Fetal islet xenotransplantation in rodents and primates

(E)-2'-deoxy-2'-(fluoromethylene) cytidine (FMdC), a novel inhibitor of ribonucleotide-diphosphate reductase, has been shown to have anti-tumor activity against solid tumors and sensitize tumor cells to ionizing radiation. Pentoxifylline (PTX) can potentiate the cell killing induced by DNA-damaging agents through abrogation of DNA-damage-dependent G2 checkpoint. We investigated the cytotoxic, radiosensitizing and cell-cycle effects of FMdC and PTX in a human colon-cancer cell line WiDr. PTX at 0.25-1.0 mM enhanced the cytotoxicity of FMdC and lowered the IC50 of FMdC from 79 +/- 0.1 to 31.2 +/- 2.1 nM, as determined by MTT assay. Using clonogenic assay, pre-irradiation exposure of exponentially growing WiDr cells to 30 nM FMdC for 48 hr or post-irradiation to 0.5 to 1.0 mM PTX alone resulted in an increase in radiation-induced cytotoxicity. Moreover, there was a significant change of the radiosensitization if both drugs were combined as compared with the effect of either drug alone. Cell-cycle analysis showed that treatment with nanomolar FMdC resulted in S-phase accumulation and that such an S-phase arrest can be abrogated by PTX. Treatment with FMdC prior to radiation increased post-irradiation-induced G2 arrest, and such G2 accumulation was also abrogated by PTX. These results suggest that pharmacological abrogation of S and G2 checkpoints by PTX may provide an effective strategy for enhancing the cytotoxic and radiosensitizing effects of FMdC.     

The alloimmune thrombocytopenic syndromes

We have summarized five thrombocytopenic syndromes caused by platelet-reactive alloantibodies. Increased awareness of these five syndromes, together with the greater availability of highly-specialized laboratory methods to detect and to characterize platelet-reactive alloantibodies, will lead to their more frequent diagnosis. It is important for the clinician to consider unusual alloimmune thrombocytopenic disorders in clinical settings in which alloantigens that could be limited to just one family could cause important disease (ie, NAT caused by a private alloantigen; theoretically, PAT or TAT related to directed donations of blood or bone marrow). These considerations underscore the need for serological investigations to involve the family members rather than to rely on standard platelet typing donor pools.     

Brain abscess in solid organ transplant recipients receiving cyclosporine-based immunosuppression

There is currently a stark therapeutic void in the treatment of evolving stroke. Although P-selectin is rapidly expressed by hypoxic endothelial cells in vitro, the functional significance of P-selectin expression in stroke remains unexplored. In order to identify the pathophysiological consequences of P-selectin expression and to identify P-selectin blockade as a potential new approach for the treatment of stroke, experiments were performed using a murine model of focal cerebral ischemia and reperfusion. Early P-selectin expression in the postischemic cerebral cortex was demonstrated by the specific accumulation of radiolabeled anti-murine P-selectin IgG, with the increased P-selectin expression localized to the ipsilateral cerebral microvascular endothelial cells by immunohistochemistry. In experiments designed to test the functional significance of increased P-selectin expression in stroke, neutrophil accumulation in the ischemic cortex of mice expressing the P-selectin gene (PS +/+) was demonstrated to be significantly greater than that in homozygous P-selectin-null mice (PS -/-). Reduced neutrophil influx was accