Subsequent Bone Marrow Transplantation Research Articles

Acute Graft versus Host Disease in Beta Thalassemia Patients Following Allogeneic Haematopoietic Stem Cell Transplantation.

To analyse the frequency, risk factors, and clinical symptoms of acute graft-versus-host disease (aGvHD) in patients with beta-thalassemia major after allogeneic haematopoietic stem cell transplantation (HSCT). Descriptive study. Place and Duration of the Study: Department of Clinical Haematology, Armed Forces Bone Marrow Transplant Centre, Rawalpindi, Pakistan, from January 2017 to December 2021. Data were obtained from patients diagnosed with bone and tissue malignancies (BTM) who had undergone haematopoietic stem cell transplantation (HSCT) and experienced aGVHD. Patients who experienced initial graft failure and individuals who underwent subsequent bone marrow transplantation were excluded. Total of 117 patients diagnosed with BTM underwent fully matched HSCT, including 76 (65%) males, and 41 (35%) females. The median age of the patients undergoing transplantation was 7.34±7.32 years and the donors' median age was 7.6±9.85 years. Among the donors, 53 (45.3%) were males and 64 (54.7%) were females. Gender disparity was observed in 46 (39.3%) instances as a female donor matched with a male recipient. A total of 106 individuals underwent bone marrow harvest (BMH); with 5 (4.3%) patients receiving peripheral blood stem cells (PBSC) and 6 (5.2%) patients receiving both BMH and PBSC. Acute GvHD was observed in 50 (42.7%) patients, including 30 (60%) males and 20 (40%) females. Grade I GvHD occurred in 32 (27.3%) individuals, Grade II GvHD in 16 (13.7%) patients, and Grade III GvHD in one (0.8%) patient. It had no statistically significant association with recipient/donor age, gender disparity, the source of the graft source, the dose of stem cells, or the presence of thymoglobulin (TG). Acute GvHD was observed in high frequency in Beta-thalassemia patients receiving morrow harvesting proportional to their gender distribution. Associated factors were GvHD prophylaxis measure, mucositis and, CMV reactivation. Beta thalassemia major patients, Acute graft versus host disease, Allogeneic haematopoietic stem cell.

Effect of short-term interferon therapy on the outcome of subsequent HLA-identical sibling bone marrow transplantation for chronic myelogenous leukemia: an analysis from the International Bone Marrow Transplant Registry

Allogeneic bone marrow transplantation (BMT) is the only curative therapy for chronic myelogenous leukemia (CML), though several studies indicate that prolonged survival can result from interferon-alpha (IFN-alpha) treatment. IFN-alpha is now often used as initial therapy for CML, before donor availability is known. Because identifying potential donors can take several weeks to months, it is important to know whether IFN-alpha adversely affects outcome of a subsequent BMT. If it does, initiation of IFN-alpha therapy might be delayed until donor availability is determined and avoided in patients for whom BMT is planned. We studied 873 patients who received HLA-identical sibling BMT for chronic-phase CML in 153 centers participating in the International Bone Marrow Transplant Registry. The object was to compare outcome in the 664 who received only hydroxyurea before BMT with outcome in the 209 who received IFN-alpha with or without hydroxyurea. The median duration of IFN-alpha therapy was 2 months (range, 1 to 39 months). Cox proportional hazards analysis was used to compare engraftment, graft-versus-host disease (GVHD), nonrelapse mortality, relapse, survival, and leukemia-free survival after adjustment for other prognostic variables. We found a higher risk of nonengraftment among patients given IFN-alpha than among those given hydroxyurea alone (2% versus 0.2%; P = 0.01). Patients who received IFN-alpha had a lower risk of relapse (relative risk, 0.17; 95% confidence interval, 0.04-0.70). Probabilities of GVHD, nonrelapse mortality, survival, and leukemia-free survival were similar in the two treatment groups. These results suggest that a short course of IFN-alpha does not adversely affect survival after a subsequent HLA-identical sibling BMT for chronic-phase CML. (Blood. 2000;95:410-415)

Protein Arginine Methyltransferase 1 (PRMT1) Dampens Self-Renewal and Promotes Differentiation of Hematopoietic Stem Cells in Mouse Models

