A partial conditioning strategy for achieving mixed chimerism in the rat: tacrolimus and anti-lymphocyte serum substantially reduce the minimum radiation dose for engraftment

Development of partial conditioning strategies to achieve reliable engraftment of allogeneic bone marrow with minimum recipient morbidity could extend the therapeutic application of bone marrow transplantation (BMT) to enzyme deficiency states, hemoglobinopathies, autoimmune diseases, and the induction of tolerance for solid organ and cellular allografts. In this study we describe a nonmyeloablative rat BMT model and examine the effect of clinically available immunosuppressants on the minimum amount of total body irradiation (TBI) required for allogeneic engraftment. Donor ACI marrow was depleted of T cells using immunomagnetic beads and transplanted to major histocompatibility complex- and minor antigen-mismatched Wistar Furth (WF) rats (ACI --> WF) conditioned with varying doses of TBI. Recipients conditioned with TBI alone required myeloablation with 1000 cGy for reliable allogeneic marrow engraftment. Administration to WF recipients of a single dose of anti-lymphocyte serum (ALS) 5 days prior to BMT together with a limited course of tacrolimus (1 mg/kg/day) resulted in engraftment of ACI bone marrow at only 500 cGy TBI. ACI --> WF recipients were stable mixed chimeras (mean donor chimerism 49% at 330 days post-BMT). Chimerism was multilineage. All recipient animals were free of graft-versus-host disease. These results suggest that a nonmyeloablative conditioning strategy based on low-dose TBI and a limited course of tacrolimus plus ALS can produce long-term mixed multilineage chimerism.     

Prevention of graft-versus-host disease in rat small bowel transplantation by recipient pretreatment with UV-B-modulated bone marrow cells

UV-B irradiation (700 J/m2) of bone marrow cells (BMC) before transplantation into lethally irradiated (1050R) allogeneic rats prevents graft-versus-host disease (GVHD) and results in stable chimerism. This study examined whether UV-B modulation of BMT is useful in the subsequent induction of tolerance to small bowel transplant (SBT) and avoids the danger of GVHD, which remains the major obstacle to successful SBT. Lethally irradiated Lewis recipients of UV-B irradiated (700 J/m2) BMT (10(8) BMC admixed with 5 x 10(6) splenic leukocytes) either from ACI or Wistar-Furth (WF) rats developed stable chimerism without any evidence of GVHD for > 360 days. Lewis recipients of UV-B ACI BMC expressed 95 +/- 6% ACI lymphoid cells at 50 and 150 days after BMT using complement-dependent cytotoxicity assay. Unmodified Lewis recipients of orthotopic ACI SBT rejected their grafts and died in 7-9 days, whereas Lewis chimeras accepted permanently (> 200 days) bone marrow donor (ACI) SBT without any evidence of GVHD when the SBT was performed at 60 or 150 days after BMT. In contrast, when SBT was performed, only 30 days after induction of chimerism with UV-B ACI BMT, the recipients developed severe GVHD and died between 17 and 21 days. The Lewis chimeras rejected third part (WF) SBT acutely and died in 7-9 days, thus demonstrating the specificity of the induction of tolerance in this model. That this immunologic unresponsiveness is not restricted by the recipient-donor rat strain combination was shown by the permanent acceptance of WF SBT without GVHD by Lewis/WF chimeric recipients. Furthermore, the Lewis chimeras that were made diabetic with STZ 28 days after BMT permanently accepted (> 300 days) BM donor-type (WF) and recipient-type (Lewis) islet cells and became normoglycemic, thus indicating tolerance to both donor and recipient Ags. The diabetic Lewis chimeras that became normoglycemic permanently accepted (> 200 days) WF SBT without any evidence of GVHD after donor-type SBT 110 days after WF islet transplantation. The apparent lack of organ-specific unresponsiveness in this model confirmed our previous observation with combined islet and heart transplants. In vitro MLR studies showed that the chimeric animals were specifically unreactive to donor- and recipient-type alloantigens. Our results demonstrate that UV-B irradiation of BMT is a promising approach to the induction of tolerance to SBT.     

Characterization of SCML1, a new gene in Xp22, with homology to developmental polycomb genes

BACKGROUND: Mixed hematopoietic chimerism induced with a nonmyeloablative conditioning regimen leads to donor-specific transplantation tolerance. Analyses of specific Vbeta-bearing T-cell families that recognize endogenous superantigens demonstrated that donor-specific tolerance is due mainly to an intrathymic deletional mechanism in these mixed chimeras. However, superantigens are not known to behave as classical transplantation antigens. We therefore used T-cell receptor (TCR) transgenic (Tg) recipients expressing a clonotypic TCR specific for an allogeneic major histocompatibility complex antigen to further assess deletional tolerance. METHODS: 2C TCR Tg mice (H2b), whose Tg TCR recognizes major histocompatibility complex class I Ld, were used as recipients of Ld+ bone marrow cells after conditioning with depleting anti-CD4 and CD8 monoclonal antibodies, 3 Gy whole-body irradiation, and 7 Gy thymic irradiation. Chimerism and deletion of CD8+ 2C recipient T cells was evaluated by flow cytometry and by immunohistochemical staining. Tolerance was tested with in vitro cell-mediated lympholysis assays and in vivo by grafting with donor skin. RESULTS: Intrathymic and peripheral deletion of 2C+ CD8-single-positive T cells was evident in mixed chimeras, and deletion correlated with the presence of donor-type cells with dendritic morphology in the thymus, and with chimerism in lymphohematopoietic tissues. Chimeras showed tolerance to the donor in cell-mediated lympholysis assays and specifically accepted donor skin grafts. CONCLUSIONS: Tolerance to transplantation antigens is achieved through intrathymic deletion of donor-reactive T cells in mixed chimeras prepared with a nonmyeloablative conditioning regimen and allogeneic bone marrow transplantation.     

A nonlethal conditioning approach to achieve engraftment of xenogeneic rat bone marrow in mice and to induce donor-specific tolerance

BACKGROUND: The supply of solid organs for transplantation will never meet the growing demand. Xenotransplantation is considered to be a potential solution for the critical shortage of allografts. However, xenograft rejection is currently not controlled by conventional immunosuppressive agents. Bone marrow chimerism induces donor-specific tolerance without the requirement for chronic immunosuppressive therapy. The aim of this study was to develop a nonlethal recipient-conditioning approach to achieve mixed bone marrow chimerism and donor-specific tolerance. METHODS: C57BL/10SnJ mice were conditioned with total body irradiation followed by a single injection of cyclophosphamide on day +2. On day 0, mice were reconstituted with untreated bone marrow cells from Fischer 344 rats. Recipients were analyzed by flow cytometry for donor bone marrow engraftment and multilineage chimerism. Donor-specific tolerance was tested by skin grafting. RESULTS: One hundred percent of recipients engrafted after irradiation with 600 cGy total body irradiation, transplantation with 80 x 10(6) Fischer 344 bone marrow cells, and injection with 50 mg/kg cyclophosphamide intraperitoneally. Donor chimerism was detectable in all engrafted animals for up to 11 months. This conditioning was nonlethal, because conditioned untransplanted animals survived indefinitely. Mixed xenogeneic chimeras were tolerant to donor-specific skin grafts but rejected third-party (Wistar Furth) grafts as rapidly as naive C57BL/10SnJ mice. In contrast, animals that received less efficacious conditioning regimens and did not exhibit detectable chimerism showed prolonged graft survival, but delayed graft rejection occurred in all animals within 10 weeks. CONCLUSION: The induction of bone marrow chimerism and donor-specific tolerance after nonlethal conditioning might be useful to prevent the vigorous cellular and humoral rejection response to xenografts.     

Osteogenic potential of allogeneic rat marrow cells in porous hydroxyapatite ceramics: a histological study

Hydroxyapatite ceramics facilitate osteogenesis but cannot induce bone formation by themselves. We studied the feasibility of bone formation supported by allogeneic bone marrow cells in porous hydroxyapatite ceramics. Coralline hydroxyapatite discs were soaked in a marrow cell suspension harvested from either ACI (RT1a), Lewis (RT1(1)), or Fischer 344 (RT1(1v)) male rats, and these discs were implanted subcutaneously into 56 male Fischer 344 rats. FK-506 (tacrolimus hydrate), an immunosuppressant, or saline was injected intramuscularly into the recipients every day for 2 weeks after surgery, and additional injections were given to 19 of the rats every 2 days for 2 more weeks. Neither of the mismatched major (ACI rat) or minor (Lewis rat) marrow cell transplants showed any bone formation without administration of FK-506. However, in rats treated with FK-506, bone formed in the pores of all the three types of ceramics implanted, which each contained the marrow cells from one of the three kinds of rats used. There were no differences among the three groups of donors with regard to the bone formation ratio. We previously reported that subcutaneous implantation of porous hydroxyapatite combined with isogeneic marrow cells resulted in consistent bone formation, even at ectopic sites. Since it would be difficult to harvest a large number of autologous marrow cells in clinical cases, we attempted to use allogeneic marrow cells and have shown the allogeneic murine marrow cells to have osteogenic potential.     

Graft-versus-host-disease-associated lymphoid hypoplasia and B cell dysfunction is dependent upon donor T cell-mediated Fas-ligand function, but not perforin function

Allogeneic bone marrow transplant recipients often exhibit a graft-versus-host-disease (GVHD)-associated immune deficiency that can be prolonged and lead to life-threatening infections. We have examined the role of donor T cell-mediated cytotoxic function in the development of GVHD-associated immune deficiency. A major histocompatibility complex-matched model of allogeneic bone marrow transplantation was employed in which lethally irradiated C3H.SW mice received a nonlethal dose of T cells from either perforin-deficient (B6-perforin 0/0), Fas-ligand (FasL)-defective (B6-gld), or normal (B6) allogeneic donor mice. T cell-depleted marrow from B6-Ly-5.1 congenic donor mice was transplanted along with the donor T cell populations to determine the effects of donor T cell-mediated cytotoxicity on engraftment. Our results demonstrate that recipients of perforin-deficient or normal allogeneic T cells exhibit profound lymphoid hypoplasia and severely reduced splenic proliferative responses to lipopolysaccharide in vitro. In contrast, GVHD-associated lymphoid hypoplasia is dramatically reduced and in vitro B cell function is intact in recipients of FasL-defective allogeneic T cells. Engraftment of myeloid and erythroid lineage cells occurs irrespective of donor T cell cytotoxic function. Although recipients of perforin-deficient or normal allogeneic T cells exhibited hematopoietic engraftment exclusively of donor origin, recipients of FasL-defective donor T cells exhibited significant mixed chimerism (Ly-5.1/Ly-5.2). Because only marrow of donor origin was transplanted, this finding suggests that Fas-mediated antirecipient cytotoxicity is required for clearance of residual hematopoietic stem cells of host origin that persist following lethal irradiation.     

Chimerism analysis in long-term survivor patients after bone marrow transplantation for severe aplastic anemia

BACKGROUND AND OBJECTIVE: Allogeneic bone marrow transplantation (BMT) is the most common treatment for young patients with severe aplastic anemia (SAA). Late graft failure represents one of the possible unfavorable outcomes in this setting. Mixed chimerism might represent a risk factor for late graft failure. We examined this relationship by studying chimerism in long-term survivor SAA patients after allogeneic BMT. METHODS: We analyzed long-term hematopoietic chimerism in 15 patients who received BMTs for SAA: 9 with an irradiation-based conditioning regimen and 6 with ATG. We used a PCR method targeting VNTR loci. Sensitivity of the technique ranged between 0.5 and 1.5%. RESULTS: All patients conditioned with radiation-based schemes showed complete donor chimerism. Conversely, out of six patients who received cyclophosphamide and ATG as a conditioning regimen, only one of them had late graft failure (day +168). In this patient, durable mixed chimera status was first detected two months after BMT. INTERPRETATION AND CONCLUSIONS: Our results suggest that in long-term survivors of SAA after BMT there is almost always complete donor chimerism in both irradiated and ATG-conditioned recipients. Mixed chimerism might predict graft failure in these patients.     

Metabolic trafficking through astrocytic gap junctions

BACKGROUND: Protocols that incorporate donor-specific cell infusions using bone marrow, spleen, or blood transfusion continue to enhance allograft survival and often lead to tolerance in experimental models. Clinical benefits from these modalities have not been as striking, leading to ongoing study in this field. We have explored culture techniques for the in vitro selection and development of cellular effectors capable of enhancing allograft survival. METHODS: Rat bone marrow or spleen cells cultured under a variety of conditions were screened for suppressor function. Bone marrow cells, nonadherent to plastic, cultured for 7 days with granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, and with or without splenocytes were found to contain predominantly myeloid lineage cells and had the ability to suppress phytohemagglutinin or mixed lymphocyte reaction-induced splenocyte proliferation. Standard donor-specific peripheral blood transfusion was compared with cultured donor-specific bone marrow cells, splenocytes, or marrow cells cultured with splenocytes (cocultured) administered intravenously at 1 x 10(7) cells/kg the day before an ACI to Lewis heterotopic heart transplant. Cyclosporine was administered at 10 mg/kg on day -1 and 2.5 mg/kg on days 0-6 relative to transplantation. RESULTS: Mean allograft survival in cyclosporine-treated animals was 8.5 days without and 16.6 days with a donor-specific blood transfusion. Cocultured cells extended allograft survival (39.5 days), whereas bone marrow or splenocytes cultured alone did not. With Percoll gradient separation, two predominant culture subfractions, one with potent suppressor function and another with stimulator function, were identified. Flow cytometric analysis showed mixed populations enriched for macrophages but also including dendritic cells in both subfractions. The suppressive fraction extended allograft survival to 20.8 days and the stimulatory fraction was less effective, yet remixing of both fractions regained the full allograft survival advantage. CONCLUSIONS: In this model, the coculture of bone marrow cells and splenocytes with granulocyte-macrophage colony-stimulating factor and lipopolysaccharide produced functionally divergent subpopulations that synergistically enhanced allograft survival. The development of cellular effectors with enhanced ability to prolong allograft survival using in vitro culture techniques is possible, and provides a new therapeutic option in the use of cell infusion-based therapies.     

Induction of donor-specific tolerance to cardiac xenografts in utero

BACKGROUND: Problems associated with heart transplantation, such as shortage of suitable organs and the side effects of immunosuppressive therapy, are especially serious for patients in the pediatric age group. Induction of donor-specific immunologic tolerance without immunosuppressive drugs would be ideal for clinical organ transplantation. In this study, we used a vascularized cardiac xenograft model to achieve donor-specific unresponsiveness without immunosuppression by manipulating the intrauterine immune response. METHODS: Lewis rats and Golden Syrian hamsters were used as the recipients and donors, respectively. Donor bone marrow cells (15 x 10(6) in 0.05 mL) were injected into each fetus of pregnant Lewis rats on days 9 (n = 2) and 16 (n = 2) of gestation. Donor hearts were heterotopically transplanted into each surviving (n = 8, n = 5) fetus of the Lewis rats at 8 weeks of age. Donor hearts were also transplanted into untreated rats as controls (n = 8). RESULTS: The mean cardiac xenograft survival time was 2.5 +/- 0.5, 7.4 +/- 4.1, and 2.8 +/- 0.8 days in the control group, gestational day 9 group, and gestational day 16 group, respectively. Chromosomal analysis of the day 9 group showed Golden Syrian hamster chromosomes as well as Lewis rat chromosomes. CONCLUSIONS: Cardiac xenograft survival was significantly prolonged by intrauterine exposure to xenograft bone marrow cells on day 9 but not on day 16 of gestation. Cardiac xenograft survival and chromosomal analysis of the recipient bone marrow suggested that chimerism was achieved between Golden Syrian hamsters and Lewis rats. Cardiac xenotransplantation may be possible by induction of donor-specific tolerance in utero.     

