Dose Of Bone Marrow Cells Research Articles

Ixazomib Impairs Dendritic Cell Function and T Cell Proliferation and Affects the Development of GvHD in a Schedule-Dependent Fashion

Background: Targeting T cells alone has yielded limited success in the prevention of graft-versus-host disease (GvHD) following allogeneic blood and marrow transplantation (BMT). Dendritic cells (DCs) play a central role in alloreactivity and therefore represent a suitable target. Proteasome inhibitors (PI), with their ability to inhibit the function and maturation of DC, have prompted investigators to examine their potential role in the prevention of GvHD. The investigational PI, ixazomib (IXZ), dissociates rapidly from 20S and is therefore truly reversible. It is also orally bioavailable. Our aim in this study was to explore its effect on healthy volunteer peripheral blood dendritic and T cells and in a pre-clinical GvHD mouse model.Methods: To characterize the effects of IXZ on healthy volunteer peripheral blood DCs, DCs were isolated using EasySep Pan-DC Pre-Enrichment Cocktail with purity over 90% (STEMCELL Technologies). DCs were then treated with IXZ at different concentrations (10-40nM) for 4 hrs and then stimulated with lipopolysaccharide (LPS) for 16 hrs. After this treatment, DCs were surface stained with antibodies against maturation markers and analyzed by flow cytometry. DC survival was evaluated with 7AAD staining and FACS analysis. To assess the effect of IXZ on the production of pro-inflammatory cytokines, DCs were incubated with IXZ at increasing concentration before or after the addition of LPS. Total pro-inflammatory cytokines in the supernatant of tissue culture were measured using EMD Millipore cytokine arrays. Standard mixed lymphocyte reaction and T cell proliferation assays were used to evaluate T cell function. At a minimum, all experiments were done in triplicate. Unpaired t test was used for statistical analysis. A p-value < 0.05 was considered significant. The B6 → BALB/c pre-clinical GvHD model was adopted to evaluate the effect of IXZ on GvHD development. Mice were transplanted in 3 groups. Group 1 received a lethal dose of total body irradiation (TBI), donor bone marrow (BM) cells, and IXZ, group 2 received TBI, donor BM cells donor splenocytes, and a vehicle, and group 3 received TBI, donor BM cells, donor splenocytes, and IXZ. The dose of BM cells and splenocytes was 5 X 106 each. IXZ was given at 1.5 mg/kg subcutaneously. Two dosing schedules were tested in 2 separate experiments: day-1 and +2 or day +1 and +4.Results: IXZ inhibited the expression of 6 DC maturation markers including CD40, CD54, CD80, CD83, CD86 and CD197 (CCR-7). The inhibition started at a concentration of 10nM and was dose-related. IXZ also decreased the percentage of total DCs simultaneously expressing multiple markers. DCs viability remained unchanged in comparison to control at a concentration of 10nM and dropped to 68% and 43%, on average with concentrations of 20nM and 40nM, respectively. IXZ significantly decreased DC production of IL-6, IL-12, and IL-23 starting at the concentration of 20nM. IL-1β was decreased at the concentration of 40 nM. Importantly, there was no significant change in the cytokine production by DCs when IXZ was added 4 hrs after LPS except for IL-1β which increased at 30nM. Starting at the concentration of 10nM, IXZ dose-dependently inhibited T cell proliferation. At 40nM IXZ abolished T cells. In our in vivo study IXZ improved GvHD scores on days +7 and +11 in group 3 in comparison to group 2 when it was given on days -1 and +2. Conversely, when IXZ was given on day +1 and +4, group 3 mice had higher scores of GvHD and worse survival outcomes when compared to group 2. There was no noticeable drug toxicity in group 1 mice.Conclusion: In summary: 1) IXZ inhibits DC maturation with relative preservation of cell viability and inhibits pro-inflammatory cytokine production in DCs when added before LPS stimulation; 2) IXZ inhibits T-cell proliferation; 3) IXZ affects GvHD development in a schedule-dependent fashion with early administration improving and late administration worsening GvHD. Additional analysis of tissue and serum samples is in progress. These results provide background for careful design of clinical trials using IXZ for the prevention of GvHD. DisclosuresAl-Homsi:Millennium Pharmaceuticals: Research Funding.

Targeting apoptosis to induce stable mixed hematopoietic chimerism and long-term allograft survival without myelosuppressive conditioning in mice

AbstractInduction of mixed hematopoietic chimerism results in donor-specific immunological tolerance by apoptosis-mediated deletion of donor-reactive lymphocytes. A broad clinical application of this approach is currently hampered by limited predictability and toxicity of the available conditioning protocols. We developed a new therapeutic approach to induce mixed chimerism and tolerance by a direct pharmacological modulation of the intrinsic apoptosis pathway in peripheral T cells. The proapoptotic small-molecule Bcl-2 inhibitor ABT-737 promoted mixed chimerism induction and reversed the antitolerogenic effect of calcineurin inhibitors by boosting the critical role of the proapoptotic Bcl-2 factor Bim. A short conditioning protocol with ABT-737 in combination with costimulation blockade and low-dose cyclosporine A resulted in a complete deletion of peripheral donor-reactive lymphocytes and was sufficient to induce mixed chimerism and robust systemic tolerance across full major histocompatibility complex barriers, without myelosuppression and by using moderate doses of bone marrow cells. Thus, immunological tolerance can be achieved by direct modulation of the intrinsic apoptosis pathway in peripheral lymphocytes—a new approach to translate immunological tolerance into clinically applicable protocols.

Amnion-Derived Multipotent Progenitor Cells Support Allograft Tolerance Induction

Donor-specific immunological tolerance using high doses of bone marrow cells (BMCs) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease remains a risk. Here, we demonstrate that the co-infusion of limited numbers of donor unfractionated BMCs with human amnion-derived multipotent progenitor cells (AMPs) 7 days post–allograft transplantation facilitates macrochimerism induction and graft tolerance in a mouse skin transplantation model. AMPs + BMCs co-infusion with minimal conditioning led to stable, mixed, multilineage lymphoid and myeloid macrochimerism, deletion of donor-reactive T cells, expansion of CD4+CD25+Foxp3+ regulatory T cells (Tregs) and long-term allograft survival (>300 days). Based on these findings, we speculate that AMPs maybe a pro-tolerogenic cellular therapeutic that could have clinical efficacy for both solid organ and hematopoietic stem cell transplant applications.

Biologics and Donor Bone Marrow Cells for Targeted Immunomodulation in Vascularized Composite Allotransplantation: A Translational Trial in Swine

Introduction Bone marrow (BM) infusion following organ transplantation is a prerequisite for potential donor-antigen-specific tolerance induction. We developed a preclinical swine model to determine the optimal dose of BM cells to achieve microchimerism. Furthermore, induction therapy was optimized by augmenting the BM infusion with biologics in the form of costimulatory blockade: cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4-Ig). Materials and Methods Yucatan miniature swine ( n = 12) underwent total body and thymic irradiation for cytodepletion. Animal groups received 15, 30, or 60 million cells per kilogram of whole unmodified BM. The optimal dose of BM cell infusion (BMT) was then applied to subsequent experiments evaluating the addition of CTLA4lg. Group 1 (control) received no treatment. Group 2 received FK506 only; group 3 received irradiation, BMT, and FK506; group 4 received FK506 and CTLA4-lg. Results Microchimerism was established in all animals after BM cell infusion; at postoperative day 9, it was significantly increased for 60 million cells per kilogram ( P = .0001). Transplanted animals in group 1 rejected the allograft 5 to 8 days after transplantation. Group 2 rejected the allograft (skin and muscle) 30 to 32 days after transplantation (2 days after cessation of immunosuppression). Group 3 rejected the skin portion of the allograft at 50, 52, and 53 days posttransplant. Remaining allograft components (muscle, bone, nerve, vessel) survived indefinitely. Group 4 animals demonstrated significantly prolonged muscle survival beyond 150 days posttransplant; the skin component survived past 150 days in two of three animals. Skin and muscle histology in all long-term surviving animals were normal. Conclusions BM cell infusion with 60 million cells per kilogram results in stable levels of microchimerism. The addition of costimulatory blockade (CTLA4lg) prolonged allograft skin survival and overall graft survival. Such targeted immunomodulatory protocols might facilitate immune tolerance and eliminate the need for multidrug immunosuppression to maintain graft survival after vascularized composite allotransplantation.

Beneficial effects of autologous bone marrow cell infusion and antioxidants/L-arginine in patients with chronic critical limb ischemia

Short-term (within 6 weeks follow-up) clinical studies indicate that implantation of bone marrow cells (BMCs) into ischemic limbs may improve peripheral ischemia. Here, the long-term safety and feasibility of intraarterial autologous BMCs with oral treatment with antioxidants and L-arginine were investigated in patients with critical ischemia on account of advanced atherosclerotic peripheral arterial disease (PAD). Eighteen patients with PAD (advanced III/IV Fontaine stages) were enrolled in this study (NCT00306085). An additional group of 18 patients taking maximal drug therapy that refused BMC therapy served as control. The BMC-treated group received two doses of BMCs in the leg arteries (time 0 and 45 days). After 30 days from the first BMC dose, patients received daily antioxidants, and L-arginine. Therapeutic neoangiogenesis was estimated by angiography and laser Doppler\capillaroscopy. Ankle brachial index improvement (DeltaABI: >0.1) was seen in 10 patients at 3 months and in 12 patients at 12-18 months. Ischemic ulcers improved in 13 patients (after 6-12 months). Although two patients underwent amputation, the mean maximum walking distance significantly increased at 3 months and was sustained up to 18 months. Among conservative patients, 10 underwent amputation in comparison with two BMC-treated patients (55.6 vs. 13.3%; P=0.014). This small study shows that intraarterial autologous BMC and antioxidants and L-arginine therapy is safe and effective in patients with advanced atherosclerotic PAD with positive effects until 18 months.

Abstract 4534: Beneficial Effects of Autologous Bone Marrow Cell Infusion in Patients with Chronic Critical Limb Ischemia

Short-term (within 6 weeks follow up) clinical studies indicate that implantation of bone marrow cells (BMCs) into ischemic limbs may improve peripheral ischemia. Here, the long-term safety and feasibility of intrarterial autologous BMCs with oral treatment with antioxidants and L-arginine was investigated in patients with critical ischemia due to advanced atherosclerotic peripheral arterial disease (PAD). The investigation was conformed with the Declaration of Helsinki. We obtained written informed consent from all patients and the Medical Ethics Committee of II University of Naples School of Medicine approved the protocol (March 2005). Moreover, we notified this study to the Italian Ministery of Health and to the NIH-controlled trial register ( NCT00306085 ). 18 patients with PAD (advanced III/IV Fontaine stages) were enrolled in this study. An additional group of 18 patients taking maximal drug therapy that refused BMC therapy served as control. The BMC-treated group received two doses of BMCs in the leg arteries (time 0 and 45 days). After 30 days from the first BMC dose, patients received daily antioxidants, and L-arginine. Therapeutic neoangiogenesis was estimated by angiography and laser Doppler\capillaroscopy. Ankle brachial index improvement (ΔABI: &gt;0.1) was seen in 10 patients at 3 months and in 12 patients at 12–18 months. Ischemic ulcers improved in 13 patients (after 6 –12 months). Although 2 underwent amputation, the mean maximum walking distance significantly increased at 3 months and was sustained up to 18 months. Among conservative patients, 10 underwent amputation in comparison to 2 BMC-treated patients. Despite some small published studies employing BMC with a short follow up in PAD patients, we provide the first evidence of long-term safety and feasibility of intrarterial autologous BMC infusion and cotreatment with antioxidants and L-arginine in patients with advanced atherosclerotic PAD. Patients received both BMCs and metabolic treatment to maximize the effect of vitamin E and L-arginine. Results showed significant improvements in the ABI, ulcer healing, maximum walking distance, microcirculation blood flow, and reduction of amputation risk, without any serious adverse reactions until 18 months.

Pbx1 Regulates Self-Renewal of Long-Term Hematopoietic Stem Cells by Maintaining Their Quiescence

Self-renewal is a defining characteristic of stem cells; however, the molecular pathways underlying its regulation are poorly understood. Here, we demonstrate that conditional inactivation of the Pbx1 proto-oncogene in the hematopoietic compartment results in a progressive loss of long-term hematopoietic stem cells (LT-HSCs) that is associated with concomitant reduction in their quiescence, leading to a defect in the maintenance of self-renewal as assessed by serial transplantation. Transcriptional profiling revealed that multiple stem cell maintenance factors are perturbed in Pbx1-deficient LT-HSCs, which prematurely express a large subset of genes, including cell-cycle regulators, normally expressed in non-self-renewing multipotent progenitors. A significant proportion of Pbx1-dependent genes is associated with the TGF-beta pathway, which serves a major role in maintaining HSC quiescence. Prospectively isolated, Pbx1-deficient LT-HSCs display altered transcriptional responses to TGF-beta stimulation in vitro, suggesting a possible mechanism through which Pbx1 maintenance of stem cell quiescence may in part be achieved.

IL-2 Pathway Blocking in Combination with Anti-CD154 Synergistically Establishes Mixed Macrochimerism with Limited Dose of Bone Marrow Cells and Prolongs Skin Graft Survival in Mice

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5×107) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day -1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.

CD8 T cell of donor splenocyte mixed with bone marrow cells is more effective than CD4 T cell for induction of donor-specific tolerance in sublethally irradiated mice

Background We previously demonstrated that even a low dose of bone marrow cells (BMCs) established donor-specific tolerance if mixed with splenocytes (SPLCs). In this study, T-cell subsets CD4 (CD4SP) and CD8 (CD8SP) of donor SPLCs were investigated for their contribution to the enhancement of BMC engraftment leading to donor-specific tolerance in sublethally irradiated mice. Methods Sublethally irradiated C57BL/6 recipient mice were intravenously injected BMCs mixing with CD4SP or CD8SP harvested from BALB/c donor mice. The degree of chimerism in the peripheral blood lymphocytes (PBLs) and in the SPLCs was analyzed using FACS, mixed lymphocyte reaction, and skin graft transplantation 3 months after injection. Results Recipients injected with 3 × 10 6 donor BMCs admixed with 10 × 10 6 donor CD8SP established chimerism. However, recipients injected with the same dose of BMCs admixed with 5 × 10 6 CD4SP, 10 × 10 6 CD4SP, and 5 × 10 6 CD8SP did not established chimerism. CD8SP contained 44% of Ly6A/E (Stem Cell Antigen-1 (Sca-1))–positive cells based on FACS analysis, whereas only 6% of CD4SP were positive for Ly6A/E. MLR supernates of donor SPLCs chimeric mice using admixture with CD8SP dominated by Th2 cytokines. In contrast, mixting with MLR supernates from failed chimera showed dominant Th1 cytokines. Conclusions CD8SP seems to make a major contribution to enhance BMC engraftment and induce donor-specific tolerance. Ly6A/E (Sca-1)–positive cells need to be further investigated for their contribution to the establishment of chimerism.

Donor-derived hematopoietic cells in organ transplantation: a major step toward allograft tolerance?

Infusion of donor-derived cells can improve organ allograft survival in animal models. Under certain conditions, it can even induce tolerance (i.e., unlimited organ survival without any maintenance immunosuppressive therapy). Use of nonmyeloablative regimens allows engraftment of donor-derived bone marrow cells, induction of mixed chimerism, and tolerance in rodents. High doses of bone marrow cells together with anti-T-cell antibodies can even result in mixed chimerism without cytoablative host conditioning. Cultured donor-derived CD34+ cells or donor-derived immature (or even mature) dendritic cells associated with monoclonal antibodies directed against co-stimulatory molecules might also induce tolerance. Among the numerous experimental protocols leading to tolerance of solid organs in animal models, how can we find our bearings in human transplantation? Numerous problems have yet to be solved: the type and amount of donor-derived cells (including stromal cells) to be used, the timing for infusion of donor cells in keeping with organ transplantation, the route of infusion (should it be intravenous, into the portal vein?), and the conditioning regimen. The first clinical trials would appear to indicate that tolerance induction in humans using donor-derived cells is a relatively safe solution that is both promising and realistic.

Rapamycin and T cell costimulatory blockade as post-transplant treatment promote fully MHC-mismatched allogeneic bone marrow engraftment under irradiation-free conditioning therapy.

Hematopoietic macrochimerism, established by bone marrow transplantation, can be used as an approach for treating autoimmune disease and inducing transplant tolerance. In this study, we investigated whether a stable, high level of fully MHC-mismatched hematopoietic macrochimerism can be induced by using irradiation-free protocols, and whether rapamycin and T cell costimulatory blockades (anti-CD40L monoclonal antibody (mAb) and CTLA4Ig) as post-transplant treatment promote bone marrow engraftment. Donor-specific blood transfusion (DST), anti-lymphocyte serum (ALS), busulfan, and cyclophosphamide were given pretransplantation. Balb/c (H-2(d)) bone marrow cells, at a dose of 4 x 10(7), were infused into each C57BL/6 mouse (H-2(b)). Rapamycin, anti-CD40L mAb, and CTLA4Ig were then administered, either alone or in combination. Without ALS or busulfan and cyclophosphamide, macrochimerism can only rarely be induced. Donor-specific transfusion (DST) enhances induction of hematopoietic macrochimerism. Rapamycin, anti-CD40L mAb and CTLA4Ig, alone or in combination, induce a stable and high level of hematopoietic macrochimerism. In the chimeric mice, donor-derived cells were detected in all lymphohematopoietic tissues and donor-specific tolerance was induced in vitro. We conclude that a stable and high level of fully MHC-mismatched hematopoietic macrochimerism can be induced in mice after transplanting a single modest dose of bone marrow cells without irradiation. Rapamycin and T cell costimulatory blockade as post-transplant treatment promote bone marrow engraftment.

Efficacy of donor splenocytes mixed with bone marrow cells for induction of tolerance in sublethally irradiated mice

Background: High dose of bone marrow cells (BMCs) has been reported to be essential to establish donor-specific tolerance. In clinical settings, a large quantity of BMCs is very difficult to be obtained. Our previous report demonstrated that even a low dose of BMCs could establish donor-specific tolerance if mixed with splenocytes (SPLCs). In the present study, various components of SPLCs were purified or removed and were investigated their contribution for enhancement of bone marrow engraftment leading to donor-specific tolerance in sublethally irradiated mice. Methods: Sublethally irradiated C57BL/6 recipient mice were intravenously injected 3×10 6 BMCs mixed with various components and various numbers of SPLCs harvested from BALB/c donor mice. One week after injection, skin grafting was performed. The degree of chimerism in peripheral blood lymphocytes (PBLs) and in SPLCs was analyzed by FACS 3 months after transplantation. Results: Recipients receiving 3×10 6 BMCs mixed with 10×10 6 T cell-enriched SPLCs established chimerism. Recipients receiving BMCs mixed with macrophage-depleted SPLCs also showed chimeirism and donor-specific tolerance. B cell-enriched SPLCs did not help small dose of BMCs to establish chimerism. Irradiated SPLCs were not effective to induce tolerance even with additional infusion to recipients. Conclusions: Active effects of splenic T cells were more important to help engraftment of small dose of BMCs than B cells, but the interaction between T and B cells might play some roles to enhance BMC engraftment. Splenic macrophages or dendritic cells might have some adverse effects against tolerance induction. Fatal graft-versus-host disease (GVHD) might be avoided by depleting adherent cells from SPLCs, so macrophages or dendritic cells were also considered as key components to induce donor-specific tolerance and prevent GVHD in this model.

Immunogenicity of Ly5 (CD45)-antigens hampers long-term engraftment following minimal conditioning in a murine bone marrow transplantation model.

