Programmed death 1 monoclonal antibody helped to treat mixed chimeric and reactivation of Epstein-Barr virus in a patient with adult-onset chronic active Epstein-Barr virus infection after allogeneic hematopoietic stem cell transplantation: A case report.

RATIONALE: Systemic forms of chronic active Epstein-Barr virus infection (CAEBV) can predispose a patient to a protracted course of fulminant hemophagocytic lymphohistiocytosis, which has a poor prognosis. Epstein-Barr virus (EBV) infection may persist even after theoretically curative hematopoietic stem cell transplantation. PATIENT CONCERNS: A female patient with CAEBV underwent chemotherapy followed by allogeneic hematopoietic stem cell transplantation from her human leukocyte antigen-matched sister. Neutrophil and platelet engraftment was observed on day +12 and +10. Full donor chimerism (DC) was achieved on Day +21. DIAGNOSES: From day +38, EBV-DNA in the blood was persistently positive, and DC declined. We attempted empirical interventions such as withdrawal of immune suppression, multiple donor lymphocyte infusion, stem cell boost, and interferon-α treatment. However, EBV-DNA copies continued to increase aggressively, whereas DC decreased rapidly and then reached a nadir of 63.27%. INTERVENTIONS: Salvage programmed death 1 (PD-1) antibody treatment was administered as salvage therapy at +69 and +84. OUTCOMES: EBV-DNA was negative on day +97 and was ultimately undetectable. Equivalently, a full and stable DC was obtained at +97. LESSONS: We summarize a case of PD-1 antibody used as salvage treatment in a post-transplant patient with CAEBV, which was eradicated and full DC was obtained. This case suggests that the PD-1 antibody appears to be a promising option for fighting EBV and mixed DCs.

Human Chorionic Gonadotropin Improves the Proliferation and Regenerative Potential of Bone Marrow Adherent Stem Cells and the Immune Tolerance of Fetal Microchimeric Stem Cells In Vitro.

Nongonadal tissues express luteinizing hormone-chorionic gonadotropin receptors (LHCG-R) which are essential for their growth during fetal development. Adult mesenchymal stem/stromal cells (MSCs) have been shown to express functional LHCG-R outside pregnancy conditions, making them susceptible to hCG stimulation. In the present study we tested the effect of hCG treatment on bone marrow (BM) derived adherent stem cells in vitro, isolated from a parous women, mother of male sons, in order to evaluate its effect on maternal MSCs and in the same time on fetal microchimeric stem cells (FMSCs), to better understand the outcomes of this safe and affordable treatment on cell proliferation and expression of pluripotency genes. Our study highlights the beneficial effects of hCG exposure on gene regulation in bone marrow adherent stem cells through the upregulation of pluripotency genes and selection of more primitive mesenchymal stem cells with a better differentiation potential. Validation of these effects on MSCs and FMSCs long after parturition in vivo represents a close perspective as it could set the premises of a new mobilization strategy for the stem cell transplantation procedures in the clinical setting.

Post-transplantation cyclophosphamide for chimerism-based tolerance.

High-dose cyclophosphamide given post-transplant (PTCy) successfully enables tolerance induction in HLA-mismatched related blood or marrow transplantation (haploBMT) manifested by low rates of graft failure, severe acute graft-versus-host disease (GVHD), and chronic GVHD. When proceeded by nonmyeloablative conditioning, PTCy has also been associated with a low incidence of nonrelapse mortality. The safety of this platform has garnered interest in expanding its use to non-malignant indications for allogeneic blood or marrow transplantation (alloBMT). After success in a preliminary Phase I/II trial, use of a PTCy-based haploBMT platform is now being explored in a large Blood and Marrow Transplant Clinical Trials Network (BMT CTN) study for sickle cell disease. These emerging data in patients with hemoglobinopathies provided the rationale for exploring the use of PTCy in combined solid organ and BM transplantation as a means of tolerance induction through donor hematopoietic chimerism with a goal to obviate the need for a lifetime of immunosuppression. Several case reports, series, and small clinical trials have now been published of combined solid organ and alloBMT in patients with hematologic malignancies who had organ failure that would have been preclusive of alloBMT in the absence of solid organ transplantation. Here we will review the pre-clinical and clinical studies supporting the use of PTCy for chimerism-based tolerance induction.

Pharmacological blockade of the CD39/CD73 pathway but not adenosine receptors augments disease in a humanized mouse model of graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation is a curative therapy for a number of hematological malignancies, but is limited by the development of graft-versus-host disease (GVHD). CD39 and CD73 form an ectoenzymatic pathway that hydrolyzes extracellular adenosine 5'-triphosphate (ATP) to adenosine, which respectively exacerbate or alleviate disease in allogeneic mouse models of GVHD. The current study aimed to explore the role of the CD39/CD73 pathway and adenosine receptor (AR) blockade in a humanized mouse model of GVHD. Immunodeficient nonobese diabetic-severe combined immunodeficiency-IL-2 receptor γnull mice were injected with human peripheral blood mononuclear cells, and subsequently injected with the CD39/CD73 antagonist αß-methylene-ADP (APCP) (50 mg kg-1 ) or saline for 7 days, or the AR antagonist caffeine (10 mg kg-1 ) or saline for 14 days. Mice predominantly engrafted human CD4+ and CD8+ T cells, with smaller proportions of human regulatory T cells, invariant natural killer T cells, monocytes and dendritic cells. Neither APCP nor caffeine altered engraftment of these human leukocyte subsets. APCP (CD39/CD73 blockade) augmented GVHD as shown through increased weight loss and worsened liver histology, including increased leukocyte and human T-cell infiltration, and increased apoptosis. This treatment also increased serum human IL-2 concentrations and decreased the frequency of human CD39-  CD73-  CD4+ T cells. In contrast, caffeine (AR blockade) did not alter GVHD severity or human serum cytokine concentrations (IL-2, IL-6, IL-10 or tumor necrosis factor-α). In conclusion, blockade of CD39/CD73 but not ARs augments disease in a humanized mouse model of GVHD. These results indicate that CD39/CD73 blockade maintains sufficient extracellular ATP concentrations to promote GVHD in this model.

Addition of melphalan to fludarabine/busulfan (FLU/BU4/MEL) provides survival benefit for patients with myeloid malignancy following allogeneic bone-marrow transplantation/peripheral blood stem-cell transplantation.

A conditioning regimen with fludarabine and myeloablative dose of busulfan (FLU/BU4) has been commonly used in allogeneic hematopoietic cell transplantation (allo-HCT). However, there are two major problems with this regimen: insufficient anti-leukemic effect, especially in advanced cases, and slow time to complete donor-type chimerism, especially T-cell chimerism. To overcome these issues, we designed a combination regimen with FLU (150 mg/m2), intravenous BU (12.8 mg/kg), and melphalan (100 mg/m2) (FLU/BU4/MEL) and conducted retrospective analyses of treatment outcomes at our institute. Forty-two patients with myeloid malignancies received allogeneic bone-marrow transplantation or peripheral blood stem-cell transplantation (allo-BMT/PBSCT) with FLU/BU4/MEL regimen. The median age of patients was 46.5 years (20-63 years). Thirteen patients (31%) did not achieve complete hematological remission at transplantation. All patients examined achieved complete whole and T-cell chimerism within 1 month after allo-HCT. The 4-year overall survival and disease-free survival rates were 66.0% [95% confidence interval (CI) 49.4-78.3%] and 59.5% (95% CI 43.2-72.6%) in all patients, and 49.4% (95% CI 19.7-73.6%) and 38.5% (95% CI 14.1-62.8%) in patients who were not in remission. In conclusion, FLU/BU4/MEL showed curative potential, even in patients with advanced myeloid malignancies, accompanied by achievement of rapid complete chimerism after allo-BMT/PBSCT.

Evaluación De Quimerismo En Pacientes Con Injerto De Células Progenitoras Hematopoyéticas En Panamá Del 2000­20 / Evaluation of Chimerism in Patients with Graft Hematopoietic Progenitor Cells in Panama from 2000­2018

