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At least 397 records · Page 22Linked to original sources
PubMed1998

Dendritic cell-based vaccines in the setting of peripheral blood stem cell transplantation: CD34+ cell-depleted mobilized peripheral blood can serve as a source of potent dendritic cells.

We are investigating the use of tumor-pulsed dendritic cell (DC)-based vaccines in the treatment of patients with advanced cancer. In the current study, we evaluated the feasibility of obtaining both CD34+ hematopoietic stem/ progenitor cells (HSCs) and functional DCs from the same leukapheresis collection in adequate numbers for both peripheral blood stem cell transplantation (PBSCT) and immunization purposes, respectively. Leukapheresis collections of mobilized peripheral blood mononuclear cells (PBMCs) were obtained from normal donors receiving granulocyte colony-stimulating factor (G-CSF) (for allogeneic PBSCT) and from intermediate grade non-Hodgkin's lymphoma or multiple myeloma patients receiving cyclophosphamide plus G-CSF (for autologous PBSCT). High enrichment of CD34+ HSCs was obtained using an immunomagnetic bead cell separation device. After separation, the negative fraction of mobilized PBMCs from normal donors and cancer patients contained undetectable levels of CD34+ HSCs by flow cytometry. This fraction of cells was then subjected to plastic adherence, and the adherent cells were cultured for 7 days in GM-CSF (100 ng/ml) and interleukin 4 (50 ng/ml) followed by an additional 7 days in GM-CSF, interleukin 4, and tumor necrosis factor alpha (10 ng/ml) to generate DCs. Harvested DCs represented yields of 4.1+/-1.4 and 5.8+/-5.4% of the initial cells plated from the CD34+ cell-depleted mobilized PBMCs of normal donors and cancer patients, respectively, and displayed a high level expression of CD80, CD86, HLA-DR, and CD11c but not CD14. This phenotypic profile was similar to that of DCs derived from non-CD34+ cell-depleted mobilized PBMCs. DCs generated from CD34+ cell-depleted mobilized PBMCs elicited potent antitetanus as well as primary allogeneic T-cell proliferative responses in vitro, which were equivalent to DCs derived from non-CD34+ cell-depleted mobilized PBMCs. Collectively, these results demonstrate the feasibility of obtaining both DCs and CD34+ HSCs from the same leukapheresis collection from G-CSF-primed normal donors and cancer patients in sufficient numbers for the purpose of combined PBSCT and immunization strategies.

Antigens, CD34↗
PubMed1981

Autologous rosette-forming T cells regulate responses of T cells. Phenotypic and functional analysis of suppressor cells generated from autologous rosette-forming T cells after autologous mixed lymphocyte reactions.

An average of 5--9% of human peripheral blood of T lymphocytes from rosettes with autologous erythrocytes (ARFT). This population responded only slightly against autologous and allogeneic non-T cells. In contrast, T cells that did not form rosettes with autologous erythrocytes (NRFT) proliferated to a greater degree in auto- and allogeneic mixed lymphocyte reactions (MLR) and also in reactions to trinitrophenyl (TNP) modified autologous non-T cells (TNP-auto-MLR) as compared with ARFT or unfractionated T cells. The ARFT populations could suppress the increased allogeneic (allo)MLR and TNP-auto-MLR of NRFT when the ARFT were added to the NRFT at the beginning of the cultures. Fluorescence-activated cell-sorter (FACS) analysis of these freshly obtained T cell fractions using monoclonal antibodies to subpopulations of T cells did not demonstrate any selective gain or less of T cell subsets in the ARFT and NRFT as compared with unfractionated T cells. But when each T cell fraction was cultured separately for a week in the presence of autologous non-T cells (auto-MLR) and the cells were again analyzed by fluorescence-activated cell sorter, there was an increase in OKT8-positive cells (suppressor/cytotoxic subset) only in the ARFT fraction. The above findings strongly suggest that suppressor T cells are generated from the ARFT fraction during an auto-MLR, these may then regulate the responses on NRFT.

