Unmatched Donors Research Articles

Solid organ donor-recipient race-matching: analysis of the United Network for Organ Sharing database.

Donor ethnicity is a prognosticator in organ transplant. However, the impact of donor/recipient race-matching is unclear. We hypothesized that there would be increased survival in donor-recipient race-matched organ recipients because of genetic and physiologic similarities. The UNOS database from 1999 to 2018 was queried for all solid organ transplantations including heart, lung, liver, kidney, and pancreas transplants. Data were sorted by donor and recipient race into matched and unmatched categories for Caucasian, African American, and Hispanic transplant recipients. After controlling for potential confounders via inverse propensity of treatment weighting, post-transplant patient and graft survival were compared between race-matched and -unmatched donor groups for each organ. Race-matched Caucasian recipients experienced 1-3% improvement in mortality across most time points in lung, liver, and pancreas transplants, while Hispanics did not benefit. Matched African American recipients experienced 4-6% improvement in patient and graft survival in liver transplant but had 7-9% worse survival rates at 5years in lung and pancreas transplants. Race-matching does not influence patient outcomes enough to factor into organ transplant offers. African American liver transplant recipients benefited the most. Matching was detrimental to African American lung and pancreas transplant recipients indicating there may be other factors influencing the outcomes of these transplants.

Aloocetra-OTS for graft versus host (GVHD) prevention and successful transplantation

Background & Aim Allocetra-OTS was developed as a cell-based therapeutic composed of unmatched donor mononuclear cells induced to an early apoptotic state. Allocetra-OTS, as do naturally occurring apoptotic cells within the body, signals to the macrophages and dendritic cells to react less aggressively to immunogenic agents, thereby resetting the system towards immuno-homeostasis. Three types of experiments showed safety and efficacy in pre-clinical studies. A single dose of Allocetra-OTS in a murine model of GvHD had a beneficial effect on GvHD prevention. Allocetra-OTS was also successful as an ancillary treatment to the standard of care for the prevention of GvHD. Finally, in a mice model with co-existing leukemia, Allocetra-OTS was effective against GvHD without interfering with the graft-versus-leukemia (GvL) effect. Methods, Results & Conclusion The clinical development program of Allocetra-OTS for GvHD consists of 2 clinical studies, a completed Phase 1/2a study (TLX-APO-101IL) for related donors (Allocetra), and a planned Phase 2/3 study (ENX-CL-01-002) for unrelated donors. In addition, Allocetra-OTS is being evaluated for sepsis in an ongoing Phase 1b open-label study. Overall, 21 subjects were exposed to at least 1 dose of Allocetra or Allocetra-OTS and no safety concerns were detected. The first GvHD study included dose-escalation of four sequential cohorts. Thirteen eligible subjects with hematologic malignancies, who received conventional myeloablative conditioning were enrolled. The results of this study suggested that a single infusion of donor early apoptotic cells (Allocetra) as prophylaxis for GvHD in myeloablative HSCT is safe and potentially effective and led to 0% (0/6) of acute high grade II-IV GvHD in the two higher dosages compared to 52% in matched historical control. The planned Phase 2/3 multi-center, open-label, 2-arm study (ENX-CL-01-002), in Israel and Germany, will evaluate the efficacy and safety of Allocetra-OTS (140 × 106 cells/kg) with or without anti-thymocyte globulin (ATG) for the prevention of GvHD in subjects undergoing HLA-matched HSCT from an unrelated donor.

Cultured thymus tissue implantation promotes donor-specific tolerance to allogeneic heart transplants.

Eighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop T cells similar to those of recipient that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell-depleted Lewis rats. The rats were then given cultured thymus tissue from F1 (Lewis × Dark Agouti ) under the kidney capsule and vascularized Dark Agouti (DA) heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naive and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with LWxDA CTTI rejected the third-party BN hearts (mean survival time 10 days); controls did not. CTTI recipients produced antibody against third-party BN donor but not against the DA thymus donor, demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus, with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.

Allocetra-Ots: Early Apoptotic Cells for Immune Homeostasis in Human Stem Cell Transplantation (HSCT) and for the Prevention of Graft Versus Host Disease (GvHD)

Allocetra-OTS was developed as a cell-based therapeutic composed of unmatched donor mononuclear cells induced to an early apoptotic state, indicated for the prevention of graft vs. host disease (GvHD) in patients receiving human stem cell transplantation (HSCT). Allocetra-OTS harnesses the naturally occurring activity of early apoptotic cells to induce an immuno-modulated state. Allocetra-OTS, as do naturally occurring apoptotic cells within the body, signals to the macrophages and dendritic cells to react less aggressively to immunogenic agents, thereby resetting the system towards immuno-homeostasis. Three types of experiments showed safety and clinical efficacy in murine models. Administration of a single dose of Allocetra-OTS in a murine model of GvHD had a beneficial effect on GvHD prevention. Allocetra-OTS was also evaluated as an ancillary treatment to the current standard of care for the prevention of GvHD, i.e. GvHD prophylaxis standard-of-care (SOC) regimen using cyclosporin A (CSA) and methotrexate (MTX) in mice. The study showed that combined treatment of immunosuppressors with Allocetra-OTS was safe and effective. Finally, in a mice model with co-existing leukemia, Allocetra-OTS was effective against GvHD without interfering with the graft-versus-leukemia (GvL) effect of the BM transplanted cells. Taken together, in mice models, Allocetra-OTS is safe and significantly improves GvHD both by itself or in addition to SOC, without reducing GvL effect. The clinical development program of Allocetra-OTS for GvHD consists of 2 clinical studies, a completed Phase 1/2a study (TLX-APO-101IL) for related donors (Allocetra), and a planned Phase 2/3 study (ENX-CL-01-002) for unrelated donors. In addition, Allocetra-OTS is being evaluated for sepsis in an ongoing Phase 1b open-label study (ENX-CL-02-001) and is included in the overall safety database. Overall, 21 subjects were exposed to at least 1 dose of Allocetra or Allocetra-OTS and no safety concerns were detected. The first GvHD study included dose-escalation of four sequential cohorts (35, 70, 140 and 210 × 106 cells/kg) and was conducted at 3 sites in Israel. Thirteen eligible subjects with hematologic malignancies, who received conventional myeloablative conditioning were enrolled. The results of this study suggested that a single infusion of donor early apoptotic cells (Allocetra) as prophylaxis for GvHD in myeloablative HSCT is safe and potentially effective and led to 0% (0/6) of acute high grade II-IV GvHD in the two higher dosages compared to 52% in matched historical control. The planned Phase 2/3 multi-center, open-label, 2-arm study (ENX-CL-01-002), in Israel and Germany, will evaluate the efficacy and safety of Allocetra-OTS (140 × 106 cells/kg) with or without anti-thymocyte globulin (ATG) for the prevention of GvHD in subjects undergoing HLA-matched HSCT from an unrelated donor.

Hematopoietic stem cell transplantation and cellular therapy in sickle cell disease: where are we now?

Sickle cell disease (SCD) is a common monogenic disorder that is characterized by an A to T substitution in the β-globin gene that leads to the production of hemoglobin S (HbS). Polymerization of HbS leads to significant morbidity including vaso-occlusion, pain, hemolytic anemia, and end organ damage. Allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative treatment; however, suitable donors are not always readily available. This study reviews the current status of allo-HCT and autologous cellular therapies for SCD. Alternative sources of allogeneic stem cells from unmatched donors such as cord blood and haploidentical donors are gaining traction. Early experience has shown that better conditioning regimens and graft-versus-host disease prophylaxis are needed before these donor sources can gain widespread use. Clinical trials are underway to determine the feasibility and efficacy of autologous transplantation with gene modified hematopoietic stem cells. Gene therapy strategies include HbS gene correction, gene addition, and hemoglobin F induction. Preliminary results are very encouraging. Matched sibling allo-HCT for patients with SCD results in more than 90% overall survival and more than 80% event-free survival. Because only 25-30% of patients have a matched sibling donor, alternative donor options such as matched unrelated donors, related haploidentical donors and unrelated umbilical cord blood donors are being considered. Clinical trials investigating various strategies for gene therapy followed by autologous transplantation are underway. One major challenge is obtaining sufficient hematopoietic stem cells for gene therapy. Studies are being conducted on the optimal mobilization regimen and collection strategy.

Increased kielin/chordin-like protein levels are associated with the severity of heart failure

BackgroundPrevious studies demonstrated that the transforming growth factor (TGF) β superfamily, including TGF-βs and bone morphogenetic proteins (BMPs), plays important roles in cardiovascular diseases. The kielin/chordin-like protein (KCP) is a secreted protein that regulates the expression and function of TGF-βs and BMPs. However, the role of KCP during heart failure (HF) remains unknown. The present study aimed to investigate the cardiac expression of KCP in human failing hearts. MethodsThe human failing heart samples from patients with dilated cardiomyopathy (DCM, n = 12) and ischemic cardiomyopathy (ICM, n = 12) were collected, and normal heart (n = 8) samples from unmatched donors were collected as controls. Collagen volume, KCP levels, and mRNA levels of several BMPs in left ventricles (LV) of all hearts were measured. ResultsThe KCP levels were significantly higher in human failing hearts than in normal hearts. KCP levels were positively associated with hypertrophy markers, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC). In addition, KCP levels were also positively associated with left ventricular end-diastolic dimension (LVEDD), collagen Iα and collagen IIIα expression but were negatively associated with left ventricular ejection fraction (LVEF). Furthermore, increased TGF-β1, BMP2/4/6/10 and reduced BMP7 levels were observed, and positive correlations between KCP and TGF-β1 and negative correlation between KCP and BMP2/7 were found, but not for BMP4/6/10. ConclusionsKCP was closely associated with heart failure. The regulation of BMP2/7 and TGF-β1 expression may be the possible mechanisms.

Universal CAR T Cells Treat Leukemia.

Two infants with relapsed, refractory B-cell acute lymphoblastic leukemia went into complete remission after being treated with CD19-targeting CAR T cells derived from an unmatched donor. The study is the first to demonstrate that a universal form of CAR T-cell therapy can be safely utilized.

298. Transfection of Neutrophils from X-Linked Chronic Granulomatous Disease Patients with gp91phox mRNA Restores Oxidase Activity with High Efficiency and Viability

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to defective phagocyte NADPH oxidase required for production of antimicrobial oxidants. Fungal or bacterial infections in CGD may persist despite intensive and prolonged antibiotic therapy. In settings where conventional antibiotic therapy and/or surgical debridement do not control infection, use of allogeneic unmatched donor granulocyte transfusions has been therapeutic. However, CGD patients treated with granulocyte transfusions may develop high titers of anti-HLA antibodies, restricting use of allogeneic granulocyte transfusions and further complicating potentially curative bone marrow transplant. Using the CGD patient as an autologous donor for granulocyte transfusions would be a helpful solution.Here we demonstrate highly efficient functional correction of the functional defect in CGD by transfection of patient's own granulocytes with mRNA while retaining viability using a scalable, cGMP-compliant MaxCyte® GT™ electroporation system capable of handling clinically relevant numbers of cells (>1010cells).Freshly collected granulocytes from normal volunteers were electroporated using the MaxCyte electroporation system with eGFP-mRNA on the day of collection. Flow cytometry of demonstrated that >90% of neutrophils expressed eGFP with viability exceeding 90% at 24 hrs after electroporation. Phagocyte NADPH oxidase activity of the normal neutrophils was retained as well as bactericidal activity against Burkolderia cepacia, a CGD bacterial pathogen. Antibacterial activity of electroporated granulocytes was comparable to unmanipulated granulocytes confirming retention of cellular function following the electroporation. Finally, electroporated eGFP positive human neutrophils injected into CGD mice were shown to remain in circulation as well as migrate to the peritoneal cavity following thioglycollate IP injection and there were no adverse effects observed in the mice.Electroporation of gp91phox mRNA into human X-linked CGD (gp91phox-deficient) patient neutrophils resulted in expression of gp91phox protein in >70% of cells with >90% viability at 24 hours after treatment. Using the dihydrorhodamine assay of phagocyte NADPH oxidase activity, >70% of neutrophils remained functionally corrected 2 days after transfection. Other studies of cellular function in vitro and in vivo with electroporated corrected CGD neutrophils are in progress. In summary, we show the feasibility of restoring the function of CGD patient-autologous neutrophils via electroporation of mRNA encoding the defective gene product. This approach may have potential clinical utility in the management of severe chronic bacterial and fungal infections in CGD but also short-term correction of other hematopoietic gene diseases.

Abstract 18918: Distinct Gene Expression Profiles of the Mitral Valve Leaflets in Barlow’s Disease and Fibroelastic Defficiency

OBJECTIVES: The mitral valve (MV) leaflets in severe MR undergo structural remodeling associated with multiple degenerative and adaptive changes. We hypothesized that the gene expression profiles of the posterior MV leaflets from patients with bileaflet prolapse (Barlow’s valves) are distinct from those with only posterior leaflet degeneration (fibroelastic deficiency or MMR), reflecting different underlying pathophysiologic processes. Methods: MVs were obtained from 12 Barlow’s patients (9 m, 3 f; age= 59.8±12.2 y, LVEF=60.4±5.8%) and 17 MMR patients (10 m, 7 f, age= 54.2±6.6 y, LVEF=59.7±5.1%) undergoing surgery for severe MR. Normal MV were obtained from 6 unmatched donor hearts (4 m, 2 f, age= 41.5±10.7 y, LVEF=57.5±8.8%). RNA was extracted and cDNA microarray was carried on the Affymetrix Gene Chip 1.1. Normalization and two-way ANOVA analysis were performed. Only genes that resulted in >1.5 fold difference and p <0.05 were considered significantly altered. RESULTS: Of ~29,000 genes examined, 973 (3.3%) were differentially expressed between the two disease groups. Principal component analysis and Hierarchical clustering of the normalized fluorescence signal values for these genes successfully differentiated all the Barlow’s from the MMR samples (Figure 1). Based on functional analysis, selected statistically significant enriched biological processes are listed in (table1). Conclusions: MV leaflets in Barlow’s and MMR exhibit distinct gene expression patterns, suggesting different pathophysiologic mechanisms are involved in the leaflet remodeling processes. Understanding such adaptive mechanisms can potentially provide therapeutic opportunities.

Allogeneic Hematopoietic Cell Transplantation For Patients With Primary and Secondary Myelofibrosis: Report Of a Single Center Experience

Introduction Myelofibrosis (MF) is a heterogenous myeloproliferative disorder arising at the level of pluripotent hematopoietic stem cells. It is characterized by reactive marrow fibrosis resulting in extramedullary hematopoeisis, cytopenias, hepatosplenomegaly and constitutional symptoms. The only curative treatment for MF patients is allogeneic hematopoietic cell transplantation (HCT). However, advanced age, comorbidities and disease-related complications are challenging factors for allogeneic HCT. Reduced intensity conditioning (RIC) regimens have lead to an increased frequency of allogeneic HCT for MF patients in recent years. We here report our own experience with allogeneic HCT in MF. Methods By searching our database of allogeneic HCT we identified a total of 55 patients with primary or secondary myelofibrosis that underwent allogeneic HCT from 1997 to 2012. Retrospective analysis of data and review of patient charts was performed. Results Among the 55 MF patients were 32 males and 23 females with a median age of 54 years (range 28-75). The median interval from diagnosis to HCT was 123 months (range 2-269). Conditioning regimens were either myeloablative (MAC, 18/55 patients (33%)) or RIC (37/55 patients (67%)). Median age was 58 years (range 45-75) for RIC und 47 years (range 28-60) for MAC. As graft source either peripheral blood stem cells (n= 46) or bone marrow (n= 9) was used from either matched related (MRD, n=17, 31%), matched unrelated (MUD, n=21, 38%) or mismatched unrelated donors (MMUD, n=17, 31%). The MAC regimen consisted of 12 Gy total body irradiation in combination with high-dose cyclophosphamide (TBI/Cy, 78%) or high-dose busulfan and cyclophosphamide (Bu/Cy, 22%). Fludarabine and busulfan were the most frequently applied drugs for RIC (Flu/Bu, n=31, 84%), in a few cases fludarabine in combination with TBI (n=1, 3%) or treosulfan-containing regimens (n=3, 8%) were used. Two patients with accelerated disease were treated with the FLAMSA-protocol, followed by busulfan and cyclophosphamide containing RIC. As graft versus host disease (GVHD) prophylaxis, a combination of calcineurine inhibitors and methotrexate or mycophenolate mofetil was used, in 80% (n=44) antithymocyte globuline was additionally applied. Kaplan Meier estimated overall survival at 4 years was 62% for all patients and 62% after RIC compared to 58% with MAC (p= 0.42) with a median follow-up of patients alive of 49 months (range 9-151). Cumulative incidence of non relapse mortalitiy (NRM) overall and for MAC and RIC was 31%. The overall survival of patients with MRD, MUD and MMUD was 60%, 61% and 61%, respectively. NRM of patients with matched and unmatched donors was not significantly different (MRD 31% versus MUD 35% versus MMUD 33%). Engraftment of neutrophils (ANC>500/µL) was achieved at a median of 16 (range 9-31) days and 23 (range 13-60) days for patients in the MAC and the RIC-group, respectively (p= 0.0051). Platelet recovery (PLT>20.000/µL) occurred at a median of 24 (range 11-51) days after MAC and 21 (range 8-41) days after RIC (p= 0.2866). Despite the significantly delayed engraftment, the risk for severe or lethal infection was lower in the RIC- than in the MAC-group (n=3 versus n=1). Graft rejection occurred only in 3 patients after RIC. Acute GVHD occurred in 31% (n=17) of the patients. As expected, patients with MAC, compared to RIC, had a higher risk of acute GVHD (71% (n=12) versus 29% (n=5), p< 0.0001). Chronic GVHD occured in 59% of the patients, with 44% extensive and 56% limited cases. Chronic GvHD was more frequent after RIC compared to MAC (59% versus 41%, p= 0.1462). Patients with an extensive GVHD only survived in 18%, whereas patients with a mild or limited GvHD survived in 39% (p= 0.2059). Overall the occurence of GVHD was associated with a poorer outcome. Conclusion Our retrospective single center study confirms existing data that allogeneic HCT following RIC may have a similar outcome compared to MAC although used in an older and more comorbid patient population. Allogeneic HCT even with MMUD thus is a potentially curative treatment option in up to 60% of patients with primary or secondary MF. Disclosures: No relevant conflicts of interest to declare.