Various techniques are available for distinguishing donor from host cells evaluating the efficacy of conditioning regimen for experimental bone marrow transplantation (BMT). Techniques include the use of extracellular immunological markers, such as Ly5 (CD45), and intracellular biochemical markers, such as glucose-phosphate-isomerase (Gpi). Because Ly5 is an extracellular protein, the disparity between donor (Ly5.1) and host (Ly5.2) antigens may induce a weak immune response whereas with Gpi, no immune response is expected. This difference may be of particular concern in experimental transplantation approaches that use minimal conditioning such as low-dose total body irradiation (TBI). Such mild conditioning may not induce the immunosuppression required to overcome host rejection of Ly5 disparate cells. To compare the relative engraftment of Ly5.1 and Gpi-1(a) donor marrow, B6 (Gpi-1(b)/Ly5.2) mice were irradiated with low-level TBI (0-6 Gy) and transplanted with several bone marrow (BM) doses (2 x 10(6)-5 x 10(7) cells). At 8, 26, and 52 weeks post-BMT, the level of donor engraftment was measured using flow cytometry (Ly5) or Gpi-electrophoresis. Lower engraftment levels were found in mice transplanted with Ly5 congenic BM in groups given low-dose TBI (< or = 4 Gy) and/or low doses of BM cells (BMC) (2 x 10(6)). However, when higher TBI or BMC doses were used, similar engraftment levels were found, suggesting sufficient immune suppression to allow equal engraftment of both sources of BM. These data suggest that even a minor phenotypic disparity between donor and host, such as Ly5, may necessitate high-dose TBI to prevent rejection. The combination of low-dose TBI or other nonmyeloablative conditioning strategies with small numbers of BMC may lead to reduced engraftment when extracellular immunological markers such as Ly5 are used for transplantation studies. Therefore, small immunological differences must be considered when using the Ly5 marker for engraftment.

Characterization of a hematopoietic stem cell with engraftment advantage

Abstract In studying hematopoietic stem cell (HSC) function in vivo, we discovered that HSCs from CXB-12/HiaJ (CXB-12) mice have 14 times the total long term repopulating ability found in the best of 11 other CXB recombinant inbred (RI) lines. This was determined using the competitive repopulation assay where bone marrow cells (BMCs) from each RI line donor were mixed with gentically marked competitor BMCs from the (BALBx B6) F1 (F1) hybrid, the mice used to produce the RI lines, and the mixtures repopulated lethally irradiated F1 recipients. After six months, percentages of donor type erythrocytes and lymphoytes gave a measure of the long term repopulating abilities of the donor RI lines relative to the F1 competitor. When high doses of BMCs were used, CXB-12 cells repopulated 6–12 times better than the F1 competitor cells, while when a low dose of BMCs were used in competitive dilution, CXB-12 donors had 2.4 times the number of HSCs as the F1 competitor, and each CXB-12 HSC repopulated 1.4 times as well. The CXB-12 HSC engraftment advantage is associated with a 50% increase in cobblestone area forming cell frequency and an one fold increase in the number of side population cells revealed by Hoechst 33342 vital dye staining. There was no effect on day 12 spleen colony forming units. The CXB-12 HSC engraftment advantage appears to result from increased HSC proliferation and differentiation abilities which are caused either by a unique recombination of parental genes or by specific mutations in the CXB-12 genome.

Cytomegalovirus inhibits the engraftment of donor bone marrow cells by downregulation of hemopoietin gene expression in recipient stroma.

Cytomegalovirus (CMV) disease after bone marrow (BM) transplantation is often associated with BM graft failure. There are two possible reasons for such a correlation. First, a poor hematopoietic reconstitution of unrelated etiology could promote the progression of CMV infection by the lack of immune control. Alternatively, CMV infection could interfere with the engraftment of donor BM cells in recipient BM stroma. Evidence for a causative role of CMV in BM aplasia came from studies in long-term BM cultures and from the murine in vivo model of CMV-induced aplastic anemia. A deficiency in the expression of essential stromal hemopoietins, such as stem cell factor (SCF), has indicated a functional insufficiency of the stromal microenvironment. It remained open to question whether CMV mediates a negative regulation of hemopoietin gene expression (the downregulation model) or whether it causes the default of a positive regulator (the lack-of-induction model). Further, even though implicitly assumed, it has never been formally documented that CMV directly interferes with the engraftment of a BM cell transplant. We addressed these problems in a murine model of CMV infection after experimental male-into-female BM transplantation. The data indicate that the downregulation model applies. Quantitation of the male-sex-determining gene tdy demonstrated an impaired engraftment of donor BM cells in the BM stroma of the female recipients. This graft failure was reflected by a diminished population of SCF-receptor-expressing hematopoietic progenitor cells and correlated with a reduced level of stromal SCF gene expression. Interestingly, high doses of BM cells protected against stromal insufficiency by a mechanism unrelated to control of infection.

