedimenting, low buoyant density, adherent cells, the physical properties of which are characteristic of activated B lymphocytes. It is suggested that these atypical cells are derived from the small newborn virgin B cell by the nonspecific effects of environmental antigenic stimuli.

Age Factors↗
PubMed1976

The nature of the cells generating B-lymphocyte colonies in vitro.

B-lymphocyte colonies are grown in semi-solid agar from mouse spleen or lymph node cells in the presence of mercaptoethanol with or without added sheep red cells. High levels of colony-forming cells were present in the spleen or normal mice and nu/nu (athymic) mice but colony-forming cells were rare in the thymus and not detected in activated T-lymphocyte populations. Colony-forming cells were theta-negative and most exhibited Fc receptors. Most colony-forming cells had the sedimentation velocity of small lymphocytes, were non-adherent and had a buoyant density similar to B-lymphocytes. Colony-forming cells were radiosensitive (Do60 rads) and sensitive to cortisone. Colony formation was potentiated by the addition of adherent spleen cells or peritoneal macrophages. It is concluded that most cells forming B-lymphocyte colonies are themselves characterisable as B-lymphocytes.

Animals↗