ALBERT

Description: Two young plantations of Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco) were examined for the presence of a symptomless endophytic fungus, Meriaparkeri Sherwood-Pike (Hemiphacideaceae). This fungus occurred in the needles of nearly every tree examined, with infection frequencies increasing with the age of the needle. Previous reports have asserted that M. parkeri is a mutualist that produces insecticidal toxins. The maternal parentage of the trees and age of the needles were highly significant predictors of infection frequencies. A detailed examination of two families of trees revealed that infection frequencies differed between sites and were positively correlated with the height of the host. Mutualism and microhabitat effects are discussed.

Description: The low yield of progenitor CD34+ cells recovered from umbilical cord blood (UCB) limits the utility of this source for transplantation in adults. This limitation has triggered investigations into how ex vivo expansion of hematopoietic stem cells (HSC) could be achieved to allow for transplantation in larger recipients. In the present study, the incubation of MNC and not selected CD34+cells in the presence of SCF 25ng/ml+MGDF 10ng/ml+FLt-3 25ng/ml+IL-6 20ng/ml, and 10% human serum in stroma-free liquid culture generated long-term expansion of transplantable UCB HSC. In vitro, HSC expansion from 13 UCB lasted 〉7 months giving 39, 1.3x104, and 4.7 x109 fold increase in total cell count after 14, 70 and 217 d of expansion as compared to d0 (105/ml). Similarly, CD34+ and CD34+/CD38− cell populations increased reaching 229 and 2.2x105 and 91 and 2.2x104 fold after 14 and 70d of expansion. Examination of cell morphology and analysis by flow cytometry showed the presence of primitive and mature cells belonging to all hematopoietic cell lineages. Similarly, multilineage colonies with recloning capacity were generated in culture. Erythroid, myeloid and mixed colonies increased by 116 and 1.8x104 fold and megakaryocytic colonies by 8 and 527 fold after 14 and 70d. Expanded cells were karyotypically normal and lacked the most common chromosome translocations seen in AML and CML t (15,17) and t (9, 22). HSC expanded for 6 and 13 weeks and cropreserved for 6–11 months were able to re-expand in liquid culture and generate colonies capable of recloning and multi-lineage differentiation. We estimated the frequency of SCID repopulating cells (SRC) in UCB samples expanded for 2 and 12 w using 1000,500,250 and 125 unselected CD34+ cells injected intravenously into sublethally irradiated NOD/SCID mice. Mice were sacrificed 20 weeks after transplantation. Human cell engraftment measured as CD45+ (HuCD45+) was detected in all mice (x3mice/dilution) (0.1–9.8%). In addition, HuCD45+ cells with multilineage phenotype were present, (CD19 (lymphoid), CD33 (myeloid), CD71and Glycophorin-A (erythroid) as well as CD34+/CD38− cells). SRC increased by 90 fold after 2 weeks of expansion and by 187 fold after 12 weeks compared to unexpanded CD34+cells. Additional proof of human cell engraftment was documented using semisolid culture (MethoCultTM GF H4434 Stem Cell Technologies). Human myeloid and erythroid colonies were generated from all dilutions, and counts ranged between 63–271/500,000 MNC. Initial studies to test the relative magnitude of UCB HSC expansion from 24-well plates to culture bags (OptiCyte TM, Baxter) using one UCB, the total cell count increased by (6.3 and 20 (bags) Vs 2.3 and 3.3 fold (wells) after 7 and 14d) and CD34+ subpopulations including CD34+/CD38−. Based on these ongoing results, a phase II clinical trial using ex vivo expanded UCB for 14d in a setting of sub ablative Conditioning is planned.

Description: Abstract 3533 Poster Board III-470 Umbilical Cord Blood (UCB) is a readily available, alternate donor source for allogeneic Hematopoietic Stem Cell (HSC) transplantation and can be used with lower stringency HLA-matching between donor and host. However, only limited numbers of cells are available and non-engraftment or delayed engraftment occurs often when recipients receive single UCB units that contain 〈 2.5 × 107 TNC/kg recipient body weight. This excludes single UCB as a donor source for many adult patients who might otherwise benefit from an UCB transplant. Both immunologic and non-immunologic mechanisms may contribute to UCB HSC engraftment failure. UCB T-cells are phenotypically and functionally immature with a high percentage of CD45RA+ T cells and T cells are known to facilitate engraftment in human HSC transplantation. Co-transplantation of irradiated T-cells supported engraftment in an immunocompetent mouse transplant model without increasing GvHD (Waller et al. Blood94: 3222,1999). The working hypothesis for his study is that the irradiated third party peripheral blood mononuclear cells (PBMNCs) induce a cytokine or cellular response that facilitates hematopoietic engraftment. Long-term culture initiating cell (LTC-IC) frequencies determined with limiting dilution assays were statistically higher when either purified UCB derived CD34+ cells or CB MNCs cells were plated with irradiated PBMNCs (Figure 1). Figure 1 Figure 1. To assess the impact of irradiated third party PBMCs on UCB engraftment we utilized sub-lethally irradiated NOD/SCID-IL2Rgnull mice in the presence and absence of irradiated third-party human PBMNCs. After sublethal irradiation (240 cGy) female 8 week-old animals were transplanted with either UCB MNCs corresponding to 8×103 (n=5) or 2.66×103 (n=9), or 8.88×102 CD34+ cells (n=9) with or without co-transplantation of 1×106 irradiated (2500 cGy) PBMNCs, and 4 animals received only irradiated PBMNCs as the control group. Engraftment levels are compared by calculating SCID repopulating cell (SRC) frequency. Co-transplantation of PB MNCs with UCB MNCs provided a steady, statistically significant increase in both short-term and long term hematopoietic engraftment (Figure 2). Figure 2 Figure 2. In these pre-clinical studies, we have confirmed in vitro effects on LTIC formation and in vivo UCB graft-enhancing effects of irradiated PBMNCs. If applied in clinic, this cellular therapy option might overcome a major barrier to successful allogeneic UCB HSC transplantation: engraftment failure, making the procedure more applicable and safer. Disclosures: No relevant conflicts of interest to declare.