An anti-CD44 antibody does not enhance engraftment of DLA-identical marrow after low-dose total body irradiation

920 cGy total body irradiation (TBI) is adequate for consistently successful engraftment of marrow from dog leukocyte antigen (DLA)-identical littermates; however, the dose is inadequate to ensure a marrow graft from DLA-nonidentical unrelated donors. Such mismatched grafts are successful only after 1800 cGy, given in three fractions. While anti-T-cell reagents enhance engraftment of DLA-identical littermate marrow after 920 cGy, they fail to be effective in the DLA-nonidentical setting. However, a monoclonal antibody (mAb) to CD44, S5, was found to be very effective in enhancing engraftment of DLA-nonidentical marrow. The current study asked whether mAb S5 was also effective in the setting of DLA-identical littermate transplants. To this purpose, the TBI dose was lowered to 450 cGy, a dose after which 70% of such grafts failed. Four dogs were treated with antibody S5, 0.2 mg/kg on days −7 though −2 (per previously published protocol), given 450 cGy TBI followed by marrow grafts from their DLA-identical littermates. All four dogs rejected their grafts; two of these died from marrow aplasia, and two survived with endogenous marrow recovery. This result was not statistically significantly different from that in 17, historical (n = 5) and concurrent (n = 12), control dogs where 11 of 17 animals rejected. Even if ten experimental animals were transplanted and all six remaining dogs engrafted, the results still would not have been significantly different from control. This result is in contrast to the successful engraftment promoted by pretreatment with antibody S5 of DLA-nonidentical unrelated dogs, consistent with the notion that different host cells are involved in graft rejection in the two disparate histocompatibility settings.     

Patellofemoral joint: kinematic MR imaging to assess tracking abnormalities

The patellofemoral joint was imaged with magnetic resonance (MR) in the axial plane while the knee was positioned from 0 degrees to 32 degrees of flexion (nine positions). These multiple sequential images obtained within the early phases of flexion of the knee were viewed in a "cine-loop" format, producing a kinematic study that clearly demonstrated the relationship of the patella to the trochlear groove. Four healthy subjects and one patient with known bilateral subluxing patellae were studied. The preliminary results suggest that kinematic MR imaging of the patellofemoral joint is potentially useful for the evaluation of patellar tracking abnormalities.     

Incidence and outcome of chronic graft-versus-host disease using National Institutes of Health consensus criteria.

Chronic graft-versus-host disease (cGVHD), a common complication after stem cell transplant (SCT), has an impact on morbidity and survival. Previous classification of cGVHD has not been reproducible or prognostic for nonrelapse mortality (NRM). Recently the National Institutes of Health (NIH) consensus criteria were proposed, but the ability of this classification to predict outcome of various subtypes of cGVHD is unknown. Patients (N = 110) undergoing an SCT for a hematologic malignancy and surviving until day 100 posttransplant from 2001 to 2003 were studied. The overall survival (OS) using a landmark analysis at day 100 was 44% versus 66% (no GVHD vs. GVHD, P = .026). The OS of patients with various types of GVHD as proposed by the NIH criteria were significantly different (P < .0001). In a univariate analyses, this was more apparent when patients with any acute features of GVHD were compared to classic cGVHD (3-year OS 46% vs. 68%, P = .033). The 3-year NRM for the entire cohort was 21%, and was not affected by presence or absence of GVHD or subtypes of GVHD. In a multivariable analysis, extensive cGVHD (hazard ratio [HR] 0.35, P = .015) and having any acute feature of GVHD after day 100 (HR 3.36, P = .0144) were significant independent predictors of survival. The OS with different NIH subtypes of GVHD after day 100 from SCT varies, and is superior for patients with classic cGVHD.     

Engraftment of dogs with Ia-positive marrow cells isolated by avidin- biotin immunoadsorption

Previous work has shown failure of engraftment in lethally irradiated dogs when autologous marrow was depleted of Ia-positive cells with an anti-Ia antibody and complement before infusion. In the current study, we have utilized an avidin-biotin immunoadsorption procedure to obtain a population of highly enriched Ia-positive cells for autologous bone marrow transplantation in dogs given lethal irradiation. Dog marrow cells (2.4 to 7.0 X 10(9) cells) that contained 8.6% to 19.9% Ia- positive cells were treated successively with monoclonal antibody 7.2, which reacts with a framework determinant of Ia-antigen, and biotin- conjugated goat antimouse immunoglobulin. These treated cells were passed over a column of avidin-Biogel (polyacrylamide) and the adherent cells removed by mechanical agitation. Seven lethally irradiated dogs were transplanted with 5.9 to 33.4 X 10(6) recovered adherent cells per kilogram of which 69.0% to 88.0% were Ia-positive. All dogs had hematologic recovery; six are alive and well with durable engraftment and one died on day 15 posttransplant. They are immunologically normal as determined by lymph node and bone marrow biopsies, lymphocyte function, and immunophenotyping of peripheral blood and bone marrow cells. These data provide further evidence that canine hematopoietic stem cells express Ia-like antigens and that these cells are capable of complete hematopoietic and immunologic reconstitution in an autologous model.     

Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector

Retroviral-mediated gene transfer is the most attractive modality for gene transfer into hematopoietic stem cells. However, transduction efficiency has been low using amphotropic Moloney murine leukemia virus (MoMLV) vectors. In this study, we investigated modifications of gene transfer using amphotropic MoMLV vectors in cell-free supernatant for their ability to increase the currently low transduction of both committed hematopoietic progenitors, granulocyte-macrophage colony- forming units (CFU-GMs), and their precursors, long-term culture- initiating cells (LTC-IC). First, based on the observation that bone marrow cells express more gibbon ape leukemia virus (GALV) receptor (Glvr-1) than amphotropic receptor (Ram-1), PG13/LN, which is a MoMLV vector pseudotyped with the GALV envelope, was compared with the analogous amphotropic envelope vector (PA317/LN). Second, progenitor cell transduction efficiency was compared between CD34 enriched and nonenriched progenitor populations. Third, the duration of transduction in vitro was extended to increase the proportion of progenitor cells that entered cell cycle and could thereby integrate vector cDNA. In 20 experiments, 1 x 10(6) marrow or peripheral blood mononuclear cells (PBMCs)/mL were exposed to identical titers of pseudotyped PG13/LN vector or PA317/LN vector in the presence of recombinant human interleukin-1 (IL-1), IL-3, IL-6, and stem cell factor (SCF; c-kit ligand) for 5 days. 50% of fresh vector supernatant was refed daily. Hematopoietic progenitor cells as measured by G418-resistant granulomonocytic colony (CFU-GM) formation were transduced more effectively with PG13/LN (19.35%) than with PA317/LN (11.5%, P = .012). In 11 further experiments, enrichment of CD34 antigen positive cells significantly improved gene transfer from 13.9% G418-resistant CFU-GM in nonenriched to 24.9% in CD34-enriched progenitor cells (P < .01). To analyze gene transfer after extended growth factor-supported long-term culture, 1 x 10(6) marrow cells/mL were cultured with IL-1, IL-3, IL-6, and SCF (50 ng/mL each) for 1, 2, and 3 weeks. Fifty percent of PG13/LN supernatant with growth factors was refed on 5 days per week. Five percent of marrow CFU-GM and 67% of LTC-IC were G418 resistant at 1 week (n = 4), 60% of CFU-GM and 100% of LTC-IC were resistant at 2 weeks (n = 2) and 74% of CFU-GM (n = 4) and 82% of LTC-IC (n = 2) were resistant at three weeks.(ABSTRACT TRUNCATED AT 400 WORDS)     

Myelosuppressive conditioning improves autologous engraftment of genetically marked hematopoietic repopulating cells in dogs