Protein arginine methylation is a common type of post-translational modification. PRMT1, the major type I protein arginine methyltransferase, catalyzes the formation of asymmetric dimethyl-arginine and is implicated in various cellular processes, including hematopoiesis and tumorigenesis. We have shown that PRMT1 expression is naturally low in hematopoietic stem cells (HSCs). However, the functions of PRMT1 in hematopoietic stem cell self-renewal and differentiation are yet to be revealed. We have found a cyanine-based fluorescent probe (E84) that can specifically label PRMT1 protein. E84 staining dynamically captures intracellular PRMT1 level and was used to separate live HSC populations with differential PRMT1 expression by flow cytometry. Subsequent bone marrow transplantation of E84high or E84low Lin−Sca1+cKit+ (LSK) cells showed that E84low LSK cells were much more advantageous in reconstituting each blood cell lineages, compared to the E84high counterparts, meaning that the stem-ness of HSCs is negatively correlated with endogenous PRMT1. Therefore, inhibition of PRMT1 was expected to enhance the number and differentiation potential of functional HSCs. The treatment of a PRMT1-specific inhibitor (MS023) to mice resulted in an enlarged LT-HSC population in bone marrow and decreased frequency of granulocyte progenitor cells. In vitro colony formation assays further demonstrated that PRMT1 is required for GMP differentiation. Then we asked whether copious expression of PRMT1 promotes the differentiation of HSC. In this line, we made a LoxP-STOP-LoxP-PRMT1 transgenic mouse model, which induces PRMT1 overexpression upon the expression of Cre recombinase from tissue-specific promoters. We established Mx1-Cre-PRMT1 (Mx1-Tg) mice. Mx1-Tg mice were injected with poly(I:C) for PRMT1 induction and analyzed at four weeks after the last dose. We found that, as predicted, LT-HSC population was reduced and the Pre-GM population was raised. Accordingly, more CFU-Gs but less GEMMs were grown on CFU assays. We further utilized this animal model to compare the blood reconstitution capabilities of bone marrow cells from Mx1-Tg vs. WT mice in the same repopulating conditions. We performed competitive bone marrow transplantation by injecting Mx1-Tg/WT (CD45.2) bone marrow plus supporting cells (CD45.1) to irradiated mice, followed by 5 doses of poly(I:C) induction. Recipient mice were analyzed during a course of approximately 16 weeks. Mx1-Tg cells were outcompeted by WT cells in reconstituting every blood lineages. Taken together, we conclude that PRMT1 promotes HSC differentiation and accelerates HSC exhaustion during the stress caused by bone marrow irradiation. To understand the mechanism on PRMT1-mediated stress hematopoiesis, we also made Pf4-Cre PRMT1 transgenic mice. When PRMT1 is specifically expressed in MK cells, the number of LT-HSCs was also reduced, implying that PRMT1 affects the self-renewal of LT-HSCs via communication between MK cells and HSCs. Mechanistically, two PRMT1 substrates - RBM15 and DUSP4 - are critical for stem cell self-renewal. We further characterized how PRMT1 activates p38 kinase pathway via directly methylating DUSP4 thus induces ubiquitylation and degradation of DUSP4. The arginine methylation site on DUSP4 has been identified. Moreover, introducing methyl-R mutated DUSP4 back to PRMT1-overexpressing cells partially rescued the loss of HSC differentiation potential. This data adds a new link between arginine methylation and protein phosphorylation mediated by MAP kinases/phosphatases. In addition, we discovered that RBM15 controls alternative RNA splicing and RNA processing in a PRMT1-dosage dependent manner. In this report, we will further address how RBM15 target genes, such as enzymes involved in fatty acid metabolic pathways, affect HSC differentiation. In summary, we report that arginine methylation is a novel regulator for the HSC differentiation via controlling p38-regulated stress pathway and metabolic reprogramming. Disclosures No relevant conflicts of interest to declare.

Treatment response with sirolimus for a pediatric patient with an EBV‐associated smooth‐muscle tumor after bone marrow transplantation

Epstein-Barr virus-associated smooth-muscle tumors (EBV-SMTs) are unique and rare neoplasms described in immunocompromised patients. The case describes a nine-year-old female with a history of acute lymphoblastic leukemia with relapse and subsequent allogeneic bone marrow transplantation who presented with multiple EBV-SMTs of the liver. EBV utilizes the mammalian target of rapamycin (mTOR) pathway for tumor growth, and sirolimus, a mTOR inhibitor, has shown to result in a short-term response. We now report an extended treatment response with sirolimus in a pediatric patient with an EBV-SMT.

