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Biomedical subjects

M Sykes

Publications and source records attributed to M Sykes.

At least 109 records · Page 6Linked to original sources
PubMed1996

Mechanism by which additional monoclonal antibody (mAB) injections overcome the requirement for thymic irradiation to achieve mixed chimerism in mice receiving bone marrow transplantation after conditioning with anti-T cell mABs and 3-Gy whole body irradiation.

A relatively nontoxic method of conditioning mice has been developed recently that allows allogeneic bone marrow engraftment and specific skin allograft tolerance induction. This regiment included anti-CD4 and anti-CD8 mAbs administered on day -5, followed by 3-Gy whole body irradiation (WBI) and 7-Gy thymic irradiation (TI) on day 0. We have recently shown that the potential toxicity of this regimen can be further reduced by replacing TI with additional anti-T cell mAb injections before and after bone marrow transplantation. Mixed chimerism and prolonged donor-specific skin graft acceptance are induced in 90% of B10 mice conditioned with anti-CD4 and anti-CD8 mAbs on days -6 and -1 and 3-Gy WBI on day 0 without TI, but only in a small fraction of mice receiving a similar regimen, except that mAbs are given on day -5 only. To determine the mechanism of tolerance induction in the former group, we compared the two groups for the extent of thymocyte depletion, for the timing of development of intrathymic and extrathymic chimerism, and for clonal deletion of host-type thymocytes with TCR recognizing superantigens presented by donor class II molecules. The results suggest that administration of a second mAb injection depletes or inactivates residual host thymocytes that are capable of causing intrathymic rejection of donor hematopoietic cells even when peripheral engraftment is achieved. The presence of donor class II+ hematopoietic cells in the thymus on day 14 correlated with marked deletion of mature host-type V beta 11+ thymocytes that recognize donor I-E plus endogenous superantigen. This suggests that tolerance is achieved primarily through a central deletional mechanism when peripheral and intrathymic host T cells are adequately inactivated or depleted by mAbs and 3-Gy WBI. In addition, the higher incidence of early failure of peripheral chimerism in mice conditioned with a single injection rather than than two mAb injections prior to bone marrow transplantation suggests that nontolerant residual host thymocytes can also induce early peripheral rejection after mAbs have cleared from the circulation. This early rejection is prevented by the longer persistence of anti-T cell mAbs observed in mice receiving two pretransplant mAb injections. Thus, administration of sufficient depleting anti-T cells mAbs followed by 3-Gy WBI allows the induction of central deletional tolerance while minimizing the toxicity of the conditioning regimen.

Animals↗
PubMed1996

Natural killer cells weakly resist engraftment of allogeneic, long-term, multilineage-repopulating hematopoietic stem cells.

Natural killer (NK) cells effect hybrid resistance, in which parental hematopoietic cell grafts are rejected by F1 recipients. NK cells can also resist engraftment of fully MHC-mismatched allogeneic marrow. However, studies of NK cell-mediated alloresistance have relied on short-term proliferation, colony, or survival assays; therefore, their results may not reflect effects of NK cells on the engraftment of allogeneic pluripotent hematopoietic stem cells (PHSC). We have now addressed the role of NK cells in resisting engraftment of these most primitive hematopoietic cells, which provide long-term repopulation of multiple hematopoietic lineages. We took advantage of a nonmyeloablative conditioning regimen that permits allogeneic marrow engraftment and induction of mixed chimerism in mice to evaluate the effect of host NK cell depletion with mAb PK136 on long-term competitive repopulating ability of allogeneic marrow. Mice were pretreated with depleting anti-CD4 and anti-CD8 mAbs, then received 3 Gy of whole body irradiation and 7 Gy of thymic irradiation prior to allogeneic bone marrow transplantation. Depending on the strain combination used, statistically significant increases in long-term allogeneic repopulation of both myeloid and lymphoid cell lineages were observed in recipients depleted of NK cells before bone marrow transplantation compared with controls. Depletion of host NK cells alone was sufficient to enhance donor PHSC engraftment. However, a statistically significant increase in allogeneic reconstitution in NK cell-depleted chimeras compared with control chimeras was not observed in every experiment, and differences were most readily apparent in a strain combination in which recipient NK cells have been shown to have high resistance to engraftment of donor short-term repopulating cells. Chronic (16 weeks) anti-NK1.1 treatment resulted in higher levels of donor-type repopulation than that in animals receiving only pretransplant NK cell depletion. Our studies demonstrate for the first time that host NK cells resist engraftment of allogeneic long-term repopulating PHSC, and provide a model for studying the elements that determine what is regarded as "self" and "non-self" by newly developing NK cells.