Periostin expression is upregulated and associated with myocardial fibrosis in human failing hearts

Background and purposePeriostin, a matricellular protein, plays an important role in cardiac development and remodeling. Its expression profile and the association with myocardial fibrosis have not been investigated in human failing hearts. This work aimed to explore the behavior and pathologic significance of periostin in signifying collagen fibrogenesis in human hearts from patients with end-stage heart failure. Methods and subjectsTissues were collected from heart transplant recipients and the control hearts were from unmatched donors. Periostin mRNA and protein were detected using quantitative real-time polymerase chain reaction and Western blotting. Immunohistochemistry staining was employed to directly detect the protein level and distribution of periostin in heart tissues. The extent of myocardial fibrosis was expressed by the percentage of Masson's trichrome staining. Gelatin zymography was used to detect the activities of matrix metalloproteinase (MMP)2 and MMP9. ResultsA low level of periostin mRNA expression was found in control hearts while not detectable at the protein level. Periostin mRNA was increased significantly in failing myocardium compared to that of controls. Periostin was distributed extensively in left ventricle and interventricular septum of the failing hearts. Correlation analysis showed periostin protein expression was positively associated with myocardial fibrosis as well as left ventricular diastolic dimension. The distribution and extent of periostin was consistent with that of myocardial fibrosis. MMP2 activity has an obvious increase about fourfold in heart tissues from HF patients. But there is no quantitative association with the expression of periostin. ConclusionsPeriostin, the distribution and expression of which were consistent with the extent of myocardial fibrosis, might be a potential biomarker of cardiac remodeling in heart failure patients.

Fluorescence In Situ Hybridization (FISH) as an Aid for the Diagnosis of Graft-Versus-Host Disease in Two Multivisceral Organ Transplant Patients

We herein describe 2 cases of adult multivisceral transplant patients who developed graft-versus-host disease manifesting predominantly as lichenoid skin papules and plaques. The diagnosis was supported by histopathology but ultimately corroborated by the utilization of the fluorescence in situ hybridization (FISH) technique using X and Y chromosome probes on unstained biopsy slides. In both cases, FISH revealed a high percentage of donor-derived cells as part of the inflammatory infiltrate in the skin biopsy. This report adds to the previous publications showing the utility of FISH in corroborating the diagnosis of graft-versus-host disease in transplant patients with unmatched sex donor.

Mitigating effects of hUCB-MSCs on the hematopoietic syndrome resulting from total body irradiation

This study evaluated the clinical and pathologic effects of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in the recovery from total body irradiation by comparing it with the effects of granulocyte-colony stimulating factor (G-CSF), an efficacious drug in the treatment of acute bone marrow radiation syndrome. BALB/c mice were treated with G-CSF or hUCB-MSCs after they were irradiated with 7 Gy cobalt-60 γ-rays. Circulating blood counts, histopathologic changes in the bone marrow, and plasma level of Flt-3L and transforming growth factor (TGF-β1) were monitored in the postirradiation period. Hematologic analysis revealed that the peripheral leukocyte counts were markedly increased in the hUCB-MSCs-treated group, whereas G-CSF-treated mice did not recover significantly. Moreover, differential counts showed that hUCB-MSC treatment has regenerative effects on white blood cells, lymphocytes, and monocytes compared with the irradiated group. Treatment with hUCB-MSCs or G-CSF significantly increased immunoreactivity of Ki-67 until 3 weeks after total body irradiation. However, at 3 weeks, the number of Ki-67 immunoreactive cells significantly increased in the hUCB-MSCs-treated group compared with the G-CSF-treated group. Furthermore, hUCB-MSC treatment significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGF-β1, whereas G-CSF treatment failed to decrease the plasma Flt-3L levels at 2 weeks after irradiation. Based on the differences in circulating blood cell reconstitution and cell density of bone marrow, the authors suggest that MSC treatment is superior to G-CSF treatment for hematopoietic reconstitution following sublethal dose radiation exposure.

