Allogeneic Bone Marrow Research Articles

Endothelial Cell Markers Kinetics Following Umbilical Cord Blood Transplantation in Adults.

In organ transplant, alloreactivity is supposed to induce modifications on the vascular endothelium. Complications occurring in the course of allogeneic haematopoietic stem cell (HSC) transplantation, such as acute or chronic graft-versus-host disease (GVHD), have been related to endothelial cell alterations. Moreover, the incidence of GVHD is significantly different following allogeneic bone marrow, peripheral blood stem cell or umbilical cord blood transplantation (BMT, PBSCT and UCBT respectively). Considering these different incidences of acute and chronic GVHD and taking into account the organ transplantation-related vasculopathy, we aimed to further clarify the vascular impact of the three aforementioned sources of HSC. We studied 21 patients (pts), 13 male and 8 female. Mean age was 42 years old (21–64). The underlying diseases were: acute leukaemia (myeloblastic 15pts; lymphoblastic 3pts), multiple myeloma (1pt), aplastic anemia (1pt) and chronic idiopathic myelofibrosis (1pt). Seven pts underwent BMT after myeloablative conditioning regimen (cyclophosphamide and busulphan or 12Gy total body irradiation-TBI), 6 pts PBSCT following non-myeloablative preparative regimen (fludarabine and 2Gy TBI) and 8 pts UCBT after reduced intensity conditioning regimen (cyclophosphamide, fludarabine and 2Gy TBI). We assessed the kinetics of plasma levels of 4 endothelial markers: soluble thrombomodulin (sTM), vWF-Ag, FVIII and the vascular endothelial growth factor (VEGF). Measurements were performed before the conditioning regimen and on days 0, 15, 30, 60, 90. Student paired t-test was used for statistical comparison. Differences were significant if p<0.05. In BMT and PBSCT we observed a significant increase of sTM on days 30 and 60 respectively, in comparison to day 0 (141 ± 42ng/ml and 143 ±13ng/ml respectively, p<0.05). No significant elevation of sTM, compared to pre-conditioning values, was shown in UCBT during the studied period. sTM in UCBT peaked on day 90 (119 ±54ng/ml, p>0.05). Similarly, vWF-Ag was significantly increased on day 0 in BMT and PBSCT (217±66% and 214±93% respectively, p<0.05 compared to pre-conditioning values), whereas in UCBT vWF-Ag levels remained almost unchanged during the studied period. FVIII was significantly elevated on day 15 in PBSCT (200±6%, p<0.05 in comparison to baseline values), but we did not observe any significant increase of FVIII in BMT and UCBT. Finally, there was no significant modification of VEGF in the 3 types of transplantation. Although GVHD was not frequent in this series, the aforementioned results correlated to clinical events. This is one of the first studies investigating the kinetics of different endothelial cell markers following UCBT and comparing them to BMT and PBSCT. A significant difference was observed in the kinetics of endothelial cell markers, according to the source of HSC. Cord blood cells seem to spare vascular endothelium, as shown by the stability of sTM and vWF-Ag plasma levels in the course of the UCBT. The presence of an active phenomenon for induction of tolerance between HSC and vascular wall of inside the cord blood population should be suggested. The low impact of UCBT on the vascular endothelium could possibly be correlated to fewer transplant-related complications and less incidence of GVHD in this type of transplantation.

Efficacy and Safety of Rituximab in Post Transplant Lymphoproliferative Disorders (PTLD): Pooled Analysis and Review of Literature.

PTLD are heterogeneous monoclonal or polyclonal neoplasm usually of B-cell origin with a variable incidence in allogenic bone marrow and solid organ transplant recipients. Due to immunosuppresion in transplant patients, PTLD follows an aggressive course with enhanced treatment-associated toxicity, leading to a overall poor clinical outcome and mortality. Rituximab is a chimeric monoclonal anti-CD20 antibody with known activity in B-cell PTLD and minimal toxicity. A few anecdotal uncontrolled studies and case reports have evaluated the use of rituximab-based therapy for PTLD. In order to define the efficacy and safety of rituximab single agent in the treatment of patients with PTLD we conducted a review of the current available literature and performed a pooled-analysis. We conducted a MEDLINE literature search using Pub med, Ovid software for the key words rituximab, anti-CD20 antibody, PTLD, hematopoetic stem cell transplant (HSCT), solid organ transplant (SOT) and lymphoproliferative disorders on all available published literature analyzed to date (January 1998 to June 30, 2007). Studies involving children (<18 years), individual case reports with less than 5 patients, patients who were treated with chemo-immunotherapy combination were excluded and our search was limited to those PTLD patients treated with rituximab single agent after failure to respond to withdrawal of immunosuppressants. References of each article were also reviewed for additional literature. Studies were also analyzed for duration of PTLD from time of transplantation, rituximab toxicity, and characteristics of responders. A total of 164 peer-reviewed publications were identified after the electronic search using the above keywords. After excluding patients based on above criteria, the pooled-analysis consisted of 308 patients described in 17 reports. Diagnosis of PTLD cases was based on histological examination and World Health Organization classification. Among the 17 reports, 3 studies included HSCT patients with n=24; the rest consisted of SOT patients. The mean age of the patients was 57; PTLD classified as late onset (>1 year) in most patients (∼70%). Rituximab was used as first line therapy after failure of immunosuppression withdrawal, and patients were initially treated with four weekly doses of rituximab at 375mg/m2. Rituximab was continued as maintenance therapy in responding patients in few studies. The complete response rate (International Workshop Criteria) was 58% (29%–78%), among SOT patients and 70% (66%–83%) among HSCT patients. Side effects were minimal and no treatment related deaths were attributed to rituximab. Long term follow up from 2 prospective studies demonstrate a durable remission in 30%–37% SOT patients at 5 years. Limited evidence exists for optimal treatment approach in PTLD. Clinical heterogeneity among small number of patients diagnosed makes it difficult to conduct randomized prospective studies in this disorder. Our study is the first pooled analysis evaluating the role of rituximab in PTLD. Minimal toxicity with good efficacy favors rituximab's use among patients with less aggressive PTLD who fail or unable to tolerate withdrawal of immunosuppression.

Fetal Immune Responses Post In Utero Transplantation of Embryonic Stem Cells, Bone Marrow and Fetal Liver Hematopoietic Stem Cells.

Introduction: We have previously shown that in utero transplanted ES cells survive and integrate into the fetus development and have established a murine model for the study of the in vivo differentiation of ES cells. The goal of this study was to monitor the fetal immune responses post in utero transplantation of ES cells, bone marrow (BM) and fetal liver (FL) hematopoietic stem cells.Materials and Methods: Murine (MHC)-mismatched ES cells genetically engineered to express yellow fluorescent protein (YFP-ES cells) were cultured on mitomycin-treated feeder layers for four days prior to in utero transplantation (IUT) in medium containing leukemia inhibitory factor. A cell dose of 5x104 YFP-ES cells ((H-2kb) was injected intraperitoneally in the fetuses of Balb/c (H-2Kd) pregnant mice at E12- E14. BM and FL hematopoietic stem cells (H-2kb) were transplanted in utero at a dosage of 1×109 cells/kg fetal body weight into E12-E14 BALB/c fetuses (H- 2Kd). Fetal immune responses were monitored by in vitro mixed lymphocyte reaction and cytotoxicity assays performed with self (Balb/c), allogeneic ES cells, BM or FL hematopoietic cells, and 3rd party (C3H cells). Cytokine levels (IL-2, IFN-gamma, IL-4 and IL-10) were determined in the cell culture supernatants from cytotoxicity assays. Liver tissue sections were prepared from in utero transplanted fetuses and examined for the presence of lymphocytic infiltration.Results: In utero transplantation of YFP-ES-cells did not induce tolerance in the fetuses and was associated with increased cytokine production compared with BM and FL groups. Microscopic examination of liver sections of ES cell transplanted group revealed the presence of marked inflammatory infiltrate.Conclusions: Embryonic stem cells transplanted in utero induce fetal immune responses and increased cytokine production associated with (MHC) upregulation.

T Regulatory Cells Obtained from Cord Blood Are Greatly Expandable and Exert Potent Suppressive Function.

