Haploidentical 'megadose' CD34+ cell transplants for patients with acute leukemia.
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Acute Disease↗
Biomedical subjects
Y Reisner
Publications and source records attributed to Y Reisner.
Haploidentical 'megadose' stem cell transplantation in acute leukemia: recommendations for a protocol agreed upon at the Perugia and Chicago meetings.
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Chicago↗
Induction of strong hepatitis B virus (HBV) specific T helper cell and cytotoxic T lymphocyte responses by therapeutic vaccination in the trimera mouse model of chronic HBV infection.
Humanized BALB/c mice (termed trimera mice) conditioned by lethal total body irradiation and bone marrow transplantation from SCID mice have been described to support rapid engraftment of human peripheral blood mononuclear cells (PBMC) and the induction of strong B and T cell responses after immunization in vivo. Moreover, these mice can be infected with the hepatitis B and C viruses (HBV, HCV). The current study employed this model to study therapeutic vaccination approaches against the HBV. Thus, strong primary Th cell responses against the HBV core (HBc) and the Borrelia burgdorferi control antigen were induced by transfer of antigen-loaded dendritic cells together with autologous PBMC from HBV-naive donors as well as by vaccination with high doses of antigen or a DNA plasmid encoding for HBcAg. Moreover, primary peptide-specific CTL responses against the immunodominant epitope HBc(18 - 27) were induced by HBc particle or DNA vaccination of chimera engrafted with HBV-naive PBMC. Finally, strong HBc-specific Th cell and antibody responses were induced by HBc or DNA vaccination of mice reconstituted with PBMC from a chronic HBV patient. Thus, since HBc represents the immunodominant antigen in self-limited HBV infection, HBc particles or DNA vectors are good candidates for therapeutic vaccination, that will be further studied in our model and clinical studies.
Animals↗
Bone marrow transplantation across major genetic barriers: the role of megadose stem cells and nonalloreactive donor anti-third party CTLS.
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Antigens, CD34↗
Evolution of action potential propagation and repolarization in cultured neonatal rat ventricular myocytes.
INTRODUCTION: Cultured neonatal rat ventricular myocytes (NRVM) reestablish gap junctions as they form synchronously and spontaneously beating monolayers, thus providing a useful model for studying activation and repolarization. METHODS AND RESULTS: We used the multielectrode array data acquisition system with 60 unipolar electrodes to investigate the functional organization of cultured NRVM, by determining propagation and repolarization patterns. Activation maps were constructed from the local activation times at each electrode. During days 3 to 8 in culture, QRS amplitude and dV/dt(max) increased with age. Concomitantly, with the culture maturation, QT interval (representing action potential duration) decreased, and T wave amplitude and slopes of the T wave ascending and descending limbs progressively increased. The changes in conduction velocity were different than those of the electrogram properties, slightly increasing during the first 3 to 5 days and gradually declining toward day 8 in culture. CONCLUSION: Establishment of uniform activation patterns in spontaneously firing or driven myocytes in monolayer cultures is accompanied by organization of activation and repolarization whose evolution appears in concert with that of a mature connexin43 staining pattern. The experimental techniques developed in this study provide useful tools to investigate the complex relations among gap junctions, conduction velocity, and propagation patterns, as well as a means to learn how gap junctional remodeling under pathophysiologic conditions predisposes the myocardium to arrhythmias.
Action Potentials↗
Haploidentical stem cell transplantation in leukemia.
In acute leukemia patients, infusing a megadose of extensively T-cell-depleted hematopoietic stem cells after an immuno-myeloablative conditioning regimen ensures sustained engraftment of full-haplotype mismatched transplants without graft-vs-host disease. Besides the conditioning regimen and the megadose of stem cells donor natural killer cell alloreactivity also plays a role in facilitating engraftment and in preventing relapse. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in 112 very high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype mismatched donor, who is immediately available, a T-cell depleted mismatched transplant should be offered, not as a last resort, but as a viable option to high risk acute leukemia patients who do not have, or cannot find, a matched donor.