Clinical Implications

HomeHypertensionVol. 65, No. 5Clinical Implications Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBClinical Implications Originally published1 May 2015https://doi.org/10.1161/HYPERTENSIONAHA.115.05488Hypertension. 2015;65:925Immune-Mediated Hypertension and Kidney Injury (page 1111)Download figureDownload PowerPointHypertension is a widespread risk factor for many diseases. Although a variety of medications are available, hypertension remains uncontrolled in many treated patients, highlighting a need for novel therapeutic options. Because of the heritability of hypertension, understanding its genetic basis may lead to better treatment and more individualized care. Human genome–wide association studies have nominated many genes for hypertension, and investigation of these genes may uncover novel mechanisms of pathology. One such gene, SH2B3, encodes an intracellular adaptor protein with no clear link to blood pressure regulation. In this issue, we demonstrate that mutation of SH2B3 in the Dahl salt–sensitive rat significantly attenuated hypertension and renal disease. Subsequent bone marrow transplantation between Sh2b3em1Mcwi mutants and Dahl SS rats revealed that the immune cells partly mediated this attenuation of pathology. Furthermore, mutation of SH2B3 significantly increased the percentage of regulatory T cells among circulating and splenic T cells. Regulatory T cells suppress inflammatory signaling of many cell types, and adoptive transfer of regulatory T cells has attenuated pathology in several models of immune-mediated disease. If hypertension is mediated by inflammation, as many recent studies suggest, then elevating the expression of regulatory T cells may prove beneficial by increasing the threshold for immune responses. Further study of SH2B3 and its signaling may reveal targetable pathways that can modulate immune cell differentiation to accommodate patients with inflammatory diseases, including hypertension.Mineralocorticoid Receptor Blockade Treats Obesity-Related Cardiac Diastolic Dysfunction (page 1082)Results of the recent Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist (TOPCAT) trial were negative about the benefit of mineralocorticoid receptor (MR) antagonism in patients with diastolic heart failure; however, the broad inclusion criteria of TOPCAT may have masked patient subgroups that might benefit from MR antagonism. Obese patients exhibit a high incidence of cardiac diastolic dysfunction, a condition that contributes to increased mortality in this population and for which no evidence-based treatment exists. Because MR antagonists are cardioprotective and obesity is associated with renin–angiotensin–aldosterone system activation, MR blockade holds promise as a treatment for obesity-related diastolic dysfunction. In this issue, Bender et al report that treatment of Zucker obese rats with a low dose of the MR antagonist spironolactone normalized established cardiac diastolic dysfunction associated with reduced cardiac fibrosis and oxidative stress, as well as improved coronary microvascular endothelial function and endothelial insulin signaling. Importantly, the cardiovascular protection reported in this study occurred in the absence of hyperkalemia, an improvement in kidney injury, or changes in blood pressure. This study highlights a context-dependent role of MR signaling as a contributor to obesity-related cardiac diastolic dysfunction and coronary microvascular dysfunction and suggests that the cardiovascular benefit of MR antagonism can be achieved with minimal effect on kidney MR. Thus, these benefits of MR antagonism warrant further clinical evaluation for therapeutic application to these conditions in obese patients.Download figureDownload PowerPointPeripheral Plasma 18-Oxocortisol in Primary Aldosteronism (page 1096)Download figureDownload PowerPointAdrenal venous sampling is currently the only reliable method to distinguish unilateral from bilateral disease in primary aldosteronism. We attempted to determine whether peripheral plasma levels of 18-oxocortisol (18oxoF) could contribute to the clinical differentiation between aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism with bilateral adrenal hyperplasia (bilateral hyperaldosteronism) in 234 patients with primary aldosteronism, including computed tomography (CT)–detectable APA (n=113) and bilateral hyperaldosteronism (n=121), all of whom underwent CT and adrenal venous sampling. All aldosteronomas were surgically resected, and the accuracy of diagnosis was clinically and histopathologically confirmed. 18oxoF and 18-hydroxycortisol were measured using liquid chromatographic tandem mass spectrometry. Receiver operating characteristic analysis of 18oxoF discrimination of adenoma from hyperplasia demonstrated a sensitivity/specificity of 0.83/0.99 at a cut-off value of 4.7 ng/dL. 18oxoF levels >6.1 ng/dL were found in 86 of 113 patients with APA (76%) but in none of 121 patients with bilateral hyperaldosteronism, 30 of whom harbored CT-detectable unilateral nonfunctioning nodules in their adrenals. In addition, 18oxoF levels <1.2 ng/dL, the lowest in aldosteronoma, were found in 52 of the 121 (43%) patients with bilateral hyperaldosteronism. Further analysis of 27 patients with CT-undetectable microscopic APAs revealed that 8 of these 27 patients had CT-detectable contralateral adrenal nodules and their highest value of 18oxoF was 4.8 ng/dL below 6.1. The peripheral plasma 18oxoF concentrations served not only to differentiate APAs but also could serve to avoid unnecessary surgery for nonfunctioning adrenocortical nodules concurrent with hyperplasia or microadenoma. Previous Back to top Next FiguresReferencesRelatedDetails May 2015Vol 65, Issue 5 Advertisement Article InformationMetrics © 2015 American Heart Association, Inc.https://doi.org/10.1161/HYPERTENSIONAHA.115.05488 Originally publishedMay 1, 2015 PDF download Advertisement

(486) - Single Center Experience With Amyloid Patients in the Current Era of Disease Specific Medications

Both amyloid light chain (AL) amyloidosis and transthyretin-related (TTR) amyloid have been viewed with reluctance to proceed with heart transplant. AL amyloid in particular has been a contraindation for heart transplant as the underlying disease process progresses. In the current era of proteasome inhibitors, AL amyloid patients may now be adequately treated and undergo heart transplant with subsequent bone marrow transplant. In our program, we have treated 7 AL amyloid patients who developed end-stage heart disease.

A Shot in the Bone Corrects a Genetic Disease

A Shot in the Bone Corrects a Genetic Disease

G408(P) Cytokine storm associated multi-organ failure with poor neurological outcome, during rituximab administration in a child with relapsed acute lymphoblastic leukaemia and ebv related lymphoproliferative disease

We present the case of a 6 year old boy, with relapsed Acute Lymphoblastic Leukaemia and subsequent matched unrelated bone marrow transplant. His Epstein Barr Virus (EBV) titres were significant 6 months post-transplant with evidence of EBV related lymphoproliferative disease. Following local protocols, Rituximab was used for therapy and within minutes of starting the infusion the patient suffered pyrexia, hypotension and seizures with subsequent multi-organ failure, due to a cytokine storm. Organ damage included fulminant hepatic failure, renal failure meriting dialysis, cardiac failure requiring inotropic support and a significant acute brain injury. There was significant neurological impairment with radiological and electrophysiological evidence of global brain cell damage. As a result of irreversible neurological injury, the focus of treatment was switched from curative to palliative. Cytokine storm is an immune mediated phenomenon, characterised by an overwhelming release of cytokines. This can produce a sepsis like response and may lead to multi-organ failure. Cytokines are normally produced by leucocytes as a response to an infective or inflammatory process and their main role is to induce leukopoeisis. Rituximab is an anti-CD20 monoclonal antibody that has been useful in treating EBV related lymphoproliferative disease. Rituximab triggers the rapid release of cytokines that may lead to a cytokine storm with maximum levels within 2 h from the start of the infusion. Cytokine storms are characterised by overproduction of immune mediators that in turn lead to cellular overactivation, increased endothelial permeability, polymorphonuclear neutrophil adhesion and migration. As there is lack of regulatory intervention, tissue congestion with activated leucocytes ensues which eventually causes parenchymal injury. Although we report multi-organ failure in this case, lungs and gut are the organs that are more commonly affected. The incidence of cytokine storm in paediatric oncology patients is not known. It has been associated with haemophagocytic lymphohistiocytosis, graft-versus-host disease following haemopoetic stem cell transplant and the use of certain monoclonal antibodies. As in this case, results of the cytokine storm can be catastrophic, despite early recognition.

Distinct susceptibility of induction of methylation of p16ink4a and p19arf CpG islands by X-radiation and chemical carcinogen in mice

Inactivation of the tumor suppressor genes p16ink4a and p19arf/p14arf by hypermethylation of promoter CpG islands occurs frequently in various tumors. The aim of this study is to investigate the difference of susceptibility of methylation induced by carcinogens between p16ink4a and p19arf. The methylation status of both genes was analyzed by denaturing high performance liquid chromatography (DHPLC) and bisulfite-sequencing, respectively. The expression level of P16 protein was analyzed by immunohistochemistry. Results showed that p16ink4a methylation was detected in the glandular stomach, small intestine and other organs of mice following X-radiation and subsequent bone marrow transplantation (BMT), but not in mock control mice. We found that the intestinal tract was the most sensitive organ for X-ray induced p16ink4a methylation. Loss of P16 protein expression was observed in the intestinal tissues of X-irradiated mice, but not in the mock control mice. Interestingly, p19arf methylation was not observed in the gastrointestinal tissues of the negative control mice following X-radiation/BMT. However, administration of N-nitrosomethylurea and/or Helicobacter felis infection promoted methylation of p19arf CpG islands in the gastrointestinal tracts, but did not promote p16ink4a methylation. In addition, p16ink4a methylation was detected not only in the X-irradiated GFP-negative tissue cells, but also in the GFP-positive bone marrow-derived cells that were transplanted into the BMT mice after X-radiation. In conclusion, the methylation susceptibility of p16ink4a and p19arf to carcinogen treatments was remarkably different: X-radiation indirectly induces systemic p16ink4a methylation, especially in the intestine; whereas N-nitrosomethylurea and/or H. felis infection induce p19arf methylation in their target organs.

Transfusion-Induced Bone Marrow Transplant Rejection Due to Minor Histocompatibility Antigens

Traditionally, alloimmunization to transfused blood products has focused exclusively on recipient antibodies recognizing donor alloantigens present on the cell surface. Accordingly, the immunologic sequelae of alloimmunization have been antibody mediated effects (ie, hemolytic transfusion reactions, platelet refractoriness, anti-HLA and anti-HNA effects, etc). However, in addition to the above sequelae, there is also a correlation between the number of antecedent transfusions in humans and the rate of bone marrow transplant (BMT) rejection-under reduced intensity conditioning with HLA-matched or HLA-identical marrow. Bone marrow transplant of this nature is the only existing cure for a series of nonmalignant hematologic diseases (eg, sickle cell disease, thalassemias, etc); however, rejection remains a clinical problem. It has been hypothesized that transfusion induces subsequent BMT rejection through immunization. Studies in animal models have observed the same effect and have demonstrated that transfusion-induced BMT rejection can occur in response to alloimmunization. However, unlike traditional antibody responses, sensitization in this case results in cellular immune effects, involving populations such as T cell or natural killer cells. In this case, rejection occurs in the absence of alloantibodies and would not be detected by existing immune-hematologic methods. We review human and animal studies in light of the hypothesis that, for distinct clinical populations, enhanced rejection of BMT may be an unappreciated adverse consequence of transfusion, which current blood bank methodologies are unable to detect.

Gads (Grb2-related adaptor downstream of Shc) is required for BCR-ABL-mediated lymphoid leukemia

Philadelphia chromosome-positive leukemias, including chronic myeloid leukemia and B-cell acute lymphoblastic leukemia (B-ALL), are driven by the oncogenic BCR-ABL fusion protein. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates both myeloid and lymphoid disease in mice receiving whole bone marrow transduced with BCR-ABL. Y177 of BCR-ABL is critical to the development of myeloid disease, and phosphorylation of Y177 has been shown to induce GRB2 binding to BCR-ABL, followed by activation of the Ras and phosphoinositide 3 kinase signaling pathways. We show that the GRB2-related adapter protein, GADS, also associates with BCR-ABL, specifically through Y177 and demonstrate that BCR-ABL-driven lymphoid disease requires Gads. BCR-ABL transduction of Gads(−/−) bone marrow results in short latency myeloid disease within 3–4 weeks of transplant, while wild-type mice succumb to both a longer latency lymphoid and myeloid diseases. We report that GADS mediates a unique BCR-ABL complex with SLP-76 in BCR-ABL-positive cell lines and B-ALL patient samples. These data suggest that GADS mediates lymphoid disease downstream of BCR-ABL through the recruitment of specific signaling intermediates.

Cord blood transplantation in Western Australia

Thirty-one umbilical cord blood transplants performed in Western Australia were retrospectively examined in order to document local experience and relevant prognostic factors. Three cord units were from human leucocyte antigen-matched siblings and the remainder were unrelated single (n= 22) or double (n= 6) cord blood transplants. Twenty patients were transplanted for malignant conditions and 11 for non-malignant conditions. Cord units contained a median of 5.6 × 107 total nucleated cells/kg and 1.4 × 105 CD34+ cells/kg. Cumulative incidence of neutrophil engraftment was 76% at day 60. Of those who did not engraft, two patients remain alive following subsequent allogeneic bone marrow transplant. There were no deaths caused by graft-versus-host disease. Overall survival at median follow up of 28 months was 62%. Two year overall survival was influenced by type of disease (non-malignant = 91 ± 9% vs malignant = 41 ± 13%, P= 0.005), total nucleated cell dose (>3.5 × 107/kg = 87 ± 9% vs <3.5 × 107/kg = 34 ± 15%, P= 0.01) and CD34 dose (>1.7 × 105/kg = 92% vs <1.7 × 105/kg = 46%, P= 0.04). Age and human leucocyte antigen match did not influence survival. Four relapses occurred, all of which were fatal. Cord blood transplantation for malignant and non-malignant disease is practised in Western Australia and outcomes are satisfactory. Trends and techniques in cord blood transplantation in this state are comparable with those observed nationally and overseas. Although numbers are small, cell dose appears to be predictive of overall survival

Mechanisms of Alloimmunization and Subsequent Bone Marrow Transplantation Rejection Induced by Platelet Transfusion in a Murine Model

For many nonmalignant hematological disorders, HLA-matched bone marrow transplantation (BMT) is curative. However, due to lack of neoplasia, the toxicity of stringent conditioning regimens is difficult to justify, and reduced intensity conditioning is used. Unfortunately, current reduced intensity regimens have high rates of BMT rejection. We have recently reported in a murine model that mHAs on transfused platelet products induce subsequent BMT rejection. Most nonmalignant hematological disorders require transfusion support prior to BMT and the rate of BMT rejection in humans correlates with the number of transfusions given. Herein, we perform a mechanistic analysis of platelet transfusion-induced BMT rejection and report that unlike exposure to alloantigens during transplantation, platelet transfusion primes alloimmunity but does not stimulate full effector function. Subsequent BMT is itself an additional and distinct immunizing event, which does not induce rejection without antecedent priming from transfusion. Both CD4(+) and CD8(+) T cells are required for priming during platelet transfusion, but only CD8(+) T cells are required for BMT rejection. In neither case are antibodies required for rejection to occur.

168 Experience of Sequential Heart and Bone Marrow Transplant for AL Amyloidosis: What Is the Optimal Waiting Time for Bone Marrow Transplant?

Cardiac transplantation is one treatment option for patients with end stage cardiac amyloidosis. Selection criteria are not accurately defined and published outcomes do not resemble those of the general transplant population. Recurrence of amyloid deposition in the heart after transplant has been reported. We report our experience with heart transplants in AL amyloidosis patients with subsequent bone marrow transplant (BMT) and 1year outcomes.

Successful Treatment of ICE-Rituximab Chemotherapy and Subsequent Bone Marrow Transplantation in a Patient With Early-relapse Burkitt Leukemia and Inverted Duplication of 1q

Although childhood acute lymphoblastic leukemias are of good prognosis than leukemias of adulthood, some chromosomal abnormalities may have negative effects on their prognosis. Inverted duplication (1q) is a chromosomal abnormality with negative effect on outcome of Burkitt leukemia and lymphomas. We report a case of CD20 Burkitt leukemia with inverted duplication (1q) mutation, who had an early relapse during NHL-BFM 95 treatment. Two courses of ICE-rituximab treatment were administered after relapse and a successful HLA-full match bone marrow transplantation was carried out. He is in follow-up for 18 months without any problem after the bone marrow transplantation. We suggest the usage of ICE protocol combined with rituximab in childhood CD20 Burkitt leukemia with poor prognostic criteria such as inverted duplication (1q) mutation.