CD4+CD25+ T-regulatory (Treg) cells have been shown to regulate self and allograft tolerance and to suppress allogeneic immune response. Studies in mouse models of bone marrow (BM) transplantation have shown that the infusion of culture-expanded Tregs can be effective in preventing and suppressing graft-versus-host disease (GVHD), while still allowing a graft-versus-leukemia effect (GVL). Cord blood (CB) represents the most recent source of hematopietic stem cell transplantation (HSCT) used in the unrelated transplant setting. Comparative studies have shown that unrelated CB transplant is an acceptable alternative to unrelated BM transplantation, characterized by a lower risk of transplant-related mortality due to GVHD. CB has been reported to contain Tregs, but minimal data are available on these cells. Aim of this study was to compare the suppressive functions of Tregs obtained from CB units with those obtained from the peripheral blood (PB) of patients who have undergone an allogeneic BM transplantation and of normal donors. Treg cells were purified from mononuclear cells obtained from CB units or PB using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and expanded for 6 days in 96-well U-Bottom plates coated with anti-CD3 and anti-CD28 MoAbs plus IL-2. To assess the suppressive functions of Treg cells, expanded CD4+CD25+ Tregs from CB units or PB were seeded with naïve autologous effector CD4+CD25- T cells stimulated with allogeneic dendritic cells (DC) pulsed with apoptotic leukemic blasts, then incubated with [3H]-thymidine and counted in a beta-counter. Suppressor activity was measured as [3H]-thymidine incorporation in the presence or absence of Tregs. The immunophenotypic analysis of Tregs from CB units - in terms of expression of surface CD4, CD25, CD62L, cytoplasmic CTLA-4 and Foxp3 - before and after expansion, did not show any specific peculiarity. CB units (n = 6) contained a proportion of Tregs comparable to those contained in the PB of allotransplanted patients (n = 8) or in the PB of normal donors (n = 8) (mean % ± SD, CB 0.28% ± 0.41; PB patients 0.4% ± 0.4; PB donors CB 0.45% ± 0.29). On the contrary, Tregs from CB units and from the PB of allotransplanted patients showed a higher expansion capacity when compared to Tregs from the PB of normal donors (mean fold increase ± SD, CB units 11.5 ± 9.0; PB patients 9.6 ± 5.0; PB donors CB 5.2 ± 2.7). Preliminary data also show that Tregs expanded from CB units (n = 2) exert a higher suppressive function on the proliferative reaction of T cells stimulated by allogeneic DC if compared to Tregs expanded from the PB of allotransplanted patients (n = 2) (mean fold reduction ± SD, CB 5.25 ± 3.7; PB patients 3.8 ± 3.3). These results demonstrate that Treg cells contained in CB units present an expansion capacity superior to that of Tregs from PB of normal donors; moreover, these expanded cells seem to exert a potent suppressive function of the proliferative effect in mixed lymphocyte reaction culture assays. These data offer further insights in the understanding of the biology of CB transplantation and may indicate a possible clinical use of expanded Tregs for the prevention and management of GVHD in the setting of CB transplantation.

Comparative Results of Gene Therapy for Adenosine Deaminase Deficiency with or without PEG-ADA Withdrawal and Myelosuppressive Chemotherapy.

Adenosine deaminase (ADA) deficiency is a form of severe combined immunodeficiency (SCID) that has long been considered a good candidate for gene therapy (GTx). In 2001–2002, we treated 4 ADA-SCID patients in a clinical trial evaluating the efficacy of 2 different retroviral vectors while continuing enzyme replacement with pegylated bovine ADA (PEG-ADA). No chemotherapy was used. All patients have been monitored for 6 years. No treatment-related serious adverse events occurred. A mild transient elevation in absolute lymphocyte count (ALC) was seen in 2 patients early post-treatment, however, no durable immunologic changes were observed. Low levels (0.1–0.7%) of vector-marked peripheral blood mononuclear cells (PBMCs) persist in 2 patients treated at the age of 4–5 years. All patients remain on PEG-ADA, prophylactic antibiotics and intravenous immunoglobulins. In 2004, we revised the protocol in order to facilitate engraftment and selective advantage of gene-corrected cells by withdrawing PEG-ADA and giving busulfan (75 mg/m^2) before GTx. In November 2005, a first patient was treated who developed unexpected prolonged bone marrow (BM) aplasia. Cytogenetics revealed trisomy 8 aberrations that were found to be present on a BM specimen obtained pre-GTx (Blood 2007; 109:503). Our second patient enrolled in January 2007. He received 5x10^6 CD34+ cells/kg that showed 40–200 units (U) of ADA activity (normal range 58–128). Over 6 months, this patient showed a slow increase in ALC (up to 750/mcL) and lymphocyte function. PBMC ADA activity has been up to 50U. The deoxyadenosine metabolite (dAXP) level has decreased to <10%, levels typically seen after allogeneic BM transplant. A third patient was treated in May 2007 and received 2x10^6 CD34+ cells/kg with ADA activity of 7-87U. Over three months, lymphocyte function has increased over pretreatment level. PBMC ADA activity has been up to ∼24U and the dAXP levels have decreased to <10%. Both patients have been off PEG-ADA since two weeks pretransplant and had mild post-GTx courses, with no documented infection, and no need for blood product transfusions. AST and ALT rose to < 5 x the upper limit of normal (ULN) in the second patient, and to < 3xULN in the third patient, and then resolved, an effect attributed to PEG-ADA withdrawal. Without myeloid growth factors, times to neutrophil count >500/mcL were 36 and 45 days, respectively. In >200 combined days of observation, there has been only one temperature above 38 °C, which resolved with acetaminophen. These data are consistent with the positive results of GTx for ADA-SCID obtained in Milan and London and show that PEG-ADA withdrawal and reduced conditioning improve the outcome of GTx for this disease. Longer follow-up should allow us to study engraftment of cells containing vector-specific sequences and conclude if either vector contributes more to recovery of lymphoid immunity.