Clinical Trials as Topic↗
Stem cell transplantation across major genetic barriers.
Megadose haploidentical transplants, mismatched at three HLA loci, engraft rapidly and durably without induction of graft-versus-host disease (GVHD). In vitro studies suggest that veto cells, contained in the population of hematopoietic progenitors, facilitate this favorable outcome. Cytotoxic T cells, not reactive against the recipient but reactive against a third party, are potent veto cells and can synergize with the stem cells and facilitate allogeneic bone marrow engraftment without GVHD. Experiments with mice deficient in FasL and Fas, with transfer of FasL gene and with anti-CD8 antibody, suggest that the veto activity associated with cytotoxic T lymphocytes (CTLs) requires simultaneous expression of FasL and CD8.
Animals↗
Human interleukin-6 facilitates hepatitis B virus infection in vitro and in vivo.
BACKGROUND AND AIM: Research on hepatitis B virus (HBV) infection in vivo has been limited due to the absence of a suitable animal model. We have developed a human-mouse radiation chimera in which normal mice, preconditioned by lethal total body irradiation and radioprotected with SCID mouse bone marrow cells, are permissive for engraftment of human hematopoietic cells and solid tissues. This resulting human-mouse model, which comprises three genetically disparate sources of tissue, is therefore termed Trimera. This study was aimed at assessing the effect of human IL-6 on HBV infection in vivo in Trimera mice. METHODS: Trimera mice were transplanted with human liver tissue fragments or with HepG2-derived cell lines, which had been previously infected ex vivo with HBV in the presence or absence of human interleukin-6 (hIL-6) and in the presence of anti-IL-6-neutralizing antibodies. RESULTS: HBV sequences appeared in the sera of animals in which the liver tissue was incubated with both HBV and hIL-6 prior to transplantation. A similar result was obtained when a human hepatoblastoma cell line (HepG2), expressing the hIL-6 receptor, was infected ex vivo with HBV in the presence of hIL-6 prior to their injection into spleens of Trimera mice. However, when liver fragments were infected ex vivo and simultaneously treated with neutralizing antibodies against hIL-6 or were incubated with HBV prior to transplantation without hIL-6, the rate of mice positive for HBV DNA in their sera was lower. Human mononuclear cells are also permissive for HBV infection in vitro: in the presence of hIL-6 the infection of these cells is enhanced; and this infection is suppressed by the chimeric protein named Hyper-IL-6, generated by the fusion of hIL-6 to the soluble hIL-6 receptor (sIL-6Ralpha, gp80). CONCLUSION: hIL-6 facilitates HBV infection in vitro and in vivo.
Animals↗
In vivo modulation of the allogeneic immune response by human fetal kidneys: the role of cytokines, chemokines, and cytolytic effector molecules.
BACKGROUND: We have recently demonstrated that human fetal renal tissue, implanted under the kidney capsule of severe immunodeficient rats, escapes early destruction by intraperitoneal infusion of allogeneic human peripheral blood mononuclear cells, compared with the rapid rejection of implants of human adult kidney tissue. Variable amounts of human mononuclear infiltrates were seen in the transplanted fetal kidney, however, prolonged survival of the fetal tissue (maintenance of graft architecture and significant growth) was independent of the cellular infiltrate. METHODS: We have used this experimental model to sequentially analyze transcript levels of interferon-gamma and interleukin (IL)-2 (T helper 1 cytokines), IL-4 and IL-10 (T helper 2 cytokines), RANTES, MIP1beta (beta chemokines) and their receptor CCR5, and Fas ligand (cytolytic effector molecule). Analysis was performed by reverse transcriptase-polymerase chain reaction, in both fetal and adult kidney grafts, after infusion of allogeneic human peripheral blood mononuclear cells. RESULTS: Transcript levels of interferon-gamma and IL-2 in the fetal grafts were markedly reduced throughout follow-up, compared with those observed in the adult implants. Peak levels of these cytokines appeared late in the rejection process. Concomitant with these findings, IL-4 mRNA was up-regulated during the early phase, whereas IL-10 mRNA persisted throughout the rejection process, indicating that a T helper 2 bias occurred in the fetal grafts. In addition, RANTES (after an early peak), MIP1beta, CCR5, and Fas ligand mRNA levels were suppressed in the fetal grafts compared with those in the adult grafts. CONCLUSIONS: These findings indicate that the immune response of kidney rejection is dependent on whether the target organ is of fetal or adult origin, and suggest that an allogeneic immune system mounts a T helper 2-biased response when the target organ is of fetal origin.