CTLA4‐Ig prevents alloantibody production and BMT rejection in response to platelet transfusions in mice

Platelet (PLT) transfusions can induce humoral and cellular alloimmunity. HLA antibodies can render patients refractory to subsequent transfusion, and both alloantibodies and cellular alloimmunity can contribute to subsequent bone marrow transplant (BMT) rejection. Currently, there are no approved therapeutic interventions to prevent alloimmunization to PLT transfusions other than leukoreduction. Targeted blockade of T-cell costimulation has shown great promise in inhibiting alloimmunity in the setting of transplantation, but has not been explored in the context of PLT transfusion. We tested the hypothesis that the costimulatory blockade reagent CTLA4-Ig would prevent alloreactivity against major and minor alloantigens on transfused PLTs. BALB/c (H-2(d)) mice and C57BL/6 (H-2(b)) mice were used as PLT donors and transfusion recipients, respectively. Alloantibodies were measured by indirect immunofluorescence using BALB/c PLTs and splenocytes as targets. BMTs were carried out under reduced-intensity conditioning using BALB.B (H-2(b) ) donors and C57BL/6 (H-2(b)) recipients to model HLA-identical transplants. Experimental groups were given CTLA4-Ig (before or after PLT transfusion) with control groups receiving isotype-matched antibody. CTLA4-Ig abrogated both humoral alloimmunization (H-2(d) antibodies) and transfusion-induced BMT rejection. Whereas a single dose of CTLA4-Ig at time of transfusion prevented alloimmunization to subsequent PLT transfusions, administration of CTLA4-Ig after initial PLT transfusion was ineffective. Delaying treatment until after PLT transfusion failed to prevent BMT rejection. These findings demonstrate a novel strategy using an FDA-approved drug that has the potential to prevent the clinical sequelae of alloimmunization to PLT transfusions.

Replacement of microglial cells using Clodronate liposome and bone marrow transplantation in the central nervous system of SOD1G93A transgenic mice as an in vivo model of amyotrophic lateral sclerosis

Disease progression of amyotrophic lateral sclerosis (ALS) is partially mediated by the toxic microenvironment established by microglia. In the present study, we used SOD1G93A transgenic mice as an in vivo ALS model and replaced microglia expressing mutant SOD1 (mSOD1) with microglia expressing wild-type SOD1 (w/tSOD1) to modulate the toxic microenvironment. Stereotactic injection of Clodronate liposome, a selective toxin against the monocyte/macrophage system, into the fourth ventricle of the brains of 12-week-old asymptomatic ALS mice reduced the number of microglia effectively in the central nervous system. Subsequent bone marrow transplantation (BMT) with bone marrow cells (BMCs) expressing w/tSOD1 and GFP leads to replacement of the endogenous microglia of the ALS mice with microglia expressing w/tSOD1 and GFP. The expression of mSOD1 in the other neural cells was not influenced by the replacement procedures, and immunological side effects were not observed. The replacement of microglia significantly slowed disease progression and prolonged survival of the ALS mice compared with the ALS mice treated by stereotactic injection of PBS-liposome and BMT with BMCs expressing mSOD1 or w/tSOD1. These results suggest that replacement of microglia would improve the neural cell microenvironment, thereby slowing disease progression. The mechanisms and functional implications of this replacement require further elucidation.

The Gads Adaptor Protein Is Required to Mediate Lymphoid Disease Downstream of BCR-ABL,