We have studied the role of different conditioning regimens for engraftment of genetically marked hematopoietic repopulating cells in dogs. Peripheral blood (PB) and/or marrow cells collected after treatment with recombinant canine stem cell factor (rcSCF) or cyclophosphamide were transduced in a vector-containing long-term culture system. Three different vector-producing cell lines with similar viral titers were used. In two of them, the neo-containing LN vector was packaged either in the PA317 cell line with an amphotropic murine retrovirus envelope or the PG13 cell line with the gibbon ape leukemia virus (GALV) envelope. The MFG/GC vector produced in PA317 cells contained the human glucocerebrosidase gene. Nineteen dogs received either no conditioning (group A, n = 5), irradiation to both humeri with 1,000 cGy (group B, n = 5), a sublethal dose of cyclophosphamide 40 mg/kg (group C, n = 4), a sublethal dose of 200 or 300 cGy total body irradiation (TBI) (group D, n = 3), or an otherwise lethal dose of 920 cGy TBI (group E, n = 3) before intravenous (groups A, C, D, E) or intramedullary (group B) infusion of the transduced autologous hematopoietic cells. Transduction efficiency of hematopoietic cells at the time of infusion into the animals was similar among the different conditioning groups. Dogs were observed for at least 6 months. PB granulocytes were obtained at least every 3 weeks after transplant and analyzed by polymerase chain reaction for the presence of the transduced genes. The percentages of positive results in dogs more than 4 weeks after transplantation were 0% without conditioning, 5% with local irradiation, 18% with sublethal cyclophosphamide, 33% with sublethal TBI, and 17% with otherwise lethal TBI. Analyzing the influence of conditioning regimens by a generalized estimating equation (GEE) technique, which considered the use of different retrovirus vectors and the number of mononuclear cells infused as potential confounding variables, we found that engraftment of genetically marked repopulating cells was significantly improved (P < .001) in dogs receiving systemic conditioning with either otherwise lethal TBI, sublethal TBI, or sublethal cyclophosphamide compared to dogs with local irradiation only or no conditioning. Within the limitation of the experimental design, these data suggest that myeloablative or myelosuppressive conditioning improves engraftment of genetically marked hematopoietic repopulating cells.     

DLA-identical bone marrow grafts after low-dose total body irradiation: the effect of canine recombinant hematopoietic growth factors

Previous studies found that bone marrow (BM) allografts from DLA- identical littermates resulted in survival of two thirds of recipient dogs after otherwise lethal doses of 450 to 600 cGy of total body irradiation (TBI) because of successful allografts or autologous recovery after rejection of the allografts. The current study asked whether survival could be further improved by treating allograft recipients with recombinant canine granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), or G-CSF/SCF. Of 21 dogs, 14 (67%) receiving allografts but no growth factors survived, 10 with successful allografts (including 5 mixed chimeras) and 4 with autologous recovery; whereas 7 animals died, 5 from infections during BM aplasia and 2 from acute graft-versus-host disease. By comparison, 30 of 34 dogs (88%) receiving hematopoietic growth factors in addition to the BM graft survived, 17 with successful allografts (including 10 mixed chimeras) and 13 with autologous recovery; whereas 4 died, all with infection related to BM aplasia after rejection of the allograft. Survival was similar for recipients of G-CSF, SCF, or the combination of G-CSF and SCF. Logistic regression analyses, which accounted for possible effects of TBI dose, showed a trend for improved survival in dogs receiving growth factors (P = .09), no change in allogeneic engraftment (P = .74), and a slight increase in autologous recovery (P = .22). In agreement with previous data, we found that grafts of BM from DLA-identical littermates improved survival of recipient dogs exposed to low but otherwise lethal doses of TBI. A further improvement in survival could be achieved by additional treatment with G-CSF, SCF, or G-CSF/SCF. Results suggest that treatment by hematopoietic growth factors along with BM grafts should be considered for victims of radiation accidents.