Leukemia inhibitory factor induces in vivo expansion of bone marrow progenitor cells that accelerate hematopoietic reconstitution but do not enhance radioprotection in lethally irradiated mice.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with distinct hematopoietic activities. In vivo treatment of mice with recombinant murine LIF induces thrombocytosis and increases the number of hematopoietic progenitor cells (HPCs) in spleen and bone marrow (BM). In this study, we applied LIF to expand HPCs in vivo prior to syngeneic BM transplantation. BALB/c donor mice were treated with recombinant human LIF at a dose of 2.5 microg/day s.c. for seven days. This resulted in a 1.6-fold increment in platelet counts from 941 to 1,470 x 10(9)/l (mean, n = 20). Mean spleen weight increased from 120 mg to 160 mg (n = 5). The total numbers of HPCs in the spleen as well as in the BM, as assessed in a CFU-GM (colony forming unit-granulocyte-macrophage) assay, were significantly higher in LIF-treated donors than in saline-treated controls (30.1 +/- 14.5 versus 7.4 +/- 5.3 x 10(3) per spleen; mean +/- SD, n = 22,p < 0.001 and 74.4 +/- 17.1 versus 55.3 +/- 16.1 x 10(3) per femur, p < 0.001). Recipient mice were lethally (8.5 Gy) irradiated and transplanted with 3 x 10(5) BM cells derived from LIF- or saline-treated donors. Hematopoietic reconstitution was monitored by tail bleeding at three-day intervals. Platelet and WBC nadir counts in control animals were reached at day 9 (31 +/- 25 x 10(9)/l for platelets and 0.40 +/- 0.10 x 10(9)/l for WBC; mean +/- SD, n = 29 per treatment group); in animals transplanted with LIF-treated BM cells, these counts were 44 +/- 25 x 10(9)/l for platelets, p < 0.05 and 0.60 +/- 0.38 x 10(9)/l for WBC, p < 0.01. In addition, platelet reconstitution was faster in recipients of LIF-treated BM cells (226 +/- 118 versus 126 +/- 62 x 10(9)/l at day 12 and 633 +/- 174 versus 434 +/- 180 x 10(9)/l at day 15, p < 0.001). Similarly, the reconstitution of WBC was also significantly enhanced. The radioprotection rate of lethally irradiated recipients with increasing cell doses of BM cells derived from LIF-treated donors was higher at all cell doses tested then of control animals, but did not reach statistical significance. These results show that in vivo treatment with LIF expands the number of committed progenitor cells and BM repopulating cells that accelerate short-term hematopoietic reconstitution without increasing radioprotection. Our data do not support a major role for LIF as a single factor inducing expansion of hematopoietic stem cells in vivo.

Multilineage hematopoietic reconstitution of supralethally irradiated rats by syngeneic whole organ transplantation. With oarticular reference to the liver.

Multilineage hematopoietic reconstitution of supralethally irradiated rats by syngeneic whole organ transplantation. With oarticular reference to the liver.

Intrathymic T cell differentiation in radiation bone marrow chimeras and its role in T cell emigration to the spleen. An immunohistochemical study.