Introducción: El Injerto de células progenitoras hematopoy éticas (ICPH) es actualmente un trata­miento para diferentes desórdenes hematológicos malignos y no malignos. El análisis del quimeris­mo post ICPH, y la cuantificación de cada población celular, deben ser monitoreados. El presente trabajo tiene como objetivo: el evaluar los res ultados de quimerismo completo y mixto en sangre periférica del receptor pos trasplante obtenidos por método cualitativo y cuantitativo del año 2000 al 2018. Material y método: El presente es un estudio descriptivo, observacional, transversal de dos mé­ todos de quimerismo efectuados a receptores y donantes de ICPH alogénico. Resultados: De los 79 pacientes estudiados por el método cualitativo: 65 (82.2%) resultaron con qui­ merismo completo y 14 (17.7%) con quimerismo mixto. No fue posible cuantificar por este método el % de células del donante y del receptor.Conclusión: El método cuantitativo es un método exacto, que determina el % de células del receptor y del donante presentes en la muestra. Con este método se evalúan un mayor número de marcadores genéticos que con el método cualitativo, y se obtienen un mayor número de loci informativos del quimerismo al compararlo con el método cualitativo.

Introduction: Hematopoietic progenitor cell grafting (ICPH) is currently a treatment for different ma­ lignant and non­malignant hematological disorders. The analy sis of post­ICPH chimerism, and the quantification of each cell population, should be monitored. The present work has as objective: to evaluate the results of complete and mixed chimerism in peripheral blood of the post­trans plant reci­pient obtained by qualitative and quantitative method from the year 2000 to 2018. Material and method: The present is a descriptive, observational, cross­sectional study of two met­ hods of chimerism performed on allogeneic ICPH recipients and donors . Results: Of the 79 patients studied by the qualitative method: 65 (82.2%) resulted with complete chi­ merism and 14 (17.7%) with mixed chimerism. It was not possible to quantify by this method the% of donor and recipient cells. Conclusion: The quantitative method is an exact method, which determi­nes the% of recipient and donor cells present in the sample. With this method, a greater number of genetic markers are evaluated than in the qualitative method, and a greater number of information loci of chimerism are obtained than with the qualitative method.

Prognostic Limitations of Donor T Cell Chimerism after Myeloablative Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia and Myelodysplastic Syndromes.

Donor T cell chimerism is associated with relapse outcomes after allogeneic stem cell transplantation (alloSCT) for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). However, measures of statistical association do not adequately assess the performance of a prognostic biomarker, which is best characterized by its sensitivity and specificity for the chosen outcome. We analyzed donor T cell chimerism results at day 100 (D100chim) after myeloablative alloSCT for AML or MDS in 103 patients and determined its sensitivity and specificity for relapse-free survival at 6 months (RFS6) and 12 months (RFS12) post-alloSCT. The area under the receiver operating characteristic curve for RFS6 was .68, demonstrating only modest utility as a predictive biomarker, although this was greater than RFS12 at .62. Using a D100chim threshold of 65%, the specificity for RFS6 was 96.6%; however, sensitivity was poor at 26.7%. This equated to a negative predictive value of 88.5% and positive predictive value of 57.1%. Changing the threshold for D100chim to 75% or 85% modestly improved the sensitivity of D100chim for RFS6; however, this was at the expense of specificity. D100chim is specific but lacks sensitivity as a prognostic biomarker of early RFS after myeloablative alloSCT for AML or MDS. Caution is required when using D100chim to guide treatment decisions including immunologic manipulation, which may expose patients to unwarranted graft-versus-host disease.

Triptolide Reduces the Required Dose of Tacrolimus by Attenuating Inflammation, Enhancing Immunosuppression, and Increasing Donor Chimerism in a Heterotopic Hindlimb Transplantation Model.

BACKGROUND: Induction of tolerance and minimizing the toxicity of immunosuppression are two fundamental goals in vascularized composite allotransplantation. Accumulating data indicate that triptolide is an agent that may have the capacity to suppress inflammation and immunologic rejection. METHODS: A heterotopic hindlimb allotransplantation model from Brown Norway to Lewis rats was established and treated with different doses of tacrolimus combined with or without triptolide. Mean survival time of the transplants was monitored, and histopathologic examination of the skin was performed. The level of inflammatory cytokine interleukin-1ß, interleukin-6, and tumor necrosis factor-á in peripheral blood was assayed. The percentage of T lymphocytes and its subsets was measured using flow cytometry. The level of recipient peripheral chimerism and the apoptosis of donor bone marrow cells were evaluated. The apoptotic related genes bcl-2 and Bax were detected by real-time polymerase chain reaction. RESULTS: The authors' results showed that triptolide not only reduces the dose of tacrolimus required for immunosuppression, but also decreased drug side effects in terms of weight gain and diarrhea. Triptolide had an obvious effect on proinflammatory cytokine expression and T-lymphocyte proliferation in the peripheral blood. Interestingly, triptolide could increase the mixed chimerism level of recipients, possibly by inhibiting the apoptosis of transplanted bone marrow cells by means of regulation of the apoptotic genes bcl-2 and Bax. CONCLUSIONS: Triptolide reduces the dose of tacrolimus required for immunosuppression by attenuating inflammation and by T-cell suppression. Furthermore, triptolide increases the chimerism level, which might contribute to acceptance of the allografts.