Prevalence of asthma and wheeze in primary school children in northern Jordan

While the existence of chimeric cells in host tissue following organ transplantation is well documented, its distribution, temporal evolution and relationship to allograft survival is less clear. To explore this phenomenon, Lewis recipients of ACI cardiac allografts representing a wide range of immunosuppressive protocols and graft survival times were examined for the presence of chimerism using a sensitive polymerase chain reaction assay. Four groups of animals were examined: untransplanted animals receiving donor specific transfusion (DST)/cyclosporine A (CsA); allograft recipients with no treatment; recipients treated with DST/CsA/supplementary immunosuppression with rejection at 21-183 days; and recipients sacrificed with functioning allografts, treated with DST/CsA/supplementary immunosuppression and surviving > 200 days. To elucidate variations in the tissue distribution of chimeric cells, bone marrow, skin, liver, spleen, and thymus were examined in each animal. Untransplanted animals receiving DST/CsA displayed no evidence of chimerism. In animals receiving a cardiac allograft but no treatment, there was extensive evidence of chimerism in four of five animals. Chimerism was also detected in seven of nine animals with intermediate graft survival at the time of rejection. In animals with long-term graft survival, only four of eight displayed chimerism. These results suggest that, without immunosuppression, early chimerism does not lead to prolonged graft survival and that, even when graft survival is moderately prolonged, these cells are not sufficient to prevent rejection. In conclusion, chimerism appears to be a common phenomenon following transplantation, is not a result of DST, and may not be necessary for maintenance of long-term graft survival.     

Stable mixed chimerism without relapse after related allogeneic umbilical cord blood transplantation in a child with severe aplastic anemia

Umbilical cord blood (UCB) cells from HLA-matched donors are used as an alternative to bone marrow for allogeneic transplantation and reports of successful UCB transplantation in patients with severe aplastic anemia (SAA) are scarce. SAA was discovered in a 4-year-old girl in February 1990. Transfusion support started in August 1990 and standard treatments were unsuccessful. The birth of an HLA-compatible brother in October 1993 permitted the cryopreservation of UCB. In December 1994 UCB transplantation was decided upon. No toxicity occurred. G-CSF was started at day 28. WBC and PMN reached 0.5 x 10(9)/l at days 33 and 37. RBC and platelet transfusion independence were reached at days 50 and 52. Mixed chimerism was demonstrated in blood cells at 1.5, 4 and 6 months after UCBT by molecular biology (VNTR). FISH studies yielded similar results at 15 and 18 months. Twenty months after UCBT, molecular biology showed full donor chimerism. Clinical follow-up (last follow-up: 32 months post transplant) is unremarkable. We suggest that CY and ATG may be a suitable regimen for related HLA-compatible UCBT in patients with SAA. Residual recipient cells can disappear even very late after UCBT, permitting the establishment of complete donor chimerism.     

Host reactive donor T cells are associated with lung injury after experimental allogeneic bone marrow transplantation

Noninfectious lung injury is common after allogeneic bone marrow transplantation (BMT), but its association with acute graft-versus-host disease (GVHD) is unclear. Using a murine BMT system where donor and host differ by multiple minor histocompatibility (H) antigens, we investigated the nature of lung injury and its relationship both to systemic GVHD and host-reactive donor T cells. Lethally irradiated CBA hosts received syngeneic BMT or allogeneic (B10.BR) T-cell-depleted (TCD) bone marrow (BM) with and without the addition of T cells. Six weeks after BMT, significant pulmonary histopathology was observed in animals receiving allogeneic BMT compared with syngeneic controls. Lung damage was greater in mice that received allogeneic T cells and developed GVHD, but it was also detectable after TCD BMT when signs of clinical and histologic acute GVHD were absent. In each setting, lung injury was associated with significant alterations in pulmonary function. Mature, donor (Vbeta6(+) and Vbeta3(+)) T cells were significantly increased in the broncho-alveolar lavage (BAL) fluid of all allogeneic BMT recipients compared with syngeneic controls, and these cells proliferated and produced interferon-gamma (IFN-gamma) to host antigens in vitro. These in vitro responses correlated with increased IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) in the BAL fluid. We conclude that alloreactive donor lymphocytes are associated with lung injury in this allogeneic BMT model. The expansion of these cells in the BAL fluid and their ability to respond to host antigens even when systemic tolerance has been established (ie, the absence of clinical GVHD) suggest that the lung may serve as a sanctuary site for these host reactive donor T cells. These findings may have important implications with regard to the evaluation and treatment of pulmonary dysfunction after allogeneic BMT even when clinical GVHD is absent.     

Induction of tolerance to an experimental cardiac allograft through intrathymic inoculation of class II major histocompatibility complex disparate antigens

Indefinite donor-specific tolerance to a cardiac allograft can be induced through pretransplantation intrathymic injection of donor spleen cells and a single intraperitoneal injection of antilymphocyte serum. This study was designed to determine whether this phenomenon was reproducible with grafts differing in either class I major histocompatibility complex only or class II MHC only. Donors of cells and hearts in all experiments were RP rats. Class I MHC disparate grafts were performed by placing an RP heart into a Lewis recipient, and class II disparate grafts were performed with RP donors and Wistar Furth recipients. Lewis (n = 10) and Wistar Furth (n = 10) recipients underwent intraperitoneal injection of 1 ml antilympocyte serum and intrathymic injection of 5 x 10(7) RP spleen cells. Three weeks later, heterotopic cardiac transplantation was done with a heart from an RP rat. Control rats had no pretreatment or received antilympocyte serum alone. Without pretreatment, RP hearts survived 7 to 9 days (mean 8 days) in Lewis recipients (n = 5) and 9 to 14 days (mean 12 days) in Wistar Furth recipients (n = 5). Antilymphocyte serum alone produced slight prolongation of graft survival. Lewis rats pretreated with class I disparate RP splenocytes and antilympocyte serum had graft survivals of 8 to 27 days (mean 14 days), not significantly different from the results with antilympocyte serum alone. Class II disparate RP grafts placed in pretreated Wistar Furth rats had significant prolongation of graft survival, with four of five grafts surviving longer than 60 days (p < 0.01 vs antilympocyte serum alone). These results suggest that a disparity at the class II locus of the major histocompatibility complex is critical for the induction of cardiac allograft tolerance after intrathymic inoculation of allogeneic cells.     

Specific tolerance induction and transplantation: a single-day protocol

Bone marrow transfusion is a well-established method for induction of mixed hematopoietic chimerism and donor-specific tolerance in animal models. This procedure, however, is inapplicable in clinical transplantation using cadaveric donors due to the interval (1 week to 7 months) between tolerance induction and organ transplantation. For clinical use, it is essential that allografts be placed at the time of bone marrow transfusion. In the present study, we performed skin transplantation within 1 hour after a nonlethal conditioning regimen. Recipient mice were treated with anti-CD3, anti-CD4, low-dose total body irradiation (3 to 6 Gy TBI) and fully mismatched or haploidentical donor bone marrow cells. Stable multilineage chimerism and specific T-cell nonresponsiveness developed. Donor skin grafts were permanently accepted. These results suggest that this single day protocol has clear potential for application in both cadaveric and living-related organ transplantation.     

Discovery of displaced canines among adolescents in the mid-section of the maxillary and mandibular apical areas and their management

Blood transfusions (BT) should be considered as transplantation of blood elements. They evoke various alterations in the immune responsiveness of blood recipients, contributing to an increased risk of infection and cancer recurrence. The exact mechanism by which blood transfusions induce a state of reduced immune responsiveness remains unclear. Relatively little is also known about immune changes occurring in lymphoid compartments other than blood following blood transfusions. In the present study, the effect of syngeneic and allogeneic blood transfusions on hemopoiesis and immune responsiveness was examined in a rat model. Transfusions of both syngeneic and allogeneic blood caused an increase in the bone marrow myeloid and lymphoid lineage cell compartments as well as a rise in the percentage of OX7+ stem cells in bone marrow. It was followed by a release of OX7+ stem cells into blood circulation, higher in the case of rats transfused with syngeneic blood. The changes in hemopoiesis were accompanied by a diminished responsiveness of blood, spleen and bone marrow lymphocytes to mitogens in both groups of rats. Data point to early changes in distribution and reactivity of bone marrow and lymphoid cells following blood transfusion.     

Hypoxia induces high-mobility-group protein I(Y) and transcription of the cyclooxygenase-2 gene in human vascular endothelium

BACKGROUND: Animals reconstituted with allogeneic whole bone marrow (WBM) are often tolerant of donor-specific solid organ grafts. Clinical application of bone marrow transplantation in solid organ transplantation has been limited, however, principally by graft-versus-host disease. We previously demonstrated that hematopoietic stem cells (HSCs) reconstitute lethally irradiated allogeneic mice without producing graft-versus-host disease. The purpose of this study was to determine whether tolerance to solid organ grafts could be induced in mice reconstituted with HSCs. METHODS: BALB/c mice were lethally irradiated and reconstituted with allogeneic C57BL/Ka, Thy-1.1 WBM or HSCs. An isolated group was given a limited number of HSCs (250 cells) and a subpopulation of allogeneic cells known to facilitate HSC engraftment (facilitators). C57BL/Ka, Thy-1.1 neonatal heart grafts were placed in reconstituted animals either at the time of hematopoietic transplant or 35 days later. Third-party C3H grafts were placed over 2 months after hematopoietic reconstitution. Tolerance was defined as the persistence of cardiac contraction for the duration of evaluation (125-270 days). RESULTS: All surviving mice that were reconstituted with C57BL/Ka, Thy-1.1 HSCs, WBM, or HSCs and facilitators were tolerant of C57BL/Ka grafts long-term. Third-party C3H grafts placed in reconstituted animals were rejected by day 12, whereas those placed in unmanipulated mice were rejected by day 9. CONCLUSION: These data indicate that tolerance to concurrently or subsequently placed solid organ grafts can be reliably achieved with limited numbers of purified HSCs in a model where immunocompetence to third-party major histocompatibility complex antigens is delayed but intact.     

Permanent and specific transplantation tolerance induced by a nonmyeloablative treatment to a wide variety of allogeneic tissues: I. Induction of tolerance by a short course of total lymphoid irradiation and selective elimination of the donor-specific host lymphocytes

The long-term success of organ transplantation is limited by complications resulting from consistent nonspecific immunosuppression. Induction of stable, donor-specific tolerance remains the main goal of transplantation immunology. In this article, a new, nonmyeloablative method is described for induction of transplantation tolerance to fully mismatched bone marrow cells (BMC), bone marrow stromal precursors, heart muscle, and skin allografts. The method is based on pretransplant conditioning with no postgraft immunosuppression, and consists of a short course (six daily fractions of 200 cGy) of total lymphoid irradiation (sTLI), followed by selective elimination of donor-specific alloreactive cells of the host escaping low-dose sTLI. Donor-specific alloreactive cells were activated by intravenous inoculation with a high dose of donor BMC (3 x 10(7) cells) 1 day after sTLI, and eliminated by a single intraperitoneal dose (200 mg/kg) of cyclophosphamide given 1 day after cell transfer. Infusion of a low number of T cell-depleted BMC (3 x 10(6) cells) after tolerogenic preconditioning converted recipients to stable mixed chimeras free of graft-versus-host disease. The same treatment provided long-lasting acceptance of heterotopically transplanted allografts of the heart muscle and of the stromal precursors to the hematopoietic microenvironment. This treatment also led to acceptance and life-long survival of full-thickness donor skin allografts. However, skin allografts survived only in mice that received donor T cell-depleted BMC after cyclophosphamide and had 20-50% donor cells in the blood. Our results suggest that after sTLI, additional selective clonal deletion of residual host cells induces a state of long-lasting specific tolerance to a wide variety of donor-derived tissues.     

Proliferative activity and loss of function of tumour suppressor genes as ''biomarkers'' in diagnosis and prognosis of benign and preneoplastic oral lesions and oral squamous cell carcinoma

Patients with hematologic malignancy or severe aplastic anemia after myeloablative chemo- and radiotherapy were given granulocyte colony-stimulating factor (G-CSF)-mobilized, cryopreserved allogeneic peripheral blood stem cells (PBSCs) from 15 healthy donors who were either human leukocyte antigen (HLA)-matched siblings (n = 13) or haploidentical offspring (2). Polymerase chain reaction-amplified short tandem repeat genotyping was used for early confirmation of donor engraftment after PBSC transplantation (PBSCT). A standard cyclosporine A/methotrexate combination was used to prevent acute graft-versus-host disease (GVHD). All donors, including one in the third trimester of pregnancy, tolerated G-CSF administration and 3-day PBSC harvesting procedures well. Engraftment was prompt for all patients; it was verified using a panel of 12 human polymorphic short tandem repeat loci from bone marrow as early as 7 days posttransplantation. This status was maintained until relapse, when mixed chimerism was detected using the polymerase chain reaction. A minimum resurgence of recipient cells to 1% of the population was required to detect chimerism. The median times to recovery of the absolute neutrophil count to greater than 0.5 x 10(9)/L and the sustained platelet count to greater than 20 x 10(9)/L without transfusion were 10 and 12 days after PBSCT, respectively. Six patients experienced acute GVHD, Grade I in two patients and Grade II in four, including two HLA-haploidentical recipients. Chronic GVHD was noticed in three of the 11 patients who were followed for at least 100 days after PBSCT. Ten patients were still alive at the latest follow-up and have been disease free for a median of 278 days (range 60-671). Five patients died from causes other than graft failure: three from leukemia relapse and two from transplant-related complications. The results confirm that G-CSF can be safely administered to healthy donors and that engraftment after allogeneic PBSCT is fast and durable. Complete chimerism can be detected early by genomic analysis. PBSCT may offer an alternative to bone marrow transplantation.     

Induction of mixed allogeneic chimerism for leukemia

Hybrid (C56BL/6 x DBA) (BDF1; H-2b/H-2d) mice bearing the P815 leukemia (H-2d) were grafted with a (CBA x C57BL/6)F1 (CBF1; H-2k/H-2b) cell suspension, comprising bone marrow cells (BMC; 25 x 10(6)/mouse) and spleen cells (SC; 55 x 10(6)/mouse) on day-4, then treated with cyclophosphamide (200 mg/kg) on day-2 and finally grafted once more with CBF1 cells (25 x 10(6) BMC + 7 x 10(6) SC) on day 0. Allogeneic cell graftings performed in this way induced durable mixed hematopoietic chimerism and significantly prolonged the survival of recipients, compared with that of leukemia-bearers grafted with syngeneic cells. The results obtained raise the possibility of using allogeneic hematopoietic tissue transplantation in combination with non-lethal cytoreductive therapy to induce a long-lasting graft-vs-leukemia effect.