A Practical and Direct Comparison of Intrinsic Metabolic Clearance of Several Non-CYP Enzyme Substrates in Freshly Isolated and Cryopreserved Hepatocytes

Human hepatocytes are a physiologically relevant tool useful in evaluating liver-related pharmacokinetics, including non-cytochrome P-450 (CYP) metabolism, due to their broad spectrum of metabolic enzyme activity. To verify the usefulness of human hepatocytes in evaluating non-CYP metabolism for drug discovery, we compared intrinsic clearance values (CL(int)) in freshly isolated and cryopreserved hepatocytes using 14 compounds primarily metabolized by non-CYP enzymes, including UDP-glucuronosyltransferase, carbonyl/aldo-keto reductase, aldehyde oxidase, flavin-containing monooxygenase, and monoamineoxidase. Cryopreservation resulted in a >20% reduction (maximum: 50%) in CL(int) in 7/14 compounds (statistically significant for 5 compounds) on comparing CL(int) values in freshly isolated and cryopreserved hepatocytes from the same donors (n = 4). However, the number of compounds with >20% CL(int) reduction decreased to 3 on comparing average of CL(int) values including un-matched donors (dolasetron: -27%, naltorexone: -32%, and phthalazine: -48%; statistically significant for phthalazine, n = 6-11). These findings suggest that fresh hepatocytes are useful in evaluating intact non-CYP enzyme activities. However, we must note that the reduction in CL(int) by cryopreservation could be rendered negligible if high-activity lots are selected for assay. We therefore recommend using cryopreserved hepatocytes for large-scale screening for non-CYP metabolism in drug discovery research considering the advantages in usability with cryopreserved hepatocytes.

Abstract 4364: Gender-Specific Regulation of Ncx in Human Heart Failure

We and others have previously reported increased protein levels of the sarcolemmal sodium-calcium exchanger (NCX) in failing (F) vs non-failing (NF) human hearts. In the non-failing cardiomyocyte, NCX transports calcium out of the cell following contraction, in an amount roughly equal to that which enters the cell through L type calcium channels upon depolarization. In the face of decreased sarcoplasmic reticulum calcium ATPase (SERCA) function in the F human heart, up-regulation of NCX is thought to be a compensatory change, helping to prevent calcium overload. It has also been demonstrated that NCX may work in reverse mode in F heart cells, providing for increased calcium influx during the action potential plateau. Recent studies have suggested that over-expression of NCX has different functional consequences in male and female animal hearts during metabolic inhibition as well as ischemia/reperfusion injury, yet studies demonstrating up-regulation of NCX in F human hearts have largely pooled male and female hearts. We have measured NCX and SERCA in a population of male and female F and NF human hearts. Nine patients were studied per group. F hearts were explanted from cardiac transplant recipients with diagnosis of dilated cardiomyopathy and NF hearts from unmatched organ donors. Immunoblotting was used to quantify NCX and SERCA, normalized to actin. NCX was increased in both male and female F hearts as compared to NF hearts, but the increase was proportionately greater in the male population (Normalized values: Male F 3.30 vs NF 1.0; Female F 2.60 vs NF 1.0). Comparison of NF hearts from males and females, however, demonstrated that levels of NCX protein are consistently higher in female than male hearts (Female NF 2.18 vs Male 1.0). SERCA protein was decreased in both male and female F hearts to equal degrees as compared to both groups of NF hearts (Male F 0.65 vs NF 1.0; Female F 0.65 vs NF 1.0), with no difference in NF levels between males and females (female NF 1.1 vs Male 1.0). These data suggest that NCX is regulated differently in male and female F human hearts, which may play a role in the ability of the heart to preserve adequate calcium cycling during cardiac dysfunction.