Antigens, Surface↗
PubMed1988

T cells recognizing polysaccharide-specific B cells function as contrasuppressor cells in the generation of T cell immunity to Pseudomonas aeruginosa.

The cellular interactions involved in the development of T cell-mediated immunity to Pseudomonas aeruginosa have been examined. T cell immunity can be generated by immunizing mice with 10 micrograms of P. aeruginosa polysaccharide (PS) plus the antimitotic agent vinblastine sulfate. Vinblastine is required to inactivate a population of Ts cells generated by immunization with 10 micrograms of PS alone. Immunization with either live bacteria or with higher dose (50 micrograms) of PS without vinblastine also generates T cell immunity; these protocols activate a population of Lyt-1+, 2-, I-J+ T cells which, like vinblastine, counteract the effect of Ts cells. Immunization with 10 micrograms PS alone fails to activate this T cell subpopulation. When administered at the time of immunization, this subpopulation can render the tolerogenic 10-micrograms immunization protocols immunogenic. Like previously described contrasuppressor T cells, this T cell subpopulation exhibits an affinity for the lectin Vicia villosa. We have determined, however, that the T cells that act as contrasuppressor cells in this system are directly activated by PS-immune B cells and not by PS Ag. Furthermore, their activity can be removed by adsorption to PS-specific B cell hybridomas. Our studies indicate an important role for B cells in the development of T cell immunity to P. aeruginosa and suggest that a complex idiotype network controls the development of this response.

Agranulocytosis↗
PubMed1988

T-cell-derived factor B151-TRF1/IL-5 activates blastoid cells among unprimed B cells to induce a polyclonal differentiation into immunoglobulin M-secreting cells.

Two distinct murine B-cell differentiation factors, designated B151-TRF1 and B151-TRF2, were described originally as B151K12 T-cell hybridoma-derived lymphokines that induce immunoglobulin (Ig) secretion by antigen-activated B cells and unstimulated B cells, respectively. In the present study, we found that a highly purified B151-TRF1 fraction prepared by reversed-phase high-performance liquid chromatography (RP-HPLC) also has the ability to cause a polyclonal differentiation of unstimulated B cells into IgM-secreting cells in the apparent absence of co-stimulant. The activity of the B151-TRF1 fraction but not the B151-TRF2 fraction on unstimulated B cells was markedly inhibited by addition of a monoclonal antibody (mAb) specific for the B151-TRF1/IL-5 to the culture. To determine whether B151-TRF1/IL-5 and B151-TRF2 act on distinct populations among unstimulated B cells, the responsiveness of neonatal B cells and adult B cells that had been fractionated by Percoll density gradient centrifugation was assessed. B151-TRF1/IL-5 predominantly acted on lower density B cells, which appeared around 3 weeks after birth in the spleen. In contrast, B151-TRF2 could activate both lower and higher density B cells almost equally and B151-TRF2-responsive B cells were already present by 1 week of age. Thus, these results suggest that B151-TRF1/IL-5 and B151-TRF2 act on distinct subpopulations among antigen-unprimed normal B cells to induce IgM-secreting cells.

Animals↗
PubMed1983

Role of the major histocompatibility complex in T cell activation of B cell subpopulations. Ir gene regulation of the T cell-dependent activation of distinct B cell subpopulations.

Antibody responses to (T,G)-A--L and (H,G)-A--L or to the TNP conjugates of these polypeptides, are regulated in the mouse by Ir genes encoded in the I-A subregion of the mouse MHC. To study the mechanism by which Ir genes function, it is essential to identify the cell interactions that are regulated by these genes. In the present report, it was specifically determined whether Ir gene function is expressed differently in the activation of B cell subpopulations that participate in two different pathways of B cell activation for in vitro antibody responses to TNP-(T,G)-A--L and TNP- (H,G)-A--L. The results of these studies demonstrate that under conditions that activate the Lyb-5- b cell subpopulation via MHC-restricted TH cell-B cell and TH cell-APC interactions, Ir gene function regulates TH cell interactions with these B cells as well as with APC. In addition, under conditions that activate the Lyb-5+ B cell subpopulation via MHC-restricted TH cell-APC but not TH cell-B cell interaction, Ir gene function regulates only TH cell interactions with APC but not with B cells.