Transplantation of allogenic cells in the therapy of patients with dilated cardiomyopathy

We carried out a pilot study on intracoronary transplantation of prenatal allogenic skeletal myoblasts and multipotent bone marrow stromal cells to patients with dilated cardiomyopathy. Intracoronary transplantation of allogenic cells is a feasible and safe procedure: there were no life-threatening rhythm and conduction disturbances, symptoms of hemodynamic instability, and thromboembolic complications. Positive clinical effect of cell transplantation persisted for 6 months and consisted in decreased content of brain natriuretic peptide and improved tolerance to physical exercises. We observed no reliable dynamics of the major echocardiographic parameters and their correlations with patient's status.

A new murine model for bronchiolitis obliterans post-bone marrow transplant.

Bronchiolitis obliterans (BO) is a major problem in lung transplantation and is also part of the spectrum of late-onset pulmonary complications that can occur after hematopoietic stem cell transplant. Better mouse models are needed to study the onset of this disease so that therapeutic interventions can be developed. Our goal was to develop a BO mouse model. Recipients were lethally conditioned and given a rescue dose of T-cell-depleted, allogeneic bone marrow (BM) supplemented with a sublethal dose of allogeneic T cells. At 2 months post-BM transplant, the lungs had extensive perivascular and peribronchiolar inflammation consisting of CD4(+) T cells, CD8(+) T cells, B cells, macrophages, neutrophils, and fibroblasts. In contrast to the acute model, histology showed airway obstruction consistent with BO. Epithelial cells of airways in the early stages of occlusion exhibited changes in expression of cytokeratins. Although the lung had severe allogeneic BM transplant-mediated disease, there was only mild to moderate graft-versus-host disease in liver, colon, skin, and spleen. High wet/dry weight ratios and elevated hydroxyproline were seen, consistent with pulmonary edema and fibrosis. Mice with BO exhibited high airway resistance and low compliance. Increases in many inflammatory mediators in the lungs of mice that develop BO were seen early post-transplant and not later at the time of BO. This new mouse model will be useful for the study of BO associated with late post-hematopoietic stem cell transplant onset and chronic graft-versus-host disease, which also leads to poor outcome in the lung transplant setting.

Association of bone marrow natural killer cell dose with neutrophil recovery and chronic graft‐versus‐host disease after HLA identical sibling bone marrow transplants

Allogeneic bone marrow (BM) transplant (BMT) outcomes have been correlated with the infused nucleated, CD34(+), and T- cell dose. The potential impact of natural killer (NK) BM infused cell dose has however not been established. We analysed the outcomes of 78 patients receiving an HLA identical BMT. A higher NK cell dose was associated with the speed of neutrophil (P = 0.05) and platelet recovery (P = 0.04). Higher nucleated cells, CD34(+), CD3(+), CD3(+)/4(+), CD3(+)/8(+) and NK cell dose were associated with a lower incidence of chronic GvHD (cGvHD) in univariate analysis. In multivariate analysis, the risk of cGvHD was increased by a lower NK cell dose [hazard ratio (HR) = 2.3 (1.2-4.4) for cell dose <0.9 x 10(6)/kg; P = 0.01] and an older age [HR = 1.4 /10 years (1.1-1.8); P = 0.002]. In addition, a higher CD3(+)/4(+) and NK cell dose were associated with a decreased incidence of viral infections (P = 0.03 and P = 0.06 respectively). No specific cell subpopulation infused dose was associated with survival. In conclusion, a higher BM NK cell dose is associated with an increased speed of neutrophil recovery and a decreased incidence of cGvHD.

Sibling stem cell donor experiences at a single institution

Allogeneic bone marrow (BM) and cytokine mobilized peripheral blood stem cell (PBSC) transplantation can be curative for patients with malignant and nonmalignant hematologic diseases. Siblings are most often selected as a donor match; however, research on sibling donors is limited and has focused primarily on conventional BM donors. This exploratory study describes the experiences of PBSC sibling donors at a single institution. Through retrospective interviews, 14 sibling donors shared their perceived needs and concerns before and after their stem cell collection. Donors identified fears about the donation procedure, and expressed the need for more information about transplant outcome and complications. The inclusion of the sibling donors themselves, rather than the report of their parents or health-care providers and the qualitative nature of the structured design allowed sibling donors to describe their concerns and thoughts without being restrained by the beliefs of the participant's parents, researcher or sibling's medical team. Suggestions for visual educational tools, psychosocial interventions and future research are provided.