Outcomes of Unrelated Umbilical Cord Blood Transplantation for X-Linked Adrenoleukodystrophy

Adrenoleukodystrophy (ALD) is an X-linked disorder caused by a defect in the metabolism of long chain fatty acids leading to demyelination, neurodegeneration, and death. The disease typically presents in young boys and adolescent boys. Allogeneic bone marrow transplantation has been used to halt progression of the disease. However, many patients lack suitable HLA- matched related donors and must rely on unmatched donors for a source of stem cells. The purpose of this study was to evaluate outcomes of unrelated donor umbilical cord blood transplantation after chemotherapy-based myeloablative conditioning and retrospectively determine if baseline studies correlate and help predict outcome. Between November 22, 1996, and November 3, 2005, 12 boys with X-linked ALD who lacked HL- matched related donors were referred to Duke University Medical Center for transplantation. These children were conditioned with myeloablative therapy including busulfan, cyclophosphamide, and antithymocyte globulin before receiving umbilical cord-blood transplants from unrelated donors. Baseline studies of neurophysiologic, neuroimaging, and neurodevelopmental status were performed and patients were subsequently evaluated for survival, engraftment, graft-versus-host disease, and neurodevelopmental outcomes. A substudy evaluated whether baseline neuroimaging and neurophysiologic studies correlated with cognitive and motor function and if these studies were predictive of posttransplantation outcomes. The umbilical cord blood grafts had normal levels of very long chain fatty acids. They delivered a median of 6.98 × 10 7 nucleated cells per kilogram of recipient body weight and were discordant for up to 4 of 6 HLA markers. Neutrophil engraftment occurred at a median of 22.9 days after transplantation. Three patients had grade II-IV acute graft-versus-host disease; 2 had extensive chronic graft-versus-host disease. Cumulative incidence of overall survival of the group at 6 months is 66.7% (95% confidence interval 39.9-93.3%). Median follow-up was 3.3 years (range 12 days to 6.3 years). As previously reported with bone marrow transplantation, symptomatic patients faired poorly with lower survival and rapid deterioration of neurologic function. This study included 3 patients transplanted at a very young age (2.6-3.5 years) before the onset of clinical symptoms who continue to develop at a normal rate for 3-5 years posttransplant. Although baseline Loes scores correlated with cognitive and motor outcome, neurophysiologic studies failed to show statistically significant differences. Transplantation of boys with X-linked ALD using partial HLA-matched umbilical cord blood yields similar results to those previously reported after bone marrow transplantation. Superior outcomes were seen in neurologically asymptomatic boys less than 3.5 years of age at the time of transplantation. Baseline Loes scores were a strong predictor of cognitive and motor outcome.

Mycobacterial infection: a difficult and late diagnosis in stem cell transplant recipients.

The Infectious Diseases Working Party of the European Blood and Marrow Transplant Group conducted a survey to obtain information about the frequency, presentation, and treatment of mycobacterial infection (MBI) in stem cell transplant (SCT) recipients. Among 29 centers, MBI was diagnosed in 0.79% of 1513 allogeneic and 0.23% of 3012 autologous SCT recipients during 1994-1998 a median of 160 days after transplantation. The mean interval between first symptoms and diagnosis was 29 days and was still longer for patients with atypical MBI or recipients of corticosteroid therapy. The prevalence of MBI was highest among those who received matched unrelated or mismatched STCs from related donors. Of 31 patients, 20 had tuberculosis, 8 had atypical MBI, and 3 had diagnoses based on histological findings only. Five patients (16%) died, all of whom had received an allogeneic SCT. Because of the increased numbers of unmatched donors and transplantation programs in countries with a high prevalence of tuberculosis, constant vigilance is required to early detect MBI in SCT recipients.

Validação do Escore de Risco do EBMT em 1.084 pacientes portadores de leucemia mielóide crônica submetidos ao transplante alogênico de células precursoras hematopoiéticas no Brasil