The Macrophage-depleting Agent Clodronate Promotes Durable Hematopoietic Chimerism and Donor-specific Skin Allograft Tolerance in Mice.

Hematopoietic chimerism is known to promote donor-specific organ allograft tolerance; however, clinical translation has been impeded by the requirement for toxic immunosuppression and large doses of donor bone marrow (BM) cells. Here, we investigated in mice whether durable chimerism might be enhanced by pre-treatment of the recipient with liposomal clodronate, a macrophage depleting agent, with the goal of vacating BM niches for preferential reoccupation by donor hematopoietic stem cells (HSC). We found that liposomal clodronate pretreatment of C57BL/6 mice permitted establishment of durable hematopoietic chimerism when the mice were given a low dose of donor BM cells and transient immunosuppression. Moreover, clodronate pre-treatment increased durable donor-specific BALB/c skin allograft tolerance. These results provide proof-of-principle that clodronate is effective at sparing the number of donor BM cells required to achieve durable hematopoietic chimerism and donor-specific skin allograft tolerance and justify further development of a tolerance protocol based on this principle.

Kidney Versus Islet Allograft Survival After Induction of Mixed Chimerism With Combined Donor Bone Marrow Transplantation.

We have previously reported successful induction of transient mixed chimerism and long-term acceptance of renal allografts in MHC mismatched nonhuman primates. In this study, we attempted to extend this tolerance induction approach to islet allografts. A total of eight recipients underwent MHC mismatched combined islet and bone marrow (BM) transplantation after induction of diabetes by streptozotocin. Three recipients were treated after a nonmyeloablative conditioning regimen that included low-dose total body and thymic irradiation, horse Atgam (ATG), six doses of anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine (CyA) (Islet A). In Islet B, anti-CD8 mAb was administered in place of CyA. In Islet C, two recipients were treated with Islet B, but without ATG. The results were compared with previously reported results of eight cynomolgus monkeys that received combined kidney and BM transplantation (Kidney A) following the same conditioning regimen used in Islet A. The majority of kidney/BM recipients achieved long-term renal allograft survival after induction of transient chimerism. However, prolonged islet survival was not achieved in similarly conditioned islet/BM recipients (Islet A), despite induction of comparable levels of chimerism. In order to rule out islet allograft loss due to CyA toxicity, three recipients were treated with anti-CD8 mAb in place of CyA. Although these recipients developed significantly superior mixed chimerism and more prolonged islet allograft survival (61, 103, and 113 days), islet function was lost soon after the disappearance of chimerism. In Islet C recipients, neither prolonged chimerism nor islet survival was observed (30 and 40 days). Significant improvement of mixed chimerism induction and islet allograft survival were achieved with a CyA-free regimen that included anti-CD8 mAb. However, unlike the kidney allograft, islet allograft tolerance was not induced with transient chimerism. Induction of more durable mixed chimerism may be necessary for induction of islet allograft tolerance.

Haploidentical Hematopoietic Cell Transplantation with Post-Transplant Cyclophosphamide in a Patient with Chronic Granulomatous Disease and Active Infection: A First Report.

PURPOSE: We describe haploidentical hematopoietic cell transplantation (HCT) with high-dose post-transplant cyclophosphamide (PTCy) in a boy with x-linked chronic granulomatous disease (CGD). METHODS: A persistent and life-threatening fungal infection was the indication for HSCT. Non-myeloablative conditioning with PTCy (50 mg/kg days 3 and 4) was used in the absence of fully matched donors. RESULTS: Engraftment occurred on day 24. The patient experienced Grade 2 graft-versus-host disease of the skin and gastrointestinal tract and CMV infection, both of which were controlled. Chimerism was 100 % at days 30 and 6 months. Cessation of antifungal therapy was consistent with cure of the infection. CONCLUSIONS: Haploidentical HCT with high-dose PTCy for CGD is feasible and succeeded even in the context of active infection.