The tracking of allogenic grafted rat bone marrow stem cells in rat liver and their role on repairing injured liver

To study the location, immigration of allogenic grafted Feridex-labeled rat bone marrow stem cells (BMSCs) in chemically-induced acute injured livers and their role on repairing the injured liver function. The rat models of chemically-induced acute hepatic injury established with CCl4 Feridex-labeled BMSCs were injected into the injured livers. Magnetic resonance imaging (MRI) examination was conducted on rat livers, the levels of ALT, AST and Fe3+ in the serum and hepatic tissues were studied 6 h before and 6 h, 1 w and 5 w after injection. Cellular necrosis, congestion in the hepatic sinusoid and infiltration of inflammatory cells were seen in the model livers. Above 90 percent of the cells were Feridex-labeled BMSCs positive by prussian blue staining and iron particles were found in their endochylema through electron microscopy. MRI examination at the sequence of SE-T2WI showed remarkably low signal changes 6 h after injecting Feridex-labeled BMSCs and the site with signal changes gradually expanded 1 and 5 w after injection. Comparatively, the changes of low signal images at each time point in the injured livers were more obvious than those of the controls at all time points, respectively. Simultaneously, pathological injuries in the livers were ameliorated and the levels of ALT and AST in serums declined: these changes in the Feridex-labeled BMSCs group were more obvious than those in the non-Feridex-labeled BMSCs group. Uniformly, there were no significant differences between the Feridex-labeled BMSCs group and the non-Feridex-labeled BMSCs group in view of histopathological examination and serological examination (including ALT, AST, Fe3+ levels) at all time points. The liver function in the model of chemically-induced acutely-injured liver may be repaired by BMSCs implantation. Traced by MRI, BMSCs in the injured liver of rats disperse at a higher rate than in the normally fed ones.

The effects of Immunosuppressive agents on NK cell function (102.18)

Abstract Natural killer (NK) cells are potent effector cells in allogeneic bone marrow and xeno transplantation. NK cells infiltrate solid organ allografts and a role for NK cells in acute and chronic rejection has been suggested. Little is known about the effects of immunosuppressive agents on NK cells Primary NK cell lines were analyzed for proliferation in the absence or presence of increasing doses (0–100 ng/ml) of cyclosporine (CSA), FK506 (FK) or Rapamycin, (RAPA). NK cells demonstrated robust proliferation both in the absence and presence of CSA and FK. In contrast, RAPA significantly (p&amp;lt; 0.05) inhibited the proliferation of NK cells in a dose-dependent manner. The effects of immunosuppressive agents on NK cells were determined in a rat liver transplant model. Peripheral NK cell levels in graft recipients treated with CSA and FK remained stable whereas there was a marked decrease in the levels of NK cells in recipients treated with RAPA. The immunosuppressive agents did not alter IFNγ production by NK cells however NK cell–mediated cytotoxicity was significantly (p&amp;lt; 0.05) inhibited in the presence of RAPA (but not CSA or FK). These data indicate that CSA and FK have little effect on NK cell function. In contrast, NK cell proliferation and function are significantly decreased in the presence of RAPA. Understanding the differential effects of immunosuppressive drugs on NK cell function is important in clinical transplantation.

Severe Food Allergy as a Variant of IPEX Syndrome Caused by a Deletion in a Noncoding Region of the FOXP3 Gene

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX; OMIM 304930) syndrome is a congenital syndrome characterized by autoimmune enteropathy, endocrinopathy, dermatitis, and other autoimmune phenomena. In the present work, we aimed to uncover the molecular basis of a distinct form of IPEX syndrome presenting at the edge of autoimmunity and severe allergy. The FOXP3 gene was sequenced, FOXP3 messenger RNA (mRNA) was quantified by real-time polymerase chain reaction (PCR), and protein expression in peripheral blood lymphocytes was analyzed by flow cytometry after intracellular staining. In coculture experiments (CD4(+)CD25(-) and CD4(+)CD25(+) cells), the functions of regulatory T cells were analyzed. Expression of interferon gamma and interleukin 2 and 4 mRNA within the inflamed intestinal mucosa was quantified by real-time PCR. Here, we describe a distinct familial form of IPEX syndrome that combines autoimmune and allergic manifestations including severe enteropathy, food allergies, atopic dermatitis, hyper-IgE, and eosinophilia. We have identified a 1388-base pair deletion (g.del-6247_-4859) of the FOXP3 gene encompassing a portion of an upstream noncoding exon (exon -1) and the adjacent intron (intron -1). This deletion impairs mRNA splicing, resulting in accumulation of unspliced pre-mRNA and alternatively spliced mRNA. This causes low FOXP3 mRNA levels and markedly decreased protein expression in peripheral blood lymphocytes of affected patients. Numbers of CD4(+)CD25(+)FOXP3(+) regulatory T cells are extremely low, and the CD4(+)CD25(+) T cells that are present exhibit little regulatory function. A new mutation within an upstream noncoding region of FOXP3 results in a variant of IPEX syndrome associating autoimmune and severe immunoallergic symptoms.

