ALBERT

Description: We evaluated the toxicity-profile, engraftment potential, and efficacy of fludarabine-based nonmyeloablative allogeneic HCT in patients with a variety of nonmalignant hematological disorders. Twenty three patients (median age 29 years; range 11–52) with nonmalignant hematological disorders including ATG refractory SAA (n=13), severe paroxysmal nocturnal hemoglobinuria (PNH: n=9), and pure red cell aplasia (PRCA; n=1) were transplanted from 5/99 – 8/2004 at the NHLBI. The majority of patients had an extensive transfusion history including 11/23 who had HLA allo-antibodies and 4/23 with allo-antibodies to RBCs. Conditioning with fludarabine (25 mg/m2 x 5 days), ATG (40mg/kg x 4 days) and cyclophosphamide (60mg/kg x 2 days) was followed by infusion of an un-manipulated G-CSF mobilized allograft from an HLA matched sibling (n=18), parent (n=2), or single antigen mismatched sibling (n=3). GVHD prophylaxis consisted of cyclosporine (CSA) either alone (n=2) or combined with mycophenolate mofetil (n=10) or mini-dose methotrexate (n=11). Despite a high prevalence of pre-transplant allo-immunization, all patients achieved sustained donor engraftment in both myeloid and T-cell lineages. Myeloid recovery (neutrophils 〉500cells/uL) occurred at a median 14 days post transplant (range 8–18 days). Conversion from mixed to full donor myeloid and T-cell chimerism occurred in all patients by 110 days post-transplant. CMV reactivation occurred in 11/21 patients at risk (KM probability 52%) without any cases of CMV disease. Grade II–IV and III–IV acute GVHD was the major transplant complication occurring in 13/23 (KM probability 60%) and 8/23 (KM probability 38%) patients respectively. Fourteen of 21 evaluable patients developed chronic GVHD (limited in 11 cases), which resolved completely with low-dose alternate day steroids and/or CSA in all but 1 case. One patient who received an allograft from his HLA matched father died 16 months post-transplant from complications related to chronic GVHD. With a median follow up of 25 months (range 1–64 months), 20/21 patients evaluable more than 100 days post-transplant survive in complete remission with full donor chimerism in all lymphohematopoietic lineages (KM probability of long-term survival 92.8 %-see figure ). Conclusion: Fludarabine-based nonmyeloablative transplantation achieves excellent donor engraftment and long-term disease free survival in heavily transfused and allo-immunized patients with ATG refractory SAA and other nonmalignant hematological disorders associated with bone marrow failure.

Description: Isolated chronic thrombocytopenia (CT) following allogeneic HCT is associated with an increased risk of transplant-related mortality and a reduction in long-term survival. Although the pathogenesis of CT is not entirely clear, it has been speculated that immune activation and cytokine dysregulation characteristic of chronic GVHD (CGVHD) play a similar role in the pathogenesis of this disorder. To better understand the impact of endogenous cytokine expression on platelets, we analyzed pro and anti-inflammatory cytokines in the serum of patients with CT following allogeneic HCT, comparing levels to healthy donors and to patients with normal platelet counts after allo-transplantation. Serum was obtained from 3 patient groups; Group 1- healthy donor controls (n=12): Group 2- post-transplant from allogeneic HCT patients with normal platelet counts (n=31): Group 3- post transplant from patients with CT (n=11). Using micro sphere-based Luminex flow cytometry, serum was analyzed for levels of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α as well as Th(1) cytokines IL1-b, IL-2, IL-12, IFN-g and Th(2) cytokines IL-4, IL-5, and IL-10. Results: Both post transplant cohorts (Group 2 and 3) had higher mean levels of GM-CSF (110 and 120 pg/ml), IFN-g (17 and 16 pg/ml), and IL-12 (318 and 202 pg/ml) compared to their respective non-transplant (Group-1) controls (GM-CSF 23 pg/ml: p

Description: The lymphatic vasculature preserves tissue fluid balance by absorbing fluid and macromolecules and transporting them to the blood vessels for circulation. The stepwise process leading to the formation of the mammalian lymphatic vasculature starts by the expression of the gene Prox1 in a subpopulation of blood endothelial cells (BECs) on the cardinal vein (CV) at approximately E9.5. These Prox1-expressing lymphatic endothelial cells (LECs) will exit the CV to form lymph sacs, primitive structures from which the entire lymphatic network is derived. Until now, no conclusive information was available regarding the cellular processes by which these LEC progenitors exit the CV without compromising the vein's integrity. We determined that LECs leave the CV by an active budding mechanism. During this process, LEC progenitors are interconnected by VE-cadherin–expressing junctions. Surprisingly, we also found that Prox1-expressing LEC progenitors were present not only in the CV but also in the intersomitic vessels (ISVs). Furthermore, as LEC progenitors bud from the CV and ISVs into the surrounding mesenchyme, they begin expressing the lymphatic marker podoplanin, migrate away from the CV, and form the lymph sacs. Analyzing this process in Prox1-null embryos revealed that Prox1 activity is necessary for LEC progenitors to exit the CV.