Increasing chimerism after allogeneic stem cell transplantation is associated with longer survival time.

Donor chimerism after allogeneic stem cell transplantation (allo-SCT) is commonly used to predict overall survival (OS) and disease-free survival (DFS). Because chimerism is observed at 1 or more times after allo-SCT and not at baseline, if chimerism is in fact associated with OS or DFS, then the occurrence of either disease progression or death informatively censors (terminates) the observed chimerism process. This violates the assumptions underlying standard statistical regression methods for survival analysis, which may lead to biased conclusions. To assess the association between the longitudinal post-allo-SCT donor chimerism process and OS or DFS, we analyzed data from 195 patients with acute myelogenous leukemia (n = 157) or myelodysplastic syndrome (n = 38) who achieved complete remission after allo-SCT following a reduced-toxicity conditioning regimen of fludarabine/intravenous busulfan. Median follow-up was 31 months (range, 1.1 to 105 months). Fitted joint longitudinal-survival time models showed that a binary indicator of complete (100%) donor chimerism and increasing percent of donor T cells were significantly associated with longer OS, whereas decreasing percent of donor T cells was highly significantly associated with shorter OS. Our analyses illustrate the usefulness of modeling repeated post-allo-SCT chimerism measurements as individual longitudinal processes jointly with OS and DFS to estimate their relationships.

Double negative Treg cells promote nonmyeloablative bone marrow chimerism by inducing T-cell clonal deletion and suppressing NK cell function.

The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft-versus-host-disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation-free condition. Our data indicate that adoptive transfer of donor-derived T-cell receptor (TCR) αß(+) CD3(+) CD4(-) CD8(-) NK1.1(-) (double negative, DN) Treg cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable-mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third-party C3H (H-2k) skin grafts. Adoptive transfer of CD4(+) and CD8(+) T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T-cell alloreactive responsiveness was reduced in the DN Treg cell-treated group and clonal deletions of TCRVß2, 7, 8.1/2, and 8.3 were observed in both CD4(+) and CD8(+) T cells. Furthermore, DN Treg-cell treatment suppressed NK cell-mediated BM rejection in a perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable-mixed chimerism and donor-specific tolerance in the irradiation-free regimen.

Nonobese diabetic natural killer cells: a barrier to allogeneic chimerism that can be reduced by rapamycin.

BACKGROUND: Induction of allogeneic hematopoietic chimerism is a promising strategy to induce tolerance to donor islets for treating type 1 diabetes. Successful induction of chimerism requires overcoming host alloimmunity. In diabetes-prone nonobese diabetic (NOD) mice, this is challenging due to their general tolerance resistance. Although the adaptive alloimmunity of NOD mice is a known barrier to allogeneic chimerism, whether NOD natural killer (NK) cells are an additional barrier has not been examined. Because NOD NK cells exhibit functional defects, they may not inhibit chimerism generation. METHODS: Antibody depletion of NK cells in vivo, or transplantation of F1 hybrid donor cells to eliminate the "missing-self" trigger of NK cells, was preformed to test the NK-mediated rejection of donor bone marrow cells. We also studied the capacity of rapamycin to block the NK cell response against allogeneic cells in vivo. RESULTS: Depleting NK cells or rendering them unresponsive to the donor greatly improved the level of chimerism obtained in NOD mice. Rapamycin significantly reduced the resistance to allogeneic chimerism mounted by NOD NK cells; however, it was much less effective than NK cell depletion by antibodies. CONCLUSIONS: Contrary to the view that NOD NK cells are defective, we found these cells to be a substantial barrier to allogeneic chimerism in the presence or absence of adaptive immunity. Moreover, rapamycin will need to be combined with other approaches to fully overcome the NK cell barrier.

Immunomodulatory effects induced by cytotoxic T lymphocyte antigen 4 immunoglobulin with donor peripheral blood mononuclear cell infusion in canine major histocompatibility complex-haplo-identical non-myeloablative hematopoietic cell transplantation.