Induced SHIP Deficiency Expands Myeloid Regulatory Cells and Abrogates Graft-versus-Host Disease

Graft-vs-host disease (GVHD) is the leading cause of treatment-related death in allogeneic bone marrow (BM) transplantation. Immunosuppressive strategies to control GVHD are only partially effective and often lead to life-threatening infections. We previously showed that engraftment of MHC-mismatched BM is enhanced and GVHD abrogated in recipients homozygous for a germline SHIP mutation. In this study, we report the development of a genetic model in which SHIP deficiency can be induced in adult mice. Using this model, we show that the induction of SHIP deficiency in adult mice leads to a rapid and significant expansion of myeloid suppressor cells in peripheral lymphoid tissues. Consistent with expansion of myeloid suppressor cells, splenocytes and lymph node cells from adult mice with induced SHIP deficiency are significantly compromised in their ability to prime allogeneic T cell responses. These results demonstrate that SHIP regulates homeostatic signals for these immunoregulatory cells in adult physiology. Consistent with these findings, induction of SHIP deficiency before receiving a T cell-replete BM graft abrogates acute GVHD. These findings indicate strategies that target SHIP could increase the efficacy and utility of allogeneic BM transplantation, and thereby provide a curative therapy for a wide spectrum of human diseases.

B-cell extrinsic CR1/CR2 promotes natural antibody production and tolerance induction of anti-αGAL–producing B-1 cells

AbstractB-1b cells produce IgM natural antibodies against α1-3Galβ1-4GlcNAc (αGal). These can be tolerized by nonmyeloablative induction of mixed chimerism using αGal-positive (αGal+) donor marrow. We assessed the role of CR1/2 in this model for induction of tolerance of B-1b cells. Mixed hematopoietic chimerism was induced in α1-3galactosyltransferase (GalT−/−) and GalT−/−Cr2−/− mice with αGal+ BALB/c marrow donors. Anti-αGal Ab and anti-αGal Ab–producing B cells became undetectable in GalT−/− chimeras, whereas they persisted in chimeric GalT−/−Cr2−/− mice. To determine whether CR1/2 expression on stromal cells and/or hematopoietic cells was critical for B-1–cell tolerance, we generated GalT−/− radiation chimeras in which CR1/CR2 was expressed on either stromal cells, hematopoietic cells, neither, or both. After induction of mixed chimerism from αGal+ allogeneic bone marrow (BM) donors, anti-αGal–producing B cells were rendered tolerant in reconstituted recipients expressing only stromal CR1/CR2. Our results suggest a possible role for follicular dendritic cells that pick up immune complexes via CR1/CR2 receptors in the tolerization of B-1b cells.

Detection of Bone Marrow-Derived Cells Expressing a Neural Phenotype in the Human Brain

Animal studies suggest that adult bone marrow cells have the potential to migrate into the brain and generate new neural cells. Because data on this physiologic repair mechanism in humans are lacking, we investigated bone marrow engraftment into the brain of bone marrow recipients after sex-mismatched transplantation. Brain sections of seven allogeneic female bone marrow recipients were examined. The Y-chromosome, which served as a natural marker of donor bone marrow-derived cells after male-to-female transplantation, was identified by in situ hybridization. The neural phenotype of Y-chromosome-positive cells was determined using neural nuclear protein (NeuN) immunohistochemistry. Y-chromosome-positive cells expressing NeuN were found within the first 3 months after transplantation in both the cerebrum and the cerebellum at a frequency of 0.003% to 0.013% of all neurons. These cells were observed only in patients with cerebral lymphocytic infiltration and graft-versus-host disease. Our data suggest that adult bone marrow cells are capable of generating cells that express the neural marker NeuN early after transplantation. Cells with this specific phenotype may contribute to tissue repair in brain regions remote from neurogenic zones.

