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Biomedical subjects

L A Turka

Publications and source records attributed to L A Turka.

At least 91 records · Page 5Linked to original sources
PubMed1994

New biologic immunosuppressive agents in transplantation.

Antigen-specific immunologic nonresponsiveness (tolerance) to alloantigens is a goal in transplantation. Most successful strategies of tolerance induction target the T cell. Activation of a quiescent T cell requires two different signals. Signal 1 is delivered when the T-cell receptor is engaged by foreign antigen presented by major histocompatibility complex molecules. Several potential pathways exist to deliver signal 2, which is also termed a costimulatory signal, although by far the best characterized pathway is through the T-cell co-receptor CD28. Blockade of either signal 1 or signal 2 may be sufficient to induce tolerance to alloantigens. Recent advances in knowledge of T-cell receptor-major histocompatibility complex interactions, in mechanisms of self-tolerance and in the identity and importance of costimulatory receptors and their ligands, have led to the development of various new strategies to block signals 1 and 2 using biologic agents. This review focuses on the most promising new modalities.

Animals↗
PubMed1994

Characterization of dermal dendritic cells in psoriasis. Autostimulation of T lymphocytes and induction of Th1 type cytokines.

Local activation of T lymphocytes is regarded as an important immunological component of psoriatic skin lesions. Within psoriatic plaques (PP) there are large numbers of dermal dendritic cells (DDCs) immediately beneath the hyperplastic epidermis surrounded by T cells. In this study we investigated the ability of DDCs isolated from PP skin to support immune responses to resting peripheral blood T cells. For comparison, other dendritic cells were obtained from blood of the same psoriatic patients, as well as DDCs from skin of normal healthy individuals (designated NN skin). All dendritic cells studied had high surface expression of HLA-DR, B7, and lymphocyte function associated antigen-1 molecules. T cell proliferative responses and cytokine production profiles to these various dendritic cells were measured in the absence and presence of PHA or bacterial-derived superantigens. In the absence of exogenous mitogens, PP skin-derived DDCs were much more effective stimulators of spontaneous T cell proliferation compared with either psoriatic blood-derived or NN skin-derived dendritic cells. Antibody blocking studies revealed involvement of HLA-DR, B7, and lymphocyte function associated antigen-1 on PP skin-derived DDCs. Cytokine profiles revealed that in the absence of exogenous stimuli PP skin-derived DDCs mediated a T cell response with high levels of IL-2 and IFN-gamma, but not IL-4 or IL-10. NN skin-derived DDCs produced a similar qualitative response, but quantitative amounts of all cytokines measured were lower. Upon addition of PHA or superantigens, both PP skin-derived and NN skin-derived DDCs mediated high levels of IL-2 and IFN-gamma production, with induction of IL-4 particularly evident for PHA reactions. Addition of conditioned medium from psoriatic dermal fragments did not enhance the autostimulatory capacity of blood-derived dendritic cells. These findings highlight the potent autostimulatory potential of PP skin-derived DDCs and suggest an important immunological contribution for these previously overlooked cell types contained within lesional skin sites.

Cell Separation↗
PubMed1993

Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets.

The relative contribution of dermal-derived immunocompetent cells to the overall immunologic response in skin has been hampered by the lack of appropriate isolation techniques. In this report, we provide a purification schema that reliably yields highly purified populations of dermal dendritic cells (DDC). These cells are motile, express high levels of class II MHC antigens that decorate their cytoplasmic dendritic processes, and lack numerous B cell, T cell, and natural killer cell antigens. Using a broad panel of 45 different antibodies, an extensive phenotypic analysis was completed, revealing distinctive profiles for subsets of DDC. Despite homogeneous light scatter profile and cytologic appearance, three subsets of DDC could be distinguished by phenotypic and functional criteria. All DDC, but not epidermal Langerhans cells, express factor XIIIa. By triple color cell staining the relative distribution of factor XIIIa positive DDC is as follows: subset 1, 65% to 70% of total DDC express neither CD1a nor CD14; subset 2, 15% to 20% of total DDC express CD1a but not CD14; and subset 3, 10% to 15% of total DDC express CD14 but not CD1a. The CD14-negative subset of DDC were shown to be as potent stimulators of allogeneic mixed lymphocyte reactions as Langerhans cells or blood-derived dendritic cells. However, DDC subsets differed in their ability to support autologous T cell proliferation in response to the mitogenic lectin PHA or bacterial-derived superantigen. In these assays, subsets 1 and 2 were significantly more potent as antigen-presenting cells compared with subset 3. Thus, normal skin contains at least three separate populations of DDC, which have distinctive phenotypic markers and immunologic capabilities.

Antigens, CD↗
PubMed1993

Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4Ig plus donor-specific transfusion.

Allograft rejection is a T cell-dependent process. Productive T cell activation by antigen requires antigen engagement of the T cell receptor as well as costimulatory signals delivered through other T cell surface molecules such as CD28. Engagement of CD28 by its natural ligand B7 can be blocked using a soluble recombinant fusion protein, CTLA4Ig. Administration of CTLA4Ig blocks antigen-specific immune responses in vitro and in vivo, and we have shown that treatment of rats with a 7-d course of CTLA4Ig at the time of transplantation leads to prolonged survival of cardiac allografts (median 30 d), although most grafts are eventually rejected. Here, we have explored additional strategies employing CTLA4Ig in order to achieve long-term allograft survival. Our data indicate that donor-specific transfusion (DST) plus CTLA4Ig can provide effective antigen-specific immunosuppression. When DST is administered at the time of transplantation followed by a single dose of CTLA4Ig 2 d later, all animals had long-term graft survival (> 60 d). These animals had delayed responses to donor-type skin transplants, compared with normal rejection responses to third-party skin transplants. Furthermore, donor-matched second cardiac allografts were well tolerated with minimal histologic evidence of rejection. These data indicate that peritransplant use of DST followed by subsequent treatment with CTLA4Ig can induce prolonged, often indefinite, cardiac allograft acceptance. These results may be clinically applicable for cadaveric organ and tissue transplantation in humans.

Abatacept↗
PubMed1993

bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death.

We report the isolation of bcl-x, a bcl-2-related gene that can function as a bcl-2-independent regulator of programmed cell death (apoptosis). Alternative splicing results in two distinct bcl-x mRNAs. The protein product of the larger mRNA, bcl-xL, is similar in size and predicted structure to Bcl-2. When stably transfected into an IL-3-dependent cell line, bcl-xL inhibits cell death upon growth factor withdrawal at least as well as bcl-2. Surprisingly, the second mRNA species, bcl-xS, encodes a protein that inhibits the ability of bcl-2 to enhance the survival of growth factor-deprived cells. In vivo, bcl-xS mRNA is expressed at high levels in cells that undergo a high rate of turnover, such as developing lymphocytes. In contrast, bcl-xL is found in tissues containing long-lived postmitotic cells, such as adult brain. Together these data suggest that bcl-x plays an important role in both positive and negative regulation of programmed cell death.

Alternative Splicing↗
PubMed1993

bcl-2 proto-oncogene expression during human T cell development. Evidence for biphasic regulation.

To ensure that the mature T cell repertoire is MHC-restricted yet not autoreactive, cortical thymocytes that express low levels of the TCR/CD3 complex along with CD4 and CD8 (double positive cells) are subjected to positive and negative selection. Surviving cells lose either CD4 or CD8 (single positive cells) and are located primarily in the thymic medulla. bcl-2, a novel proto-oncogene that promotes cell survival by inhibiting programmed cell death (apoptosis), may be an important protein in regulating cell survival during thymocyte development. We have examined the expression of bcl-2 during T cell development by using human thymocytes. Consistent with previous studies, human thymic tissue sections stained for bcl-2 revealed occasional bcl-2+ cells within the thymic cortex and intense staining of virtually all medullary thymocytes. More quantitative western blot analysis and S1 nuclease protection assay revealed that single positive thymocytes contained approximately 2 to 3 times the level of bcl-2 protein and 3 to 4 times the level of bcl-2 mRNA as double positive thymocytes. Flow cytometric analysis of purified double positive thymocytes revealed that minimal amounts of bcl-2 protein was in fact detectable in most cells, although a small subpopulation (10-20%) contained higher levels. In contrast, brighter staining for bcl-2 was observed in virtually all single positive thymocytes. Surprisingly, CD4-CD8- thymocytes (both CD3- and CD3+) expressed the same amount of bcl-2 as did the single positive thymocytes. Because a large percentage of CD3-CD4-CD8- cells are cycling, we examined the effect of mitogenic stimulation on bcl-2 expression by double positive thymocytes by using western blot analysis. bcl-2 expression in double positive thymocytes could not be induced by cell cycle entry following stimulation with PMA and ionomycin. Our data demonstrate that bcl-2 expression is biphasic during T cell development. Both CD3-CD4-CD8- and CD3+CD4+ and CD3+CD8+ thymocytes express high levels of bcl-2. Therefore, diminished bcl-2 expression in double positive thymocytes seems to be the result of specific down-regulation in order to facilitate the selection CD4+CD8+ thymocytes.

Cell Differentiation↗
PubMed1993

IMP dehydrogenase inhibitors as immunomodulators.

IMP dehydrogenase is a key enzyme in the de novo pathway of purine biosynthesis and is responsible for catalyzing the first step in the formation of guanine ribonucleotides from inosine monophosphate. Mizoribine, an immunosuppressive agent in wide-spread clinical use in Japan, has been demonstrated to inhibit this enzyme. We have investigated the effects of mizoribine on human T cell activation. Stimulation of purified human peripheral blood T lymphocytes with phorbol ester and ionomycin leads to a five-fold increase in guanine ribonucleotide levels over 72 hours. The addition of mizoribine to these cultures at concentrations that are achieved in vivo leads to a dose-dependent inhibition of proliferation and concomitant 50% decrease in guanine ribonucleotide levels, an effect that is reversible with the addition of guanosine, which repletes the GTP pool. Similar effects are seen with direct stimulation via the CD3/T cell receptor complex. Inhibition of proliferation occurs at the G1/S interface of the cell cycle and is additive to that produced by cyclosporine. In order to determine whether inhibition of IMP dehydrogenase is a common mechanism of immunosuppression for drugs such as azathioprine and 6-mercaptopurine that interfere with purine biosynthesis, we compared the effects of these agents on the metabolism of purified T lymphocytes. The results of these studies demonstrate that mizoribine and mycophenolic acid, a highly specific inhibitor of IMP dehydrogenase, inhibit proliferation directly by the depletion of guanine ribonucleotides; 6-mercaptopurine, on the other hand, has a mixed effect on adenine and guanine ribonucleotide pools, whereas azathioprine inhibits proliferation by a mechanism completely independent of its effects on the purine metabolic pathway. We conclude from these studies that inhibitors of IMP dehydrogenase have potential as specific immunosuppressive agents.

Azathioprine↗
PubMed1993

Elevated proliferating cell nuclear antigen levels in immature thymocytes. Dissociation from cell cycle progression.

Entry into and progression through the cell cycle is associated with a tightly regulated program of gene expression in mature T cells. One such gene product, proliferating cell nuclear Ag (PCNA), is the auxiliary protein of DNA polymerase delta, and is induced during late G1 and early S phase after stimulation of resting (G0) cells. Blockade of PCNA production has been found to inhibit cell division suggesting that PCNA plays an important role in cell proliferation. The extent to which PCNA and other proliferation-related gene products are similarly regulated in thymocytes has been largely undetermined. Here, we report that immature double positive (CD4+CD8+) thymocytes express high levels of PCNA protein and mRNA relative to mature single positive (CD4+CD8- or CD4-CD8+) thymocytes or peripheral blood T cells. Elevation of PCNA expression among double positive thymocytes is not the result of increased numbers of cycling cells in this subpopulation, being specifically observed in double positive thymocytes with normal diploid DNA content and resting (G0) levels of RNA. Unlike mature thymocytes and T cells, mitogenic stimulation did not induce an increase in PCNA expression in double positive thymocytes. These data indicate that immature thymocytes express high levels of PCNA in the absence of cell cycle progression, thus providing evidence for differential regulation of proliferation related pathways during lymphoid development. Altered expression patterns of these genes in immature vs mature thymocytes may contribute to the process of thymic selection.

Animals↗
PubMed1993

Accessory cell function of keratinocytes for superantigens. Dependence on lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction.

A growing body of evidence points to a role for epidermal keratinocytes as active participants in immunologic reactions. Inasmuch as certain T cell-mediated skin diseases, such as psoriasis and atopic dermatitis, are triggered by microbial infection, we asked whether multipassaged human keratinocytes could provide the costimulatory signals necessary to induce autologous T cell proliferation in response to bacterial-derived super-antigens. On exposure to IFN-gamma, keratinocytes are induced to express HLA-DR and HLA-DQ class II MHC Ag, and the lymphocyte function-associated Ag-1 counter-receptor intercellular adhesion molecule-1 (ICAM-1). This change in keratinocyte phenotype is accompanied by the ability of these cells to support T cell proliferation induced by two different bacterial-derived superantigens, staphylococcal enterotoxins A and B. Superantigen-driven proliferation in the presence of IFN-gamma-treated keratinocytes was significantly inhibited (70-90% reduction) by mAb against the LFA-1 alpha- or beta-chain or ICAM-1. Proliferation was not inhibited by mAb against the CD28 ligands BB-1 or B7, even though these keratinocytes express BB-1. In addition to previous defined roles for class II MHC Ag, stimulation of LFA-1 on the T cells by ICAM-1 on the keratinocytes also plays an important costimulatory role in this superantigen-mediated response. The accessory cell capability of keratinocytes was not unique to superantigen driven responses as PHA, as well as anti-CD3 mAb also induced vigorous T cell proliferation when IFN-gamma-treated keratinocytes were added. However, IFN-gamma-treated keratinocytes consistently failed to provoke an allogeneic response. These data demonstrate that 1) keratinocytes can serve as accessory cells for T cell proliferation using a variety of different stimuli, 2) the LFA-1/ICAM-1 interaction plays a major role in keratinocyte-mediated costimulation, and 3) previous reports in which IFN-gamma-treated keratinocytes failed to support T cell proliferation to nominal or alloantigens, may reflect impaired Ag presentation via class II MHC molecules, rather than lack of necessary costimulatory signals. These findings highlighting the accessory cell function of keratinocytes may have implications for our understanding of the pathogenesis of immunologic disorders of the skin.

Antibodies, Monoclonal↗
PubMed1993

Activated keratinocytes present bacterial-derived superantigens to T lymphocytes: relevance to psoriasis.

In the past, epidermal keratinocytes were felt to be primarily concerned with the barrier function of skin. During inflammatory and immune-mediated skin diseases, keratinocytes were only portrayed as being passive/inert targets for noxious agents produced by infiltrating leukocytes. This innocent bystander and/or 'brick and mortar' conceptualization of the keratinocyte must now be significantly modified to take into account the growing body of experimental in vitro and in vivo results that substantiate re-classification of keratinocytes as fully fledged members of the immune system (i.e. immunocytes). Because keratinocytes produce important primary cytokines, adhesion molecules, and mononuclear cell chemotactic factors; as well as functioning as accessory cells for resting T lymphocytes, they can initiate and perpetuate the inflammatory and immunological reactions in the skin which contribute to the pathobiology of psoriasis. This review will emphasize the dynamic contribution that epidermal keratinocytes make to cutaneous immunohomeostasis, with particular focus on the potential role of bacterial derived superantigens and their ability to stimulate resting T cell proliferation when presented by cytokine-activated keratinocytes.

Adult↗
PubMed1993

Excision products of TCR V alpha recombination contain in-frame rearrangements: evidence for continued V(D)J recombination in TCR+ thymocytes.

Cortical thymocytes that express a TCR on their surface continue to express mRNA for the recombination activating genes RAG-1 and RAG-2. As the expression of these genes appears to be necessary and sufficient for V(D)J recombination to occur, this finding suggests that productive TCR gene rearrangement may not by itself terminate TCR recombination. To further study this question, we have utilized a polymerase chain reaction-based strategy to isolate and sequence excision products of secondary TCR alpha gene rearrangement events. Our data shows that TCR alpha excision products from TCR+ thymocytes contain in-frame V-J joints and thus support the concept that V(D)J recombinatorial activity may continue even in thymocytes which have undergone productive TCR gene rearrangement.

Base Sequence↗
PubMed1993

Discordant expression of CD28 ligands, BB-1, and B7 on keratinocytes in vitro and psoriatic cells in vivo.

The process for optimal T-cell activation requires not only engagement of the T-cell receptor/CD3 complex, but also the delivery of additional co-stimulatory signals that synergize with the primary response mediated through the T-cell receptor. Thus, the regulated expression of ligands for such co-stimulatory molecules can be critical in determining whether a cell can effectively activate T cells following the presentation of a foreign antigen. The CD28 antigen has recently been shown to mediate such co-stimulatory signals by interacting with the B7/BB-1 molecule expressed on activated B cells and monocytes. We show in this study that activated keratinocytes, both in vitro and in vivo display a discordance in expression between B7 and BB-1 based on differential monoclonal antibody (MAb) reactivity. Activated keratinocytes in vitro, as well as psoriatic keratinocytes and epithelial cells in the thymus, are reactive with the BB-1 MAb but not anti-B7 MAbs. These BB-1 positive cells fail to express detectable B7 messenger RNA by Northern blot analysis. Furthermore, keratinocytes bind specifically to CD28-transfected COS7 cells, and this binding is inhibited by anti-CD28 and anti-BB-1 but not B7 MAbs. These studies suggest: 1) that the MAb against BB-1 binds a functional epitope on a molecule distinct from B7 as detected on activated keratinocytes in vitro and in vivo and 2) that keratinocytes in skin and epithelial cells in thymus can express cell-surface molecules that might mediate T-cell co-stimulation via CD28.

Antibodies, Monoclonal↗
PubMed1992

T-cell activation by the CD28 ligand B7 is required for cardiac allograft rejection in vivo.

Organ graft rejection is a T-cell-dependent process. The activation of alloreactive T cells requires stimulation of the T-cell receptor/CD3 complex by foreign major histocompatibility complex (MHC)-encoded gene products. However, accumulating evidence suggests that, in addition to T-cell receptor occupancy, other costimulatory signals are required to induce T-cell activation. Previously, the CD28 receptor expressed on T cells has been shown to serve as a surface component of a signal transduction pathway that can provide costimulation. In vitro, interaction of CD28 with its natural ligand B7 expressed on the surface of activated B cells or macrophages can act as a costimulus to induce proliferation and lymphokine production in antigen receptor-activated T cells. We now report evidence that stimulation of T cells by the CD28 ligand B7 is a required costimulatory event for the rejection of a MHC-incompatible cardiac allograft in vivo. These results demonstrate that the B7/CD28 activation pathway plays an important role in regulating in vivo T-cell responses.

Animals↗
PubMed1992

Isolation of a cDNA clone encoding a zinc finger protein highly expressed in T-leukemia lines.

A cDNA clone encoding a novel zinc finger protein expressed in lymphoid cells has been isolated. This protein contains 5 repeats of the C2H2 motif previously described in the Drosophila gap gene, Krüppel, which is involved in embryo segmentation. Northern blot analysis showed that the messenger RNA (mRNA) encoding this protein is expressed at high levels in a variety of T-leukemia cell lines, at lower levels in some B cells, but is not observed in nonlymphoid cells. Within the T lineage, the mRNA is found at high levels in both alpha beta and gamma delta T cells. These data suggest that this cDNA, designated Hkr-T1, represents a gene that may contribute to the determination of the differentiation and the specificity within lymphoid cells.

Amino Acid Sequence↗
PubMed1992

Defective signal transduction by the CD2 molecule in immature T-cell receptor/CD3- thymocytes.

The CD2 accessory molecule mediates an activation pathway in mature T cells, transducing signals similar to those observed following stimulation of the T-cell receptor/CD3 (TCR/CD3) complex. CD2 is also one of the earliest cell surface markers to appear during thymic ontogeny and has been proposed to be a stimulatory pathway for immature thymocytes that have not yet expressed TCRs on their surface (TCR/CD3-). To examine this hypothesis highly purified TCR/CD3- human thymocytes were stimulated using mitogenic combinations of anti-CD2 monoclonal antibodies or individual biotinylated anti-CD2 monoclonal antibodies crosslinked with avidin. TCR/CD3+ thymocytes responded readily to either stimulus as determined by anti-phosphotyrosine immunoblotting, and the pattern of tyrosine phosphorylated substrates was similar to that of mature T cells. In contrast, TCR/CD3- thymocytes responded weakly and with a distinct substrate pattern. In addition, the altered signal transduced by CD2 in TCR/CD3- thymocytes did not lead to a rise in intracellular calcium, failed to induce interleukin 2 receptor expression, and did not serve as a comitogen with phorbol ester or interleukin 2, functions that were all intact in TCR/CD3+ thymocytes. Failure of TCR/CD3- thymocytes to respond to CD2 stimulation was not due to an intrinsic defect in these cells as they responded normally to phorbol ester plus calcium ionophore. In TCR/CD3- thymocytes, CD2 stimulation also failed to affect steady-state mRNA levels of the recombination-activating genes RAG1 and RAG2, whereas in TCR/CD3+ cells activation of the CD2 pathway terminated their expression. Together, these data support the concept that CD2 engagement does not deliver a stimulus to TCR/CD3- thymocytes and suggests that this molecule may not directly participate in the earliest stages of thymic development.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Antibody and B7/BB1-mediated ligation of the CD28 receptor induces tyrosine phosphorylation in human T cells.

CD28 is an adhesion receptor expressed as a 44-kD dimer on the surface of a major subset of human T cells. The CD28 receptor regulates the production of multiple lymphokines, including interleukin 2 (IL-2), by activation of a signal transduction pathway that is poorly understood. Here we show that ligation of CD28 by a monoclonal antibody (mAb) or by a natural ligand, B7/BB1, induces protein tyrosine phosphorylation that is distinct from T cell receptor (TCR)-induced tyrosine phosphorylation. CD28-induced protein tyrosine phosphorylation was greatly enhanced in cells that had been preactivated by ligation of the TCR, or by pretreatment with phorbol esters. Rapid and prolonged tyrosine phosphorylation of a single substrate, pp100, was induced in T cells after interaction with B7/BB1 presented on transfected Chinese hamster ovary (CHO) cells. Anti-B7 mAb inhibited B7/BB1 receptor-induced tyrosine phosphorylation, indicating that B7-CD28 interaction was required. CD28-induced tyrosine phosphorylation was independent of the TCR because it occurred in a variant of the Jurkat T cell line that does not express the TCR. Herbimycin A, a protein tyrosine kinase inhibitor, could prevent CD28-induced tyrosine phosphorylation and CD28-induced IL-2 production in normal T cells. The simultaneous crosslinking of CD28 and CD45, a tyrosine phosphatase, could prevent tyrosine phosphorylation of pp100. These results suggest that specific tyrosine phosphorylation, particularly of pp100, occurs directly as a result of CD28 ligand binding and is involved in transducing the signal delivered through CD28 by accessory cells that express the B7/BB1 receptor. Thus, this particular form of signal transduction may be relevant to lymphokine production and, potentially may provide a means to study the induction of self-tolerance, given the putative role of the costimulatory signal in the induction of T cell activation or anergy.

Antibodies, Monoclonal↗