Adult↗
Reduced hepatitis B virus surface antigen-specific Th1 helper cell frequency of chronic HBV carriers is associated with a failure to produce antigen-specific antibodies in the trimera mouse.
In chronic hepatitis B virus (HBV) infection weak antiviral immune responses are associated with viral persistence. We studied possible immune deficits underlying the lack of serum antibodies of such patients against the HBV surface antigen (HBsAg) in a novel human/mouse chimeric model. A hepatitis B surface antigen (HBs) vaccination of Balb/c mice engrafted with peripheral blood mononuclear cells (PBMC) of naturally HBV-immunized donors induced high frequencies of human HBsAg-specific B and T helper 1 (Th1) cells. These responses were associated with high serum anti-HBs antibody levels of the subclasses immunoglobulin G1 (IgG1) and IgG2 that are driven by interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). In contrast, PBMC of chronic HBV carriers transplanted into the chimera failed to produce anti-HBs antibodies after vaccination with HBsAg and exhibited a deficit of antigen-specific Th1 cells. A possible influence of HBsAg or viremia was excluded by the lack of viral replication in such chimera. The observed T-cell defect was specific for HBsAg, as the B- and T-cell responses to tetanus toxoid (TT) were fully retained. Thus, our study shows that viral persistence in chronic HBV carriers is associated with an HBsAg-specific Th1 cell defect, which likely is responsible for the insufficient neutralizing anti-HBs-antibody response and is not reversed by HBs vaccination. Alternative approaches to induce HBs-specific Th1 cell responses might represent a future therapeutic option.
Animals↗
Transplantation tolerance induced by "mega dose" CD34+ cell transplants.
Early studies in murine models and more recent clinical data in heavily pretreated leukemia patients have shown that escalation of hematopoietic progenitor cells can overcome major genetic barriers and enable rapid and durable engraftment of haploidentical 3-loci mismatched transplants without graft-versus-host disease. In vitro studies suggest that veto cells within the progenitors population most likely mediate this facilitating effect. Leukemia relapse is relatively low in patients with acute myeloid leukemia (AML) but is greater in adults with acute lymphoblastic leukemia (ALL). Donor NK cells most likely mediate the resistance to relapse in patients with AML who are recipients of haploidentical transplants. Immune reconstitution in adults but not in children is slow as in adult recipients of HLA matched unrelated bone marrow transplants. The "mega dose" concept was also shown recently to be useful for tolerance induction in sublethally irradiated mice, so as to effectively overcome the marked resistance presented by the large number of lymphocytes surviving the sublethal conditioning. Thus, allogeneic chimeras generated by transplantation of large doses of Sca1+Lin- cells, permanently accept allogeneic donor type skin grafts. However, the numbers required to attain this desirable goal may not be easily collected from human donors. Nonalloreactive T cells synergize with murine Sca1+Lin- cells and might, therefore, enable achievement of engraftment of haploidentical transplants in sublethally conditioned patients.
Adult↗
Tolerance induction by 'megadose' transplants of CD34+ stem cells: a new option for leukemia patients without an HLA-matched donor.
Early studies in murine models and more recent clinical data in heavily pre-treated leukemia patients have shown that escalation of the dose of hematopoietic progenitor cells can overcome major genetic barriers and enable rapid and durable engraftment of haploidentical, three-locus-mismatched transplants without graft-versus-host disease. In vitro studies suggest that veto cells within the progenitor population most probably mediate this facilitating effect.