128: High prevalence of bone loss in long term survivors following allogeneic bone marrow and peripheral blood stem cell transplantation

128: High prevalence of bone loss in long term survivors following allogeneic bone marrow and peripheral blood stem cell transplantation

Maribavir

Maribavir is a novel benzimidazole riboside compound that is currently in clinical development with ViroPharma for the prevention of cytomegalovirus (CMV) infections in transplant patients. Maribavir is thought to inhibit viral DNA assembly and inhibit egress of viral capsids from infected cell nuclei. This mechanism is different to other available CMV treatments that inhibit CMV DNA polymerase. Maribavir, originally developed by GlaxoSmithKline, was licensed to ViroPharma in August 2003. Glaxo was conducting phase I/II clinical studies in the EU and the US in 2001, but discontinued development because the company felt that there was no longer a significant clinical need for maribavir as advances in the treatment of HIV have lead to improved immune systems in patients, resulting in a reduction in the incidence of CMV and CMV retinitis. In August 2003, ViroPharma acquired exclusive worldwide rights (excluding Japan) to develop and commercialise maribavir for the prevention and treatment of CMV infections related to transplant and congenital transmission and in patients with HIV infection. ViroPharma paid GlaxoSmithKline a $US3.5 million upfront licensing fee and will pay additional milestones based on defined clinical development and regulatory events. The company will also pay royalties on product sales in the US and the rest of the world, excluding Japan.A phase III study with maribavir as a prophylactic agent in patients undergoing allogeneic stem cell transplant who are CMV seropositive has been initiated and another phase III trial in solid organ transplant patients is planned. The stem cell transplant trial will take approximately 18 months to complete enrolment and the solid organ transplant trial, which won't be as large as the stem cell transplant trial, will be completed within this time-frame also. ViroPharma hopes to file an NDA for maribavir in 2009. Maribavir was granted fast-track status by the US FDA in February 2006 for the prevention of CMV infection in allogeneic bone marrow and solid organ transplant patients. Maribavir has received orphan drug status in the US for the prevention of CMV viraemia and disease in at-risk populations. ViroPharma has conducted a dose-ranging phase II clinical study designed to evaluate the antiviral activity, safety and pharmacokinetic profile of maribavir for the prevention of CMV infection in patients who have undergone allogeneic stem cell transplantation. The randomised, double-blind, placebo-controlled, dose-ranging study was conducted at 13 transplant centres across the US. Patients (n = 111) were randomised 3 : 1 to receive maribavir in three ascending dose groups (100mg bid, 400mg qd, 400mg bid) or placebo for up to 12 weeks. Enrolment in the phase II trial was completed in November 2005. Preliminary results have been reported. In February 2004, ViroPharma announced the initiation of a clinical programme to develop maribavir for prevention of CMV infection in transplant patients. A phase I drug-drug interaction and safety study in healthy volunteers, designed to evaluate the potential for maribavir to affect the blood levels of various other drugs that are metabolised by the liver, was conducted in the US. Results of this study were reported in March 2005. A second phase I study was initiated in March 2004 to evaluate the pharmacokinetic profile of a single 400mg dose of maribavir in patients with varying levels of renal functional impairment, compared with subjects with normal renal function. Maribavir has been tested in several phase I studies by GlaxoSmithKline, in which the drug demonstrated antiviral activity, oral bioavailability and an acceptable safety and tolerability profile.The patents covering maribavir held by GlaxoSmithKline expire in 2015.

Promise and Challenges of Human Regulatory T Cells in the Clinic

Regulation of immune reactions is critical in ealth and disease. Nowhere is this clearer than folowing allogeneic hematopoietic cell transplantation HCT). Here the beneficial effects of graft-versusumor reactions are clearly demonstrated, as well as he detrimental morbidity and mortality of unconrolled graft-versus-host disease (GVHD). Control of mmune reactions is complex and involves many facors including blood flow, micro-environmental interctions, cytokines, chemokines and both pro-inflamatory and anti-inflammatory molecules. In addition t has become well recognized that specialized popuations of cells, termed regulatory T cells, are critically nvolved in controlling immune reactions. Although he field of regulatory T cell biology has been exlored for many years, the more recent identification f specific populations of cells capable of regulating mmune responses with defined phenotype has opened ur re-examination of the potential impact of these ellular populations on immune function. To date, a umber of different cell populations have been charcterized with defined ability to regulate immune rections, which include two major cell populations, amely those of CD4 CD25 regulatory T cells Treg) [1] and abTCR NK-T cells which express an nvariant T cell receptor and are CD1 reactive [2]. hese cellular populations have generated enormous nterest in the field of HCT since animal models have emonstrated the potential benefit of regulatory T ell biology.

Donor CD4 + T-cell production of tumor necrosis factor alpha significantly contributes to the early proinflammatory events of graft-versus-host disease

Tumor necrosis factor alpha (TNFalpha) is an old foe in allogeneic bone marrow transplantation (allo-BMT) promoting acute graft-versus-host disease (aGVHD). We investigated to what extent donor T cells contribute to TNFalpha production. Lethally irradiated B6D2F1 mice were transplanted with bone marrow (BM) and T cells from syngeneic B6D2F1 or allogeneic B6 donors and assessed for cytokine production, aGVHD, and survival. Analysis of serum TNFalpha kinetics in recipients of allogeneic B6 wild-type BM and wild-type T cells revealed that TNFalpha levels peaked around day 7 after allo-BMT, whereas TNFalpha was undetectable in syngeneic controls. TNFalpha was produced by both host and donor cells. Further exploration showed that specifically donor CD4(+) but not CD8(+) T cells were the primary donor cell source of TNFalpha at this early time point; numbers of TNFalpha expressing splenic CD4(+) T cells were higher than CD8(+) T cells 7 days after allo-BMT, and maximal serum TNFalpha levels were detected following allo-BMT with only CD4(+) T cells compared to levels found in allogeneic recipients of only wild-type CD8(+) or to only CD4(+) TNFalpha(-/-) T cells. Concurrent with increased TNFalpha levels, early clinical aGVHD and mortality were more severe following allo-BMT with either wild-type CD4(+) and CD8(+) or CD4(+) T cells only. Our data demonstrate that in addition to residual host cells donor CD4(+) T cells significantly contribute to the proinflammatory cytokine milieu engendered early after allo-BMT through the production of TNFalpha. These findings support strategies focusing on TNFalpha neutralization as primary treatment for aGVHD.

FTY720 Inhibits Graft-Versus-Host Disease (GVHD) and Allogeneic Bone Marrow (BM) Graft Rejection Independent of Effects on T Cell Egress from Lymphoid Tissue.

FTY720 (FTY), a sphingosine-1-phosphate receptor agonist, inhibits lymphocyte egress from lymphoid tissues although the complete mechanism of its immunomodulatory effects is not well understood. We studied FTY in murine GVHD and engraftment models as a single agent and in combination with known beneficial strategies. FTY (3 mg/kg orally d0-28) inhibited GVHD, increasing the median survival time (MST) of lethally irradiated C57BL/6 (B6) mice given BALB/c BM and a moderate T cell dose from 30d to 71d (p<.001) or a higher T cell dose from 7d to 32d (p<.006). FTY was additive with ex vivo activated and expanded CD4+CD25+ regulatory T cells (Tregs) for GVHD inhibition. In contrast to control mice which all died of GVHD by d70 (MST = 37d), mice receiving FTY or Tregs or both had 100 day survival rates of 50%, 71% and 100%, respectively. The additive effects of FTY and Tregs were reproduced in a second strain combination. Although we hypothesized that FTY trapped Tregs in the lymph nodes allowing for increased contact time for Tregs to inhibit GVHD effector T cells, imaging studies of green fluorescent protein (GFP)+ Tregs did not support this hypothesis. Moreover, imaging studies of GFP+ effectors did not indicate that FTY inhibited donor T cell egress from lymphoid tissues. To study alloantigen-specific responses, B6 TCR Tg CD8+ and CD4+ T cells that are reactive against BALB/c alloantigen were adoptively transferred into B6 rag mice prior to sublethal irradiation and infusion with BALB/c BM. FTY reduced the expansion of Tg CD8+ and CD4+ T cells in the spleen 10d after BMT by 80% and 78%, respectively. In addition to GVHD inhibition, FTY also promoted donor BALB/c BM engraftment in sublethally (5.0 Gy) irradiated B6 mice although donor chimerism was not stable in all mice. In contrast to 4 of 26 water-treated controls, all 29 FTY-treated mice (3 mg/kg d0-13) were engrafted at 1 month as assessed by PBL phenotyping (ave of 9% vs 68% donor. p<.001). However, by 3 months, the engraftment rate in FTY-treated mice had decreased to 12 of 28 mice. All 9 mice receiving FTY for an additional 2 weeks (d0-28) had high donor chimerism levels (>96%) at 1 month but chimerism was lost in 3 mice by 6 months after BMT. In a different strain combination, even a 2 wk FTY course increased donor chimerism that remained stable for 6 months. Imaging studies revealed that FTY did not prevent the egress of adoptively transferred host-type GFP T cells in allogeneic BM recipients. FTY was also tested in combination with anti-CD40L mAb, a potent engraftment-promoting agent in mice. In contrast to anti-CD40L mAb, FTY did not promote engraftment in mice conditioned with very low levels of irradiation (0.5–2.0 Gy) although the combination of anti-CD40L mAb and FTY resulted in superior stable engraftment rates and levels compared to anti-CD40L mAb as a single agent. Interestingly, FTY increased donor chimerism in syngeneic, as well as, allogeneic recipients. These data indicate that FTY is efficacious as a single or adjunct agent for the prevention of GVHD and graft rejection and further indicate that inhibition of lymphocyte egress was not essential for the beneficial effects of FTY in BMT recipients.