Description: AMD3100 (AMD) is a bicyclam compound that inhibits the binding of stromal cell derived factor-1 to its cognate receptor CXCR4 present on CD34+ hematopoetic progenitor cells. Recently, investigators have shown that substantial numbers of CD34+ cells are released into circulation following a single injection of AMD, making this a potentially attractive mobilization agent for both autologous and allogeneic hematopoietic cell transplantation. Mononuclear cells transplanted in G-CSF (G) mobilized allografts mediate both desirable and undesirable post-transplant events (i.e. Graft-vs-tumor and GVHD); therefore, to determine the suitability of AMD mobilized products for allografting, we assessed the cellular content of apheresis collections obtained from the same donors mobilized with AMD vs G. Between 11/03–07/04, 6 healthy donors (male=3, female=3), median age 43 years (range 18–57), underwent a 15–25 liter apheresis following G mobilization (10mcg/kg/d x 5 days); 66–143 (median 82) days later, the same donors underwent repeat apheresis 6 hours following a single subcutaneous injection of AMD (240mcg/kg); the apheresis blood volume processed after AMD mobilization was matched to the volume processed after G mobilization in 5/6 donors. Data on peripheral blood (PB) and apheresis cellular content with both mobilization agents are shown in Table-1. AMD was well tolerated (no 〉 grade I toxicities) and effectively mobilized CD34+ cells in the majority of donors who had a previous successful G mobilization. Both drugs significantly increased PB CD34+ counts and the total WBC count, and absolute neutrophil counts (ANC), monocyte counts (AMC) and lymphocyte counts (ALC) above pre-mobilization baselines. In the PB, the increase in WBC count, ANC, and the CD34+ counts were significantly higher after G mobilization compared to AMD. In contrast, there was a trend towards a higher blood ALC increase following AMD administration compared to G. Apheresis collections mobilized with AMD contained similar numbers of mononuclear cells and CD3+ T-cells, higher numbers of CD19+ B-cells and lower numbers of monocytes and CD34+ cells compared to G mobilized collections; whether prior G mobilization negatively impacted the CD34+ cell content in AMD mobilized grafts can not be determined from this study. One patient failed mobilization with both G (CD34+ pre-count 6 /uL) and AMD (CD34+ pre-count 6 /uL); a trial investigating the efficacy of combining AMD with G in patients who fail to mobilize with G alone is currently being pursued. A detailed phenotypic analysis of lymphocyte subsets mobilized with G vs AMD3100 will be presented in a separate analysis.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder of hematopoietic stem cells characterized by RBC susceptibility to complement-mediated lysis. Infections related to neutropenia, bleeding associated with thrombocytopenia, and thrombosis all contribute to morbidity and mortality. Although allogeneic hematopoietic cell transplantation (HCT) can be curative, the high-risk of treatment-related mortality with myeloablative HCT precludes this approach for most patients with severe disease. We previously reported in vitro and in vivo data showing PNH cells could be killed by allo-reactive donor T-cells recognizing minor histocompatibility antigens expressed on both normal and GPI negative cells. Here we present updated data on a cohort of 11 patients with severe PNH who received a NST at the NHLBI from 5/99 through 6/2004. Eligibility included a diagnosis of PNH associated with one or more of the following :1) Transfusion dependence (n=9) 2) Prior thrombotic episodes (n=4) 3) Recurrent debilitating hemolytic crisis (n=7). Patients received a T-cell replete G-CSF mobilized blood stem cell transplant from an HLA-matched family donor following nonmyeloablative conditioning with cyclophosphamide (120mg/kg) and fludarabine (125mg/m2). Patients with a significant transfusion history had horse ATG (40mg/kg/day x 4) added to the conditioning regimen (n=9). CSA either alone (n=1) or combined with either MMF (n=4) or mini-dose methotrexate (n=6) was used as GVHD prophylaxis. The median % of GPI anchored protein negative neutrophils pre-transplant was 83% (range 13%–99%). Blood samples obtained post-transplant were analyzed by FACS to determine the percentage of persisting GPI negative neutrophils (CD15+/CD66b−/CD16−). Chimerism was also assessed post-transplant in T-cell and myeloid fractions by PCR assay of polymorphic short tandem repeats (STR). Neutrophil recovery occurred at a median 15 days (range 10–19). STR analysis revealed donor engraftment occurred in both myeloid and T-cell lineages in all patients. Self-limiting febrile hemolytic reactions associated with ATG administration (6/9 patients) and grade II-IV acute GVHD (n=5) were the most common complications associated with transplantation. With a median follow-up of 458 days (range 31–1917), all patients survive either in remission (n=8) or with declining GPI negative populations (n=3); GPI negative neutrophils were detected in all patients at engraftment but gradually declined until no longer detectable (

Description: G-CSF is currently the preferred agent to mobilize peripheral blood stem cells (PBSC) for allogeneic hematopoietic cell transplantation. AMD3100, a selective antagonist of SDF-1, has recently proven to rapidly mobilize hematopoietic stem cells in both humans and mice. It is currently unknown whether GVHD will differ in recipients of T-cell replete allografts mobilized with AMD3100 versus G-CSF. Therefore, we investigated the effects of AMD3100 on GVHD in a murine model of PBSC transplantation in which Balb/c recipients received 15x106 splenocytes from allogeneic MHC matched B10.d2 mice following 950cGy of irradiation. Splenocytes from donor mice were harvested six hours after a single subcutaneous injection of AMD3100 (100μg). Controls consisted of Balb/c recipients of splenocytes harvested after five daily subcutaneous injections of G-CSF (10μg) or saline. In addition, one Balb/c cohort received splenocytes mobilized with the combination of G-CSF and AMD3100. Significantly higher numbers of stem cells (KLS cells; cKit+, lineage-, sca-1+) were mobilized after AMD3100 compared to saline controls (mean=32,300±3,900 vs. 14,700±4,900; p=0.02). The absolute number of KLS cells was significantly lower after AMD3100 mobilization compared to G-CSF (mean=52,400±8,600; p=0.03). No difference in number of KLS cells was observed in mice mobilized with AMD3100 and G-CSF compared to G-CSF alone. Splenocytes from G-CSF mobilized B10.d2 donors contained a significantly (p