Animals↗
Anti-third party CD8+ CTLs as potent veto cells: coexpression of CD8 and FasL is a prerequisite.
Several bone marrow cells and lymphocyte subpopulations, known as "veto cells," were shown to induce transplantation tolerance across major histocompatibility antigens. Recently, it has been suggested that anti-third party CTLs depleted of alloreactivity are endowed with marked veto activity and therefore might potentially facilitate bone marrow allografting without graft versus host disease (GVHD). The veto mechanism is still obscure. While early studies emphasized the role of CD8-mediated apoptosis, more recent evidence indicates a role for Fas-FasL. In the present study we show, by using blocking anti-CD8 antibody, by generating CTLs from FasL or perforin mutated mice, and by gene transfer of FasL, that the veto activity of anti-third party CD8+ CTLs is dependent upon the simultaneous expression of both CD8 and FasL.
Animals↗
Successful haploidentical bone marrow transplantation in Fanconi anemia.
A 10-year-old girl with Fanconi anemia and severe aplastic anemia underwent a haploidentical BMT from her mother due to lack of a matched family donor. T cell depletion was done by positive selection of CD34 cells with immunomagnetic beads. Due to graft rejection a second haploidentical BMT from the father was successfully undertaken. No immunosuppression was given after the transplant. Immunological reconstitution took approximately 6 months, with no GVHD or severe infections. Such a transplant, containing a large purified CD34 cell fraction with a minimal number of added T cells, should be considered as the treatment of choice for patients with Fanconi anemia if no HLA matched donor is available.
Antigens, CD34↗
Preclinical evaluation of two human anti-hepatitis B virus (HBV) monoclonal antibodies in the HBV-trimera mouse model and in HBV chronic carrier chimpanzees.
Two human monoclonal antibodies (mAbs) against hepatitis B surface antigen (HBsAg) generated in the Trimera mouse system are described. Both mAbs 17.1.41 and 19.79.5 are of the IgG1 isotype and have high affinity constants for HBsAg binding in the range of 10(-10) mol/L. Monoclonal antibody 17.1.41 recognizes a conformational epitope on the a determinant of HBsAg whereas mAb 19.79.5 recognizes a linear one. The 2 mAbs bind to a panel of hepatitis B virus (HBV) subtypes with distinct patterns. The neutralizing activity of these antibodies was tested in 2 different animal model systems. Administration of each mAb to HBV-Trimera mice, a system that provides a mouse model for human hepatitis B infection, reduced the viral load and the percentage of HBV-DNA-positive mice in a dose-dependent manner. These 2 mAbs were more effective than a polyclonal antibody preparation (Hepatect; Biotest Pharma, Dreieich, Germany) in both inhibition of HBV liver infection and reduction of viral load. A single administration of a mixture of these mAbs into HBV chronic carrier chimpanzees resulted in immediate reduction in HBsAg levels followed by recurrence to initial levels within few days. Thus, these mAbs may be potential candidates for preventive therapy or in combination with other antiviral agents against HBV. Further studies in humans are needed to assess these mAbs in various clinical indications.
Animals↗
Autologous T cells control B-chronic lymphocytic leukemia tumor progression in human-->mouse radiation chimera.
B-chronic lymphocytic leukemia (B-CLL) is characterized by the clonal accumulation of CD5+ B cells. It has been suggested that CLL cells may be regulated by inhibitory and growth-promoting signals exerted by autologous T cells. We have recently described a model for human B-CLL in which peripheral blood mononuclear cells (PBMCs) are transplanted into the peritoneal cavity of lethally irradiated mice radioprotected with bone marrow from mice with severe combined immunodeficiency. In this model, adoptive transfer of low-stage PBMCs leads to marked engraftment of T cells or combined T and CLL cell engraftment, whereas infusion of high-stage PBMCs leads to dominance of CLL cells with a miniscule level of T-cell engraftment. This mutual exclusive pattern of engraftment indicated that T cells might control the expansion of tumor cells in the peritoneum of recipient BALB/c mice. In the present study, we further investigated this question and we demonstrate that in vivo T-cell depletion, using OKT3 antibody, markedly enhances the engraftment of B-CLL cells from patients with early-stage disease. In mice receiving PBMCs from 11 donors with advanced-stage disease, the results were more heterogeneous. In five patients the results were similar to those observed in early stage, whereas in two cases no CLL cell engraftment was found in the absence of T cells. The addition of purified T cells to PBMCs led to a substantial decrease of CLL engraftment in three advanced-stage cases. These results strengthen the working hypothesis that autologous T cells can actively suppress the expansion of the pathological cells in human-->mouse radiation chimera. This effect is prominent in early-stage disease, whereas in advanced stage suppressive and/or stimulatory effects may occur in different patients. The interaction of T cells with tumor cells and the potential of autologous T cell/immune-therapy in CLL can be further explored in this model.