Animals↗
PubMed1984

Ia-restricted interaction of normal lymphoid cells and SJL lymphoma (reticulum cell sarcoma) leading to lymphokine production. III. Relative roles of reticulum cell sarcoma and normal lymphoid cells in lymphokine production.

Production of interleukin 2 (IL-2) in mixed lymphocyte cultures of SJL/J lymph node (LN) and gamma-irradiated, syngeneic lymphoma cells of transplantable reticulum cell sarcoma (gamma-RCS) was abolished by pactamycin pretreatment of gamma-RCS. LN cells needed for this interaction were Lyt-2 T-cells, not adherent to Sephadex G-10. The effect of separation of T-cells and gamma-RCS after the first 24 hours of culture suggested that both components contributed to the IL-2 production. Exogenous IL-2, but not interleukin 1 (IL-1), restored the ability of pactamycin-treated gamma-RCS to induce syngeneic T-cell proliferation. The inability of T-cells from "nonresponder" F1 hybrids of SJL to proliferate to gamma-RCS was not corrected by addition of IL-1 or IL-2. Interferon (IFN) production also required the presence of untreated gamma-RCS and LN cells, but it was dependent on a Sephadex G-10 and plastic adherent cell in LN. IFN-free supernatant of LN cells plus gamma-RCS induced IFN production in fresh normal lymphoid cells, suggesting a possible indirect induction of this lymphokine. In addition, unirradiated RCS cells (la+ cells) produced immune IFN over a period of 3 weeks in vitro, after which the cells lost their viability. Prolonged IL-2 production was not observed in these cultures. The possible biological importance of the lymphokine production during the exaggerated syngeneic mixed lymphocyte reactions induced by RCS cells is discussed.

Animals↗
PubMed1993

Basket cells or shadow cells of Gumprecht: a scanning electron microscope study, and the correlation between percentages of basket cells, and cells with altered chromatin structure (dense cells), in chronic lymphocytic leukemia.

For over 50 years the received wisdom has been that the shadow cells of Gumprecht otherwise known as basket cells (BC) are in artefact, produced during preparation of films when a drop of blood is spread on a slide. The assumption has been that they are therefore of no significance. They are commonly seen in blood films from patients with lymphoproliferative syndromes and particularly in chronic lymphocytic leukemia (CLL). In 96 patients with CLL a statistically significant correlation existed between the basket cells observed in films and the lymphocytes with dense chromatin (DC) determined by flow-cytometry. There was no statistically significant correlation between the number of BC and DC, and the anatomic-clinical stage of the disease.

Chromatin↗
PubMed1999

Antigen presentation by T cells versus professional antigen-presenting cells (APC): differential consequences for T cell activation and subsequent T cell-APC interactions.

We compared the effects of antigen (Ag) presentation by T cells and professional antigen-presenting cells (APC) on T cell proliferation, cytokine production and surface molecule expression. Ag presentation by T cells (T-T presentation) induced an initial T cell activation phase as measured by proliferation and IL-2 production. These activated T cells became anergic upon antigenic restimulation by professional APC, as shown by a failure to proliferate or produce IL-2 or IFN-gamma. Interestingly, such T cells were not intrinsically defective in their signal transduction pathways since they did proliferate and produce cytokines upon restimulation with mitogenic stimuli. Flow cytometric analysis revealed a more profound TCR and CD3 down-regulation during T-T presentation than during APC-T presentation. However, no up-regulation of CD80, CD86, CD45RC and OX40 (CD134) was observed on T cells during T-T presentation or subsequent antigenic restimulation of anergic T cells in the presence of professional APC, whereas increased expression of these molecules was observed during professional APC-T presentation of non-anergic T cells. The impaired expression of co-stimulatory and activation molecules on T cells after T-T presentation of Ag might lead to altered interactions between T cells and professional APC upon antigenic restimulation. We propose that T cell anergy is a functional consequence of these altered T cell-APC interactions.

Amino Acid Sequence↗
PubMed1987

T cell activation by anti-CD3 antibodies: function of Fc receptors on B cell blasts, but not resting B cells, and CD18 on the responding T cells.

Mouse anti-human CD3 (T3) antibodies can induce T cell proliferation in the presence of Fc receptor (FcR)-bearing accessory cells. Depending on whether the particular antibody can interact with the FcR, it can be mitogenic or otherwise. Previously, some of us (Smith, K. G. C. et al., Eur. J. Immunol. 1986. 16:478) examined human T cell responses to the murine anti-CD3 antibody switch variants UCHT1 (IgG1) and UCHT1B (IgG2b). Using a novel xenogeneic system with mouse macrophages (M phi) and an anti-FcR antibody, 2.4G2, we obtained direct evidence for accessory function of FcR in these responses. However, mouse B cells which also possess FcR were not accessory cells. Here we show that resting B cells do not inhibit anti-CD3 responses in the presence of other accessory cells, and they do not synergize with them. They appear to be inert in these responses but this is not simply because of their radiosensitivity. In contrast, B cell blasts proved to be potent stimulators of responses with UCHT1, UCHT1B and OKT3 (IgG2a). All three responses were inhibited by 2.4G2, whereas we have shown previously that the OKT3 response with M phi was not, in keeping with the known specificities of B cell and M phi FcR. These findings are discussed in relation to the molecular cloning of FcR, and we consider the possibility that distinct FcR could be expressed on resting and activated B cells. A report that anti-CD18 (LFA-1) antibodies blocked the UCHT1 response with human monocytes raised the possibility that this molecule might also be involved in accessory function. However, we show that this inhibition is in fact at the level of the T cell, since anti-human, but not anti-mouse CD18 antibodies, inhibited proliferative responses and clustering with both human and mouse accessory cells. Our results demonstrate that the principal contribution of accessory cells to anti-CD3 responses may be the provision of an FcR, and that CD18 is most probably required at the level of the T cell.

Animals↗
PubMed1995

Stimulation of human immunodeficiency virus type 1 infected cells with superoxide enhances the chemotactic motile response of CD4+ human T cells: implication for virus transmission by cell-to-cell interaction.

We previously showed that superoxide (O2-) significantly enhanced human immunodeficiency virus type 1 (HIV-1)-induced syncytia formation in co-cultured infected and uninfected human T cells. In this study, we describe a novel chemotactic response of uninfected CD4+ T cells by stimulating infected T cells with O2-. Syncytia formation was amplified only when persistently infected cells were stimulated by O2-. When the infected cells in lower well of microplate were cultured with uninfected cells in the upper well of a Boyden chamber with 8.0 microns pores, uninfected cell migration to the porous membrane was significantly amplified by stimulating infected cells with O2-. In contrast, similar functions were slight under the same assay conditions in the presence of known chemokines such as human RANTES and macrophage inflammatory protein 1 (MIP-1 alpha and beta), which all activate T lymphocytes. In addition, it is unlikely that the O2(-)-induced chemotactic response is due to soluble HIV-1 proteins from infected cells or to amplified expression levels of cell surface functional molecules such as CD4 and LFA-1 (CD11a and CD18) as well as HIV-1 Env gp120 on uninfected and/or infected cells. Thus, an unknown chemotactic factor could be generated from infected T cells by stimulation with O2- and it might contribute to viral transmission by activating cell-to-cell interactions.

Antigens, Surface↗
PubMed1975

Concanavalin A receptors on normal rat liver cells, on rat liver cells in vivo transformed by diethylnitrosamine and on Zajdela ascites hepatoma cells of the rat: morphokinetic analysis of cell surface dynamics.

Comparative electron microscopic investigations were performed in living cultures of normal rat liver cells, of rat liver cells in vivo transformed by diethylnitrosamine and on Zajdela ascites hepatoma cells of the rat concerning the mobility of the Concanavalin A cell surface receptors. The cells were incubated in Concanavalin A and peroxidase and subsequently washed. They were then reincubated for various periods at +37 degrees C in PBS prior to fixation. In the case of the Zajdela ascites hepatoma cells the cells were reincubated after Concanavalin A incubation followed by fixation and peroxidase incubation. The cytochemical procedure allowed us to show differences in the mobility of Concanavalin A surface receptors between normal and transformed rat liver cells. The cell surface label disappeared completely within 15 min of reincubation in the transformed cells, whereas in normal cells the same degree of loss in surface label was visible after 120 min reincubation. In both cases an internalization of labelled plasma membrane areas occurred. After complete disappearance of cell surface label in diethylnitrosamine transformed cells a complete relabelling of the cell surface occurred after 60 min reincubation caused by an exocytosis.

Animals↗
PubMed2026

Differential cell signaling testing for cell-cell communication inference from single-cell data by dominoSignal.

MOTIVATION: Algorithms for ligand-receptor network inference have emerged as commonly used tools to estimate cell-cell communication from reference single-cell data. Many studies employ these algorithms to compare signaling between conditions and lack methods to statistically identify signals that are significantly different. We previously developed the cell communication inference algorithm Domino, which considers ligand and receptor gene expression in association with downstream transcription factor activity scoring. We developed the dominoSignal software to innovate upon Domino and extend its functionality to test statistically differential cellular signaling. RESULTS: This new functionality includes the compilation of active signals as linkages from multiple subjects in a single-cell data set and testing condition-dependent signaling linkage. The software is applicable for analysis of single-cell data sets with multiple subjects as biological replicates as well as with bootstrapped replicates from data sets with few or pooled subjects. We use simulation studies to benchmark the number of subjects in compared groups and cells within an annotated cell type sufficient to accurately identify differential linkages. We demonstrate the application of the Differential Cell Signaling Test (DCST) in the dominoSignal software to investigate consequences of cancer cell phenotypes and immunotherapy on cell-cell communication in tumor microenvironments. These applications in cancer studies demonstrate the ability of differential cell signaling analysis to infer changes to cell communication networks from therapeutic or experimental perturbations, which is broadly applicable across biological systems. AVAILABILITY: dominoSignal is available through Bioconductor at https://www.bioconductor.org/packages/release/bioc/html/dominoSignal.html.

Cell Communication↗
PubMed1996

Cell-mediated immunity to pseudorabies virus: cytolytic effector cells with characteristics of lymphokine-activated killer cells lyse virus-infected and glycoprotein gB- and gC-transfected L14 cells.

We examined cytolytic cells that lyse pseudorabies virus (PRV)-infected cells in pigs. In vitro stimulation of peripheral blood mononuclear cells from PRV-immune pigs with live PRV generated cells that lysed PRV-infected immortalized B cells. Several lines of evidence indicated a major contribution of non-major histocompatibility complex (MHC)-restricted cytolytic cells, which displayed characteristics of natural killer (NK) or lymphokine-activated killer cells: cytolysis was non-MHC-restricted, depended on CD2+CD4-CD8bright- (or CD2+CD4-CD8dull+) cells, was strongly augmented by in vitro antigenic stimulation and was not limited to virus-infected cells, i.e. the NK cell-susceptible target cell line K562 was also lysed. Cytolytic cells were also generated by in vitro antigenic stimulation with UV-inactivated PRV. Target cells transfected with and stably expressing PRV gB or gC were lysed to the same degree as PRV-infected target cells.

Animals↗
PubMed2001

Effect of estradiol and FBS on PRL cells, GH cells, and PRL/GH cells in primary cultures of pituitary cells from prenatal rats.

The effects of estradiol (E2) treatment on prolactin (PRL) cells, GH cells, and PRL/GH cells in immature pituitary cells were determined using primary cultures from prenatal rats and immunocytochemistry with fluorescent antibodies. Anterior pituitaries obtained from fetuses on day 22 of pregnancy were monodispersed and cultured in chemically defined medium or medium containing 10% fetal bovine serum (FBS). After pre-incubation for 24 hr, E2 (final concentrations were 0 M, 10(-8) M, 10(-7) M, and 10(-6) M) was added into each medium. After 72 hr of incubation, cells were subjected to immunocytochemistry. E2 stimulated the increase of PRL cells in a dose-dependent manner, and the PRL cell percentage cultured with FBS in all groups was significantly higher than that cultured in chemically-defined medium. PRL/GH cells also responded to E2 in the same manner as PRL cells. E2 was not effective in proliferating GH cells, and GH cell percentage significantly decreased with the addition of FBS into the medium. These results suggest that E2 is dose-dependently capable of increasing immature PRL cells and/or PRL/GH cells in vitro. Moreover, there is a factor(s) in FBS that regulates the increase of these cells.

Cell Division↗
PubMed1987

Interstitial cell heterogeneity in rat testes. I. Purification of collagenase-dispersed Leydig cells by unit gravity sedimentation and demonstration of binding sites for gonadotropin in light cells versus enhanced steroidogenesis in heavier cells.

Two testicular interstitial cell fractions, light and heavier, biochemically and morphologically distinct were obtained by a unit gravity sedimentation procedure. Binding sites for 125I-labeled human chorionic gonadotropin (hCG) were preferentially localized in the light cell fraction (apparent Kd = 2.02 X 10(-10) M; Bmax = 1.17 X 10(-5) nmol/2 X 10(6) cells). These cells did not synthesize testosterone in response to hCG, but the basal release of testosterone was higher than by cells in the heavier fraction (2.49 +/- 0.02 ng/2 X 10(6) cells in the light versus 0.22 +/- 0.00 ng/2 X 10(6) cells in the heavier fraction). The cells in the heavier fraction bound little or no hCG. The binding data from this fraction did not obey saturation kinetics, but testosterone levels were elevated 700-800% in the presence of hCG (i.e. basal value 0.22 +/- 0.00 ng/2 X 10(6) cells versus 1.81 +/- 0.04 ng/2 X 10(6) cells in hCG-stimulated cells). Electron microscopy revealed that heavier cells had features typical of Leydig cells such as large ovoid nucleus with peripherally located heterochromatin, numerous mitochondria with tubular cristae, some lipid droplets, extensively developed smooth endoplasmic reticulum, and well developed Golgi complex. The cells in the light fraction contained an ovoid nucleus with one or more deep infoldings, and their most notable cytoplasmic feature was the presence of numerous vacuoles of varying sizes and shapes. Based upon this and the investigation which follows (Bhalla, V.K., Flasch, M.V., Browne, E.S., Sohal, G.S., and Sharawy, M.M. (1987) J. Biol. Chem. 262, 5322-5332), we conclude that occupancy of high affinity hCG binding sites, generally assumed to be coupled to steroidogenesis, is not necessarily related to the elicitation of this biological response.

Animals↗
PubMed1987

Interstitial cell heterogeneity in rat testes. II. Purification of cells by Percoll and metrizamide gradient centrifugation with preferential localization of gonadotropin binding sites in light cell fraction and hormone-induced steroidogenesis in heavier cell fraction.

The ability of 125I-labeled human chorionic gonadotropin (125I-labeled hCG) to bind and stimulate steroidogenesis was studied in light cells (density, 1.053-1.065 g/cm3) and heavier cells (density, 1.090-1.110 g/cm3) purified from collagenase-dispersed rat testicular interstitial cells by unit gravity sedimentation (Bhalla, V.K., Rajan, V.P., Burgett, A.C., and Sohal, G.S. (1987) J. Biol. Chem. 262, 5313-5321). Preferential localization of gonadotropin binding sites was demonstrated on light cells, and the heavier cells produced testosterone in response to hCG without occupancy of high affinity (Kd = 2.02 X 10(-10) M) binding sites. In this study, established methods for interstitial cell purification involving gradient centrifugation were utilized to demonstrate the cell heterogeneity. Light cells bound hCG with high affinity (Kd = 3 X 10(-10) M) without manifestation of steroidogenic response. The heavier cells responded to hCG with elicitation of steroidogenesis, but the occupancy was negligible. Stimulation of steroidogenesis by hCG in heavier cells was dose and time dependent. Dibutyryl and bromo cyclic AMP (1 mM) also promoted steroidogenesis comparable to a level stimulated by the tropic hormone (700% stimulation). The concept of spare receptors was tested in purified cell fractions. Upon cell purification, no saturable high affinity binding sites were observed in the heavier cell fraction. Autoradiographic analyses at the electron microscopical level supported this conclusion. Our data suggest that target cell activation is not preceded by hormone occupancy of high affinity binding sites. A model for defining the functional domains of the physiological receptor for hCG is presented.

Animals↗
PubMed1990

Determination of apical membrane polarity in mammary epithelial cell cultures: the role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions.

The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. We have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembraneous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. In such cultures the boundary between apical and basal domains was observed at the point of cell contact with the substratum. Immunocytochemical analysis of these cell-substratum contacts revealed the absence of a characteristic basement membrane containing laminin, collagen (IV), and heparan sulfate proteoglycan. However, serum-derived vitronectin was associated with the basal cell surface and the cells were shown to express the vitronectin receptor on their basolateral membranes. Additionally, treatment of cultures with antibodies against the vitronectin receptor caused cell detachment. We suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS-O while colchicine and acrylamide did not. We hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.

Actin Cytoskeleton↗
PubMed1991

EBV cell-lines (LCL) and E- cells as stimulator cells for limiting dilution analysis (LDA) of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). Comparison of their stimulator capacity and ability to generate specific alloreactivity.

The choice of stimulator cells is crucial in determining the frequency of alloreactive cells by limiting dilution analysis (LDA). In humans, E- cells or EBV-lymphoblastoid cell lines (LCL) are available for this purpose. The E- cells have been mostly used until now, but they appear to stimulate much less than LCL in other models. Consequently, we undertook a systematic comparison of the efficiency of both types of cell for the LDA of alloreactive IL-2-secreting cells (IL-2-SC) and cytotoxic precursors (CTLp). We show that LCL are stronger stimulator cells both for IL-2 secretion and for cytotoxicity induction. However, high levels of non-allogeneic reactivity were also induced. Therefore, to measure the frequency of specific alloreactive IL-2-SC and CTLp, T cells were depleted of such irrelevant reactive cells by a suicide technique: culture with autologous LCL in the presence of BUDR and use of the remaining live cells as responding effectors in LDA. Using this methodology, no non-allogeneic reactive T cells remain in the responding cells: after restimulation by autologous LCL, no IL-2-SC could be seen and no cytotoxic activity could be observed against autologous, irrelevant or LAK sensitive targets. In contrast, the number of IL-2-SC and CTLp against allogeneic targets was preserved. Therefore with this methodology, the strong stimulator capacity of LCL could be used whereas non-specific activation could be avoided in order to assess the frequency of allo-specific IL-2-SC and CTLp.

Bromodeoxyuridine↗