Abstract 3750Chronic Myeloid Leukemia is mediated by the BCR-ABL chromosomal translocation. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates a robust myeloproliferative disease that is fatal in recipient animals within 21–28 days that have received transduced marrow from donor mice primed with 5-fluorouracil. Structure/function experiments revealed that mice receiving bone marrow expressing BCR-ABL Y177F generate a T cell lymphoma with a latency of 73–116 days, demonstrating that the SH2 proteins that bind to BCR-ABL pY177 are critical for promoting myeloproliferative disease.There are three members of the Grb2 adaptor protein family: Grb2, Gads and Grap. Biochemical experiments suggest that the Grb2 adaptor protein binds to BCR-ABL Y177 and acts to couple BCR-ABL to the Gab2 adaptor protein. In turn, Gab2 links to activation of the Ras and PI 3 kinase signal transduction pathways. Previous studies from our group revealed that Gads binds to BCR-ABL in vitro. We hypothesized that Gads plays an important role downstream of BCR-ABL. To test this objective, we performed retroviral transduction-bone marrow transplantation experiments utilizing bone marrow from wild type and Gads-deficient mice.Thymocytes from Gads−/− mice exhibit defective proliferation in response to CD3 activation and deficits in positive and negative selection. The loss of Gads results uncoupling of Slp-76 and Lat, and impaired PLCγ phosphorylation in thymocytes. No other prominent phenotypes have been documented in this animal model.We show that retroviral transduction-bone marrow transplantation experiments completed with 5-fluoruracil primed bone marrow display a similar latency in BCR-ABL expressing bone marrow from wild type or Gads-deficient mice.Retroviral transduction-bone marrow transplantation experiments can also be completed with normal (unprimed) bone marrow which generates a mixed syndrome comprising myeloproliferative disease, B cell Acute Lymphoid Leukemia and macrophage tumours with a latency from 30–70 days in mice transduced with BCR-ABL. Strikingly, we observe that the lymphoid disease is absent in experiments completed with Gads−/− bone marrow expressing BCR-ABL and all mice succumb to myeloproliferative disease at 21 days. These data suggest that Gads is required for lymphoproliferative disease in this model.We have examined signaling events in the CML-T1 and LAMA-84 CML cell lines that co-express GRB2 and GADS in order to develop models to account for our observations. GRB2 and GADS both co-immunoprecipitate BCR-ABL in both cell lines. However, there is remarkable specificity in the complexes that bind to each adaptor protein. For example, GRB2 co-immunoprecipitates the SHC adaptor protein (but not GAB2), whereas GADS co-immunoprecipitates GAB2 and SLP-76 from both CML cell lines. We are currently performing experiments to confirm these observations in BCR-ABL expressing Acute Lymphoid Leukemia patient specimens.Our data suggests that BCR-ABL mediates Y177-dependent signaling via Grb2-dependent activation of Ras and PI 3 kinase in a myeloproliferative model of CML. However, in keeping with the important role that Gads plays in maintaining lymphocyte homeostasis, Gads mediates lymphoproliferative disease downstream of BCR-ABL, through the recruitment of specific signaling intermediates. Disclosures:No relevant conflicts of interest to declare.

Minor Antigens on Transfused RBCs Crossprime CD8 T Cells but Do Not Induce Full Effector Function

HLA-matched bone marrow transplantation (BMT) is a cure for nonmalignant hematological disorders; however, rejection rates are high and correlate with the number of antecedent transfusions. Recently, using murine models, we reported that minor antigens (mHAs) in transfused leukoreduced red blood cell (RBC) or platelet units induce rejection of subsequent BMT. To study RBCs as an immunogen, we utilized transgenic donors that express a model mHA selectively on RBCs (HOD mouse). Transfusion of HOD blood did not induce BMT rejection of marrow that shared mHAs with the HOD RBCs. Similarly, no endogenous anti-HOD CD8(+) T-cell response was detected with antigen-specific tetramer reagents. Adoptively transferred OT-I T cells rapidly expanded after HOD blood transfusion; however, only a semi-effector phenotype was observed (tumor necrosis factor-α and interferon-γ secretion, but essentially no Granzyme B). After initial expansion, OT-I T cells contracted rapidly to very low levels. A similar trend was observed by in vivo CTL assay, with only transient lytic activity. Together, these data indicate that RBCs may not be the component of RBC units that induces BMT rejection, and suggest that contaminating platelets or leukocytes may be responsible.

Rituximab and cyclosporine therapy for accelerated phase Chediak–Higashi syndrome

A 19-month-old male with Chediak-Higashi syndrome developed Epstein-Barr virus (EBV)-associated accelerated phase. Real-time polymerase chain reaction showed high EBV-DNA levels in plasma and peripheral blood mononuclear cells. His condition was refractory to conventional treatments for hemophagocytic lymphohistiocytosis, including corticosteroids, cyclosporine, and etoposide. In situ hybridization revealed higher proportion of EBER-1-positive cells in CD19+ cell fraction than in CD8+ cell fraction. Complete remission was achieved by combination therapy with rituximab and cyclosporine; subsequent bone marrow transplantation was successful. Combination therapy with rituximab and cyclosporine could be effective in patients with EBV-infected T and B cells.