Immunohistochemical studies were made on the regeneration of T cells of host- and donor-type in the thymus and spleen of radiation bone marrow chimeras by using B10- and B10.BR-Thy-1 congenic mice. Both the thymic cortex and the medulla were first repopulated with thymocytes of irradiated host origin, restoring the normal histologic appearance by days 11 to 14, regardless of the H-2 compatibility between the donor and the host. In Thy-1 congenic chimeras, thymocytes of donor bone marrow origin, less than 100 cells in one thymic lobe, were first recognized at day 7, when the thymus involuted to the smallest size after the irradiation. The thymocytes of donor-type then proliferated exponentially, showing a slightly faster rate when higher doses of bone marrow cells were used for reconstitution, reaching a level of 100 million by day 17 and completely replacing the cortical thymocytes of host origin by day 21. The replacement of cortical thymocytes started from the subcapsular layer in a sporadic manner. The replacement of medullary thymocytes from host- to donor-type occurred gradually between days 21 and 35, after the replacement in the cortex was completed. In the spleen, about 1 million survived cells were recovered at day 3 after the irradiation, and approximately 60% of them were shown to be host-type T cells that were observed in the white pulp areas. The host-type T cells in the spleen increased gradually after day 10, due to the influx of host-type T cells from the regenerating thymus. Thus a pronounced increase of T cells of host-type was immunohistochemically observed in the splenic white pulp between days 21 and 28, when thymocytes of host-type were present mainly in the thymic medulla. These host-type T cells were shown to persist in the spleen for a long time, as long as 420 days after the treatment. Phenotypically, they were predominantly Lyt-1+2+ when examined at day 28, but 5 mo later, they were about 50% Lyt-1+2+ and 50% Lyt-1+2-. Donor-type T cells in the spleen began to appear at about day 14 in chimeras that were transplanted with a larger dose of bone marrow cells, whereas this was slightly delayed in those grafted with a smaller dose of bone marrow cells, starting at about day 28.(ABSTRACT TRUNCATED AT 400 WORDS)

Abnormalities of myeloid progenitor cells after "successful" bone marrow transplantation.

We studied recovery of peripheral blood- and bone marrow-derived myeloid progenitor cells (CFU-G,M) in 29 patients who received bone marrow transplants 2 mo to 8.5 yr previously. All patients had normal levels of peripheral blood neutrophils, normal bone marrow cellularity, and a normal myeloid-erythroid ratio. Both peripheral blood- and bone marrow-derived CFU-G,M were markedly reduced compared with normal controls and bone marrow donors [5 +/- 1/10(6) vs. 37 +/- 4/10(6) (P less than 0.001) and 23 +/- 5/2 x 10(5) vs. 170 +/- 21/2 x 10(5) (P less than 0.001)]. Five patients had no detectable CFU-G,M even when 10(6) bone marrow cels were plated. These abnormalities of CFU-G,M were unrelated to age, sex, diagnosis, conditioning regimen, dose of bone marrow cells transplanted, and presence or absence of graft-vs.-host disease. Patients who received either autotransplants or transplants from identical twins also had decreased or absent CFU-G,M indicating that allogeneic factors and posttransplant immune suppressor with methotrexate or corticosteroids were not major determinants of this abnormality. Co-culture of normal or donor peripheral blood or bone marrow mononuclear cells with recipients peripheral blood or bone marrow mononuclear cells, purified T cells, or serum failed to show any evidence of active CFU-G,M suppression. Furthermore, the abnormality of CFU-G,M could not be corrected by the addition of normal syngeneic (donor) hematopoietic cells or serum. Depletion of T-cells from recipient bone marrow by physical techniques resulted in marked increase in CFU-G,M (36 +/- 13 vs. 138 +/- 36; P less than 0.05). The abnormality could be reproduced in vitro by readdition of autologous T cells. In contrast to results with T cell depletion by physical techniques, T cell depletion with a monoclonal anti-T antibody (B7) and complement had no effect. These data indicate that most-transplant recipients have a marked abnormality in CFU-G,M when these cells are cultured in vitro. In at least some of these patients, the decreased cloning efficiency of CFU-G,M appears to be mediated by a suppressive effect of autologous T cells.

Effect of insulin and its derivatives on incorporation of [1-14C]glycine into tissue proteins

Lethally irradiated DBA/l mice or (C57Bl X DBA/l1 F1 hybrid mice were injected with therapeutically effective doses of isologous bone marrow cells; simultaneously syngeneic lymph node cells from either intact (control) animals or mice survived after sublethal irradiation were transplanted. In control the viability of the recipients was not affected by the presence of lymphoid cells in the mixed transplant. In contrast, the beneficial action of the bone marrow cells was abolished (killing-effect) by the lymphoid cells from mice sacrificed 6 to 12 months after the irradiation (600--700 r). The manifestation of the killing-effect depended on the number of the transplanted lymphoid cells and on the dose of the bone marrow cells in the transplant. The killing-effect was not revealed when the lymphoid cells were obtained from the donors on the 30th day after irradiation. The results suggest the autosensitization of the organism at the late postirradiation periods.