Animals↗
PubMed1996

Chimerism and central tolerance.

With adequate depletion or inactivation of the pre-existing immune system and establishment of conditions permitting donor hematopoietic stem cell engraftment, a robust state of central deletional tolerance to allogeneic or xenogeneic donors can be induced. Advances have been made in the ability to achieve this permissive state without toxic, myeloablative conditioning, thus bringing this approach closer to clinical application.

Animals↗
PubMed1996

Long-term engraftment of precultured post-5-fluorouracil allogeneic marrow in mice conditioned with a nonmyeloablative regimen: relevance for a gene therapy approach to tolerance induction.

Introduction of MHC class I Kb cDNA via recombinant retrovirus into B10.AKM (Kk) bone marrow cells (BMC) has been shown to confer specific hyporesponsiveness to B10.MBR (Kb) allogeneic skin grafts in lethally irradiated B10.AKM recipients of the transduced syngeneic BMC. We have recently developed a nonmyeloablative conditioning regimen that allows engraftment of fully MHC-mismatched allogeneic bone marrow and the induction of donor-specific tolerance. We ultimately plan to adapt this nonmyeloablative regimen for the use of retroviral transfer of the Kb gene to syngeneic marrow. As a step toward this goal, we have assessed the effects of our current BMC transduction protocol on engraftment of class I mismatched marrow in mice prepared using the nonmyeloablative regimen. BMC from B10.MBR (KbIkDq) mice treated 2 days earlier with 5-fluorouracil (5-FU) were cultured for 4 days with rIL-3 and rIL-6, and then injected into B10.AKM (KkIkDq) recipients conditioned with anti-CD4 and anti-CD8 mAbs, 7 Gy of thymic irradiation and 3 Gy of whole body irradiation. Engraftment was comparable to that of freshly prepared normal B10.MBR marrow. All recipients of 10(6) precultured BMC developed long-term multilineage mixed WBC (white blood cells) chimerism, and six of seven of these animals showed long-term specific tolerance to B10.MBR skin grafts. Four of seven recipients of 2 x 10(5) precultured BMC showed long-term repopulation by the donor of > 1% of multiple WBC lineages and four of five recipients showed specific tolerance to B10.MBR tail skin. These data suggest that our previously described nonmyeloablative conditioning regimen could be applicable to the gene therapy approach for the induction of donor-specific transplantation tolerance.

Animals↗
PubMed1996

Skin graft tolerance across a discordant xenogeneic barrier.

Specific T-cell tolerance may be essential for successful xenotransplantation in humans. Grafting of thymectomized, T cell-depleted normal mice with xenogeneic fetal pig thymus and liver (FP THY/LIV) tissue results in the recovery of functional CD4 antigen-positive cells. We have tested T-cell tolerance by skin grafting. Donor-matched pig skin survived permanently (> 200 days), whereas allogeneic mouse skin was rapidly rejected. Nontolerant control mice rejected pig skin within 26 days. Both porcine and murine histocompatibility class IIhigh cells were detected in long-term thymus grafts, and T-cell repertoire analyses suggested that tolerance to both donors and recipients developed, at least in part, by intragraft clonal deletion. This study demonstrates the principle that tolerance, measured by the stringent criterion of skin grafting, can be induced across a widely disparate species barrier.

Animals↗
PubMed1996

Immunobiology of transplantation.

Despite major advances resulting from the development of new immunosuppressive drugs in recent years, the field of clinical transplantation is currently limited by inadequate organ availability, chronic rejection, and complications of chronic, nonspecific immunosuppressive therapy. Because of the organ shortage, extensive research has recently focused on the potential to use donors from other species, i.e., xenotransplantation. The best way to avoid the problems of chronic rejection and complications of immunosuppressive therapy, and to overcome the considerable immunologic barriers to xenotransplantation, would be to induce a state of systemic tolerance to the donor in the recipient. Recent developments in the understanding of mechanisms of central and peripheral T cell tolerance have led to new strategies for inducing tolerance in experimental models, some of which are already being evaluated clinically.

Adaptation, Physiological↗
PubMed1996

Alloresistance to K locus class I-mismatched bone marrow engraftment is mediated entirely by CD4+ and CD8+ T cells.