Aged↗
Donor-type chimerism determination by competitive polymerase chain reaction (PCR) in a primate model for bone marrow transplantation.
BACKGROUND: Evaluation of the outcome of successful bone marrow transplantation (BMT) and in-depth studies of transplantation biology rely increasingly on accurate detection of donor origin cells in the transplanted recipients. This study describes a quantitative competitive polymerase chain reaction (PCR) assay for accurate evaluation of chimerism after allogeneic BMT in a cynomologous primate model, based on detection of monkey Y-specific DNA. METHODS: A competitor standard was generated via PCR using a mutagenic primer that makes the competitor DNA 22 bp less than the wild type monkey Y-specific DNA. The mutated form can still be amplified by the primer pair for the detection of monkey Y-specific DNA. A fixed amount of sample subjected to chimerism detection was co-amplified with a range of competitor DNA using a touch down program and hot start PCR technique. The PCR products were analyzed by computing densitometry. The ratio of competitor/target (Y-specific) DNA for each sample pair was calculated. RESULTS: Using DNAs prepared from an artificial mixture of male and female cells, a set of standard curves has been obtained and the sensitivity of the established quantitative PCR was found to be 25 pg of male DNA, which corresponds approximately to 0.005 fg competitor DNA. A DNA sample taken from a female monkey, transplanted with purified CD34+ stem cells from a male monkey donor 26 days after BMT, was subjected to the competitive PCR with 10% male DNA as a control; the level of male DNA in this sample was calculated to be around 50%. CONCLUSIONS: This quantitative PCR assay offers both a high degree of specificity as well as a very accurate and sensitive evaluation of chimerism in a sex-mismatched monkey BMT model.
Animals↗
Induction of donor-type chimerism and transplantation tolerance across major histocompatibility barriers in sublethally irradiated mice by Sca-1(+)Lin(-) bone marrow progenitor cells: synergism with non-alloreactive (host x donor)F(1) T cells.
Induction of transplantation tolerance by means of bone marrow (BM) transplantation could become a reality if it was possible to achieve engraftment of hematopoietic stem cells under nonlethal preparatory cytoreduction of the recipient. To that end, BM facilitating cells, veto cells, or other tolerance-inducing cells, have been extensively studied. In the present study, we show that BM cells within the Sca-1(+)Lin(-) cell fraction, previously shown to be enriched for early hematopoietic progenitors, are capable of reducing specifically antidonor CTL-p frequency in vitro and in vivo, and of inducing split chimerism in sublethally 7-Gy-irradiated recipient mice across major histocompatibility complex barriers. The immune tolerance induced by the Sca-1(+)Lin(-) cells was also associated with specific tolerance toward donor-type skin grafts. The minimal number of cells required to overcome the host immunity remaining after 7 Gy total body irradiation is very large and, therefore, it may be very difficult to harvest sufficient cells for patients. This challenge was further addressed in our study by demonstrating that non-alloreactive (host x donor)F(1) T cells, previously shown to enhance T-cell-depleted BM allografts in lethally irradiated mice, synergize with Sca-1(+)Lin(-) cells in their capacity to overcome the major transplantation barrier presented by the sublethal mouse model.
Animals↗