BACKGROUND AIMS. Previously, cytotoxic T lymphocyte antigen 4 (CTLA4) immunoglobulin (Ig) has been shown to allow sustained engraftment in dog leukocyte antigen (DLA)-identical hematopoietic cell transplant (HCT) after non-myeloablative conditioning with 100 cGy total body irradiation (TBI). In the current study, we investigated the efficacy of pre-transplant CTLA4-Ig in promoting engraftment across a DLA-mismatched barrier after non-myeloablative conditioning. METHODS. Eight dogs were treated with CTLA4-Ig and donor peripheral blood mononuclear cells (PBMC) prior to receiving 200 cGy TBI followed by transplantation of granulocyte-colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells from DLA haplo-identical littermates with post-grafting immunosuppression. A control group of six dogs was conditioned with 200 cGy only and transplanted with grafts from DLA haplo-identical littermates followed by post-grafting immunosuppression. RESULTS. In vitro and in vivo donor-specific hyporesponsiveness was demonstrated on day 0 before TBI in eight dogs that received CTLA4-Ig combined with donor PBMC infusions. Four of five dogs treated with increased doses of CTLA4-Ig achieved initial engraftment but eventually rejected, with a duration of mixed chimerism ranging from 12 to 22 weeks. CTLA4-Ig did not show any effect on host natural killer (NK) cell function in vitro or in vivo. No graft-versus-host disease (GvHD) was observed in dogs receiving CTLA4-Ig treatment. CONCLUSIONS. Non-myeloablative conditioning with 200 cGy TBI and CTLA4-Ig combined with donor PBMC infusion was able to overcome the T-cell barrier to achieve initial engraftment without GvHD in dogs receiving DLA haplo-identical grafts. However, rejection eventually occurred; we hypothesize because of the inability of CTLA4-Ig to abate natural killer cell function.

A prospective dose-finding trial using a modified continual reassessment method for optimization of fludarabine plus melphalan conditioning for marrow transplantation from unrelated donors in patients with hematopoietic malignancies.

BACKGROUND: Because of the less graft-facilitating effect by bone marrow (BM), we need to assess a dosage of conditioning more accurately particularly in combination with reduced-intensity conditioning. Thus we examined that modified continual reassessment method (mCRM) is applicable for deciding appropriate conditioning of allogeneic BM transplantation. PATIENTS AND METHODS: The conditioning regimen consisted of i.v. fludarabine (125 mg/m2) plus an examination dose of i.v. melphalan. The primary endpoint was a donor-type T-cell chimerism at day 28 with successful engraftment defined as >90% donor cells. Five patients per dose level were planned to be accrued and chimerism data were used to determine the next dose. RESULTS: Seventeen patients were enrolled at doses between 130 and 160 mg/m2. The dose was changed from 160 to 130 mg/m(2) (second level) after five full-donor chimerisms. With one patient of 0% chimera in the second level, the dose was increased to 135 mg/m2 (third level). Following five full-donor chimerisms in the third level, the study was complete as projected. CONCLUSIONS: mCRM was shown to be a relevant method for dose-finding of conditioning regimen. The melphalan dose of 135 mg/m2 was determined as the recommended phase II dose to induce initial full-donor chimerism.

GSK-3ß inhibition promotes engraftment of ex vivo-expanded hematopoietic stem cells and modulates gene expression.

Glycogen synthase kinase-3ß (GSK-3ß) has been identified as an important regulator of stem cell function acting through activation of the wingless (Wnt) pathway. Here, we report that treatment with an inhibitor of GSK-3ß, 6-bromoindirubin 3'-oxime (BIO) delayed cell cycle progression by increasing cell cycle time. BIO treatment resulted in the accumulation of late dividing cells enriched with primitive progenitor cells retaining the ability for sustained proliferation. In vivo analysis using a Non-obese diabetic/severe combined immunodeficient (NOD/SCID) transplantation model has demonstrated that pretreatment with BIO promotes engraftment of ex vivo-expanded hematopoietic stem cells. BIO enhanced the engraftment of myeloid, lymphoid and primitive stem cell compartments. Limiting dilution analysis of SCID repopulating cells (SRC) revealed that BIO treatment increased human chimerism without increasing SRC frequency. Clonogenic analysis of human cells derived from the bone marrow of transplant recipient mice demonstrated that a higher level of human chimerism and cellularity was related to increased regeneration per SRC unit. Gene expression analysis showed that treatment with BIO did not modulate the expression of canonical Wnt target genes upregulated during cytokine-induced cell proliferation. BIO increased the expression of several genes regulating Notch and Tie2 signaling downregulated during ex vivo expansion, suggesting a role in improving stem cell engraftment. In addition, treatment with BIO upregulated CDK inhibitor p57 and downregulated cyclin D1, providing a possible mechanism for the delay seen in cell cycle progression. We conclude that transient, pharmacologic inhibition of GSK-3ß provides a novel approach to improve engraftment of expanded HSC after stem cell transplantation.