Abstract This study is a retrospective analysis of 1084 patientstransplanted in ten Brazilian centers in the period fromFebruary 1983 to March 2003. The main objective was tovalidate the EBMT risk score in chronic myeloid leukemiapatients submitted to allogeneic hematopoietic stem celltransplantation.The risk score is based on five variables: recipientage, disease stage, donor type [HLA-identical related,matched unrelated or unmatched donor], donor-recipientgender combination, and interval from diagnosis totransplantation. The total of the points allocated to eachof the different criteria stratifies the patients in risk scoregroups of from zero to seven.The mean age of the evaluated patients in this studywas 33 years old (from 6 to 58 years), with 647 (59.7%)male and 437 (40.3%) female. At the time of transplantation,898 (82.8%) were in the chronic phase, 146 (13.5%) in theaccelerated phase and 40 (3.7%) in the blastic phase. Onehundred and fifty-one (13.9%) of the patients were under20 years old, 620 (57.2%) between 20 and 40 years old and313 (28.9%) were older than 40. The transplantation wasHLA-identical related in 1025 (94.6%) of the recipients andnon-related in only 59 (5.4%) of the cases. The donor-recipient gender combination was female into male in 283(26.1%) of the transplantations, whilst other combinationsoccurred in 801 (73.9%) cases. The interval from diagnosisto transplantation was less than 12 months in 223 (20.6%)patients and greater than 12 months in 861 (79.4%). In 179(16.6%) patients, the risk score was 0 or 1, in 379 (36.6%)the score was 2, in 345 (31.8%) it was 3, in 135 (12.5%) itwas 4 and in the remaining 28 cases (2.6%) the score was5 or 6. There were no cases with a score of 7.The overall survival rate was 49.23% at a median of67 months. The disease free survival rate was 50.36%, thetransplant-related death rate was 45.18% and the incidenceof relapse was 24.66%.Patients with risk scores of 0 and 1 or 2 in relation tothe overall survival rate did not present with significantdifferences (58.5% - 55.4%: Relative Risk 19). The overallsurvival rate was significant in relation to risk score of 3and greater (44.3%: Relative Risk 53). The criterion, diseasestage, was considered significant. The chronic phase gavean overall survival rate of 54.7%; the accelerated phase27.7% (relative risk of 81) and the overall survival rate inthe blastic phase was 14% (relative risk of 211). The donor-recipient gender combination was also significant with anoverall survival rate of 40.5% (relative risk 33) when thetransplant was female into male. Other combinationspresented with a rate of 52.4%. There were no significantdata of overall survival in relation to the other three criteria.The risk score was significant in respect to thedisease free survival, transplant-related death with scoresof 3 and higher, and incidence of relapse with scores of 5or 6 with values of 45.6%, 48.9% and 73.3% respectively.In isolation, the disease phase was significant in the threeparameters and donor-recipient gender combination onlyin relation to disease free survival and transplant-relateddeath. The results indicate that the potential of overallcure of patients submitted to transplantation isapproximately 50%, and that the EBMT score as it wasdesigned is a decision making tool for indication oftransplantation. Additionally, the results identify thedisease stage and the donor-recipient gender combinationas the two most important independent criteria. With theresults of this independent study, we can confirm that theEBMT risk score for chronic myeloid leukemia is valid andshould be used for clinical decision making whenindicating transplantation.

Amotosalen: Allogeneic Cellular Immunotherapies system, INTERCEPT Plasma System, INTERCEPT Platelet System, S 59.

Amotosalen: Allogeneic Cellular Immunotherapies system, INTERCEPT Plasma System, INTERCEPT Platelet System, S 59.

Comparison of pretransplant transfusion with or without cyclosporin inhibition on post transplant outcome

The likelihood of blood group compatible but HLA unmatched (random) donor blood to alter the outcome post transplant is controversial. Pediatric patients who require transfusion pretransplant may be sensitized. In an effort to analyze this risk we compared patients who received cyclosporin with transfusion to those transfused without calcineurin inhibition, and to patients non-transfused control patients. 51 infants (< 1 year) transplanted for complex congenital heart disease were included. Thirty transplanted infants (2.4 mo/old) did not receive a transfusion prior to transplant (Group A). Eleven infants (4.1 +/- 2.5 mo/old) received 1.6 transfusions prior to transplant (Group B) and 10 infants (3.9 +/- 1.7 mo/old) received 1.7 transfusions bracketed by 2 mg/kg of cyclosporin separated by 12 hours (Group C). Prior to transplant all received a similar induction protocol (either antithymocyte serum or antithymocyte globulin) post transplant. All patients received a similar immunosuppression (cyclosporin/Imuran, no steroids). The mean follow-up was 4 +/- 2.5 years in Group A, 6 +/- 1.7 years in Group B, and 2 +/- 1.5 years in Group C. The recipient blood type was similar in all three groups, as was blood type transfused (Groups B & C). The incidence of infection pretransplant and post-transplant was similar. There were 25 rejections in Group A (0.83 episodes/patient/year). In Group B, there were 10 episodes of rejection (0.91 episodes/patient/year), and in Group C, 6 episodes of rejection (0.6 episodes/patient and graft survival rate was similar in Groups A and B (76% versus 73%). Group C patients had 100% graft survival (p < 0.05). The duration of follow-up is longest in Groups A and B, which may influence the difference in graft survival. In summary patients transfused in the presence of calcineurin inhibition had the lowest rejection rate and highest survival. This suggests that this strategy should be evaluated further in patients requiring transfusion prior to orthotopic heart transplantation in infancy.