Clinical application of approaches to inducing transplantation tolerance that involve bone marrow reconstitution will require achievement of engraftment without major toxicity to the recipient. These requirements are likely to vary according to the type of histoincompatibility between donor and recipient. We have attempted to determine the minimal conditioning required to achieve lasting mixed allogeneic chimerism and tolerance in the presence of a class I MHC disparity by evaluating the host elements that resist alloengraftment. We based our approach on a regimen that was shown to induce mixed chimerism in fully MHC-mismatched strain combinations. Recipient B10.AKM (KkIkDq) mice were treated with 7 Gy thymic irradiation (TI) and 3 Gy whole body irradiation (WBI) and received either anti-CD8 mAb alone or anti-CD4 plus anti-CD8 mAbs before transplantation of K locus-disparate B10.MBR (KbIkDq) marrow. All (27 of 27) animals receiving both mAbs showed lasting multi-lineage mixed chimerism and donor-specific tolerance. In contrast, five of 22 (23%) recipients pre-treated with anti-CD8 mAb alone in the same experiments failed to develop lasting multilineage mixed chimerism, suggesting that the CD4 T cell subset also participates in resistance to class I-mismatched marrow engraftment. We next attempted to determine whether or not host non-T cell elements resist allogeneic engraftment by comparing the minimum number of syngeneic vs allogeneic BMC required to achieve lasting multilineage mixed chimerism. Titrated numbers (10(6) to 10(7)) of B10.MBR (KbIkDq) bone marrow cells were administered to B10.AKM recipients treated with anti-CD4 and -CD8 mAbs, 3 Gy WBI and 7 Gy TI. All recipients of each marrow dose developed lasting multilineage mixed chimerism and showed specific tolerance to B10.MBR skin grafts. The level of donor-type repopulation in recipients of each dose was not lower than that observed in similarly irradiated recipients in an Ly5 congenic, otherwise syngeneic, BMT system. Together, our results suggest that CD4+ T cells contribute to resistance to K locus class I-mismatched marrow allografts and that resistance is mediated only by CD4 and CD8 T cells, with no role for non-T cell host elements.

Animals↗
PubMed1996

Hematopoietic cell transplantation for the induction of allo- and xenotolerance.

Durable tolerance can be reliably achieved by inducing engraftment of allogeneic or xenogeneic hematopoietic cells in recipients initially depleted of T lymphocytes. Engraftment of pluripotent hematopoietic stem cells (PPHSC) provides a constant supply of donor antigen to ensure the ongoing central deletion of donor-reactive T cell clones, resulting in a permanent state of donor-specific tolerance. Because of the toxicity of myeloablative therapy used to achieve allogeneic PPHSC engraftment, this approach has not yet been applied in humans. However, a non-myeloablative, relatively non-toxic conditioning regimen allowing allogeneic or concordant xenogeneic bone marrow engraftment and tolerance induction has recently been developed in a murine model. Host pre-treatment with depleting doses of anti-CD4 and anti-CD8 mAbs (and in the xenogeneic combination, anti-Thy 1.2 and anti-NK 1.1 mAbs), followed by 3 Gy whole body irradiation (WBI) and 7 Gy of thymic irradiation (TI) allows engraftment of allogeneic or xenogeneic rat bone marrow cells with mixed, multilineage lymphohematopoietic chimerism and donor-specific skin graft tolerance. TI can be omitted from the regimen if additional mAb treatments are given. Engraftment of allogeneic PPHSC is associated with early migration of donor bone marrow-derived cells to the host thymus, resulting in deletion of developing thymocytes with reactivity to donor antigens. Maintenance of long-term tolerance is purely due to this deletional mechanism. In the xenogeneic rat-->mouse species combination, mixed chimerism is also associated with deletional T cell tolerance, and also leads to tolerance at the level of B cells that make natural antibodies. In the discordant pig-->mouse species combination, we have found that physiologic preference for host hematopoiesis is a major barrier to achieving donor hematopoietic reconstitution. Pig-specific hematopoietic cytokines can at least partially overcome this barrier. Furthermore, if normal, immunocompetent mice are thymectomized, then receive T- and NK-cell-depleting mAbs, and a fetal swine thymus is grafted, murine CD4 T cells recover in the swine thymus, and demonstrate specific tolerance to the xenogeneic swine donor. These T cells appear to be able to recognize antigen in the context of host MHC, and demonstrate immunocompetence. Our studies have demonstrated for the first time that donor-specific skin graft tolerance can be induced across a discordant species barrier.