Ultraviolet radiation and handling medium osmolarity affect chimaerism success in zebrafish.

The effects of a predefined ultraviolet radiation dose (0.529 mW/cm2 for 30s) together with two different micromanipulation medium osmolarities (30 mOsm/kg vs 300 mOsm/kg) were tested on embryo survival at different developmental stages and on the somatic (skin) and germ-line chimaerism rates. Somatic (13%, 6/47 adults) and germ-line chimaerism (50% pigmented F1 larvae) were detected only in the UV-treated recipient embryos micromanipulated in a 300 mOsm/kg medium. From the results obtained, we concluded that the conditions cited above were the most suitable to improve somatic and germ-line chimaerism rates in zebrafish.

Safety and efficacy of targeted busulfan therapy in children undergoing myeloablative matched sibling donor BMT for sickle cell disease.

Busulfan influences engraftment and toxicities during hematopoietic stem cell transplantation (HSCT). We report our single-institution experience of targeted busulfan therapy for myeloablative, matched sibling donor (MSD) HSCT for sickle cell disease (SCD) and assess the relationships of busulfan levels to engraftment and toxicities. Twenty-seven patients with SCD underwent MSD HSCT from 1993 to 2007, 25 with busulfan measurements. The conditioning regimen was busulfan (initial dose 0.875 mg/kg for 16 doses), CY and antithymocyte globulin. Busulfan area under curve (AUC) was determined with the first dose, and dose adjustments were made to target the desired AUC range. Median AUC was 963 µmol min/L (range 780-1305 µmol min/L). Engraftment occurred in all cases: 21 (84%) full donor chimerism (> 95% donor cells), 4 (16%) partial donor chimerism. Hepatic veno-occlusive disease (VOD) occurred in 8 (32%) patients. Lower AUC was seen with partial donor chimerism (862 ± 73 µmol min/L) versus full donor chimerism (AUC 1018 ± 122 µmol min/L) (P = 0.022). VOD was not associated with busulfan AUC (P = 0.153). Of 25 patients, 24 survived with median follow-up of 4.9 years. Our experience shows that targeting busulfan AUC above the range used in previous multicenter trials appears safe and may contribute to sustained engraftment in SCD.

Myeloablative irradiation and granulocyte colony-stimulating factor prolong the survival of chimeric limb allografts in a rodent model.

BACKGROUND: Limb allografts consist of different types of tissues with varying antigenicities. One of the key components is the bone marrow. Removal and reconstitution of this tissue may allow better control of limb allograft rejection. The authors evaluated the efficacy of a new protocol for bone marrow removal that uses pretransplant graft irradiation, granulocyte colony-stimulating factor, and temporal FK506, with particular reference to the dose requirement and toxicity of irradiation. METHODS: In total, 57 whole-limb allotransplants from LacZ transgenic rats to Lewis rats were performed. Graft irradiation with various doses ranging from 250 to 1500 R was administered just before transplantation. Granulocyte colony-stimulating factor was given for 4 days, whereas FK506 was used for 28 days. The level of intra-bone marrow chimerism of the graft was evaluated by semiquantitative polymerase chain reaction. RESULTS: Rejection-free survival of grafts was increased significantly in groups that received more than 1000 R of pretransplant irradiation and granulocyte colony-stimulating factor, whereas grafts that received less than 500 R of irradiation showed no prolongation in survival. Bone marrow in the myeloablative irradiation groups was quickly reconstituted by recipient-derived cells. In the group treated with 1500 R of irradiation, the graft tissue was damaged and this led to infection of the recipient. In the 1000-R group, two of eight recipients showed rejection-free graft survival of more than 1 year without any immunosuppression. CONCLUSIONS: The authors study found that removal of allogeneic bone marrow in the limb graft and rapid reconstitution with recipient marrow cells reduced the recipient's immune response. Graft rejection was delayed significantly but not completely prevented.