Animals↗
PubMed1996

CAG repeat mutation in the mouse IL-2 gene enables concurrent assessment of T- and B-cell-specific gene expression by northern analysis.

The unique presence of CAG triplet repeats in the mouse IL-2 gene, lending the IL-2 cDNA a second specificity to MHC class II invariant chain (Ii) mRNA, coincides with another unusual genetic feature of the mouse, the lack of Ii gene upregulation in stimulated T cells. While the former anomaly allows simultaneous measurement of IL-2 and Ii mRNAs by Northern analysis, the latter condition ensures that Ii mRNA remains a T-cell-independent, specific marker of Ii positive, primarily B cell function. Thus, Northern analysis using CAG repeat containing IL-2 cDNA enables concurrent assessment of T and B cell activities in the spleen or in other mixtures of mouse lymphocytes.

Animals↗
PubMed1995

Inhibition of graft-versus-host disease by interleukin-2 treatment is associated with altered cytokine production by expanded graft-versus-host-reactive CD4+ helper cells.

In a fully MHC plus multiple minor antigen-mismatched murine bone marrow transplantation (BMT) model, we have demonstrated that a short course of high dose IL-2, begun on the day of BMT, protects against graft-versus-host disease (GVHD). This inhibitory effect is directed against donor CD4+ cells. To determine whether the mechanism of IL-2-induced GVHD protection involves clonal deletion or anergy of host-reactive donor T helper cells (Th), we performed limiting dilution analyses to measure the frequency of activated Th that reacted to donor, host, and third-party antigens in GVHD control and IL-2-protected mice. Marked and specific expansion of host-reactive Th was observed to a similar extent in GVHD control and IL-2-protected mice by day 5 after BMT, and the number of these cells in the spleen increased by several orders of magnitude between days 3 and 5 after BMT, which suggests that recirculation from other tissues occurred in this period. A high proportion (approximately 80%) of donor T cells expressed CD25 in both GVHD control and IL-2-protected mice on day 4 after BMT, which suggests a high level of bystander T cell activation. Since marked quantitative differences in the GVH response were not observed between GVHD control and IL-2-protected mice, we assessed both groups for qualitative differences in the Th response. Spleen cells isolated in the first 8 days after BMT were cultured with host-type, donor-type, or third-party stimulators or without stimulators, and cytokines were measured in supernatants harvested at 24 hr. GVHD was associated with marked increases in supernatant IFN-gamma levels from day 3 to day 6 after BMT, and with increases in IL-2 levels compared with naive A/J controls or syngeneic BMT controls stimulated with host antigens. Production of these cytokines was specifically induced by host-type antigens. Supernatants from spleens of IL-2-treated mice showed delayed kinetics of IFN-gamma production, and tended to contain higher levels of IL-4 in response to host antigen compared with GVHD controls on days 2 and 4 after BMT. Both IL-4 and IFN-gamma were produced almost exclusively by CD4+ cells in spleens of GVHD control and IL-2-protected mice on day 4. However, no consistent difference was observed between the groups in supernatant IL-2 or IL-10 levels, ruling out a simple Th1 to Th2 switch.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Interleukin-12 inhibits murine graft-versus-host disease.

Interleukin-12 (IL-12) is a potent immunostimulatory cytokine and an inducer of type-1 T-helper cell activity and of cytotoxic T lymphocyte and natural killer cell function. We report here the paradoxical observation that a single injection of 4,900 IU of recombinant murine IL-12 inhibits acute graft-versus-host disease (GVHD) in a fully major histocompatibility complex (MHC) plus multiple minor antigen-mismatched bone marrow transplantation (BMT) model (A/J-->B10). The protective effect was enhanced by administration of T-cell-depleted host-type BM cells, and complete donor-type lymphohematopoietic reconstitution was observed in most animals. Treatment with a protective course of IL-12 led to increased serum interferon-gamma (IFN-gamma) levels as compared with those for GVHD controls at early time points, when IFN-gamma was produced predominantly by host-type natural killer cells, but led to almost complete inhibition of the later GVHD-associated increase in serum IFN-gamma levels, when IFN-gamma is produced predominantly by CD4+ T cells. Furthermore, IL-12 treatment was associated with marked alterations in the kinetics of donor T-cell expansion. Reductions in donor CD4+ and CD8+ T cells were observed in the spleen on day 4 post-BMT, but a marked increase in donor CD8+ cells was observed on day 7. Unlike broadly immunosuppressive methods for inhibiting GVHD, which are associated with loss of antileukemic effects, IL-12 has the potential to mediate antileukemic effects of its own; therefore, the GVHD-inhibitory effects of IL-12 described here suggest a potential application for this cytokine in clinical BMT.

Animals↗
PubMed1995

Specific unresponsiveness to a retrovirally-transferred class I antigen is controlled through the helper pathway.

To investigate the potential role for gene therapy in induction of tolerance to solid organ grafts, we used a murine model based on the introduction of an allogeneic MHC class I gene (H-2Kb) into hematopoietic cells of congenic animals differing only at the class I locus. The H-2Kb gene was placed into a retroviral vector under the control of regulatory sequences of the myeloproliferative sarcoma virus long terminal repeat. Transplantation of H-2Kb retrovirus-transduced autologous bone marrow into B10.AKM (Kk Ik Dq) recipients resulted in detectable levels of H-2Kb RNA and cell surface protein in lymphoid and myeloid lineages. H-2Kb expression was significant, although variable, in bone marrow Mac1+ cells, in splenic B cells, and in CD4+/CD8+ thymocytes 8 wk post-bone marrow transplantation. The recipients of the H-2Kb-transduced bone marrow showed specifically delayed rejection of B10.MBR (Kb Ik Dq) skin grafts disparate only for Kb. However, B10.MBR skin grafts were rapidly rejected when grafted simultaneously with skin grafts from B10 (Kb Ib Db) mice, a strain bearing additional third party alloantigens in association with Kb. Our experiments suggest that the hyporesponsive state induced by using the retrovirally mediated gene transfer model is characterized by the persistence of H-2Kb-specific cytolytic T cell precursors, which may be inactive because of deficient T cell help. Tolerance to Kb induced by this approach may therefore be restricted to T helper cell lineages.

3T3 Cells↗
PubMed1995

Mixed allogeneic chimerism and renal allograft tolerance in cynomolgus monkeys.

We have developed a nonmyeloablative preparative regimen that can produce mixed chimerism and renal allograft tolerance between MHC-disparate nonhuman primates. The basic regimen includes ATG, nonmyeloablative total-body irradiation (TBI, 300 rads), thymic irradiation (TI, 700 rads), and donor bone marrow infusion. Kidney allografts from MHC-mismatched donors were transplanted with various manipulations of the preparative regimen. Monkeys treated with the basic regimen alone (n = 2) rejected allografts by day 15. With the addition of cyclosporine (CsA) for one month (n = 3), one monkey developed multilineage mixed chimerism and renal allograft tolerance thereafter (> 430 days). To reduce the toxicity of the preparative regimen, TBI was fractionated to 150 rads on two successive days in subsequent studies. All monkeys receiving this modified regimen (n = 4) developed multilineage chimerism with fewer side effects and accepted renal allografts long-term with no further immunosuppression (196 days, 198 days, > 150 days, and > 40 days). In long-term survivors, donor-specific nonreactivity was confirmed by MLR and skin transplantation. Three monkeys treated with the basic regimen plus CsA but with only 150 rads of TBI (n = 1) or no TBI (n = 2) did not develop multilineage chimerism and grafts were rejected (day 40-50) soon after the CsA discontinuation. Monkeys treated with the same regimen, but without DBM (n = 2), rejected kidney allografts by day 52. Therefore, at least transient engraftment of DBM appears to be essential for induction of donor specific tolerance in this monkey model.

Animals↗
PubMed1995

A randomised trial comparing the safety and efficacy of the Zoladex 10.8-mg depot, administered every 12 weeks, to that of the Zoladex 3.6-mg depot, administered every 4 weeks, in patients with advanced prostate cancer. The Dutch South East Cooperative Urological Group.

A new longer-acting depot formulation containing 10.8 mg Zoladex administered every 12 weeks was compared to the 3.6-mg Zoladex depot administered every 28 days, in a randomised trial in patients with advanced prostatic carcinoma in which pharmacodynamic efficacy and safety were assessed. Effective induction of mean serum testosterone suppression into the surgically castrate range by 21 days and maintenance of suppression for the duration of therapy was achieved with both the 3.6-mg and the 10.8-mg depot formulations. The Zoladex 10.8-mg depot was well tolerated both locally and systemically. This new formulation which is equivalent to three successive 3.6-mg depots will provide a more convenient dosing regime for both patient and doctor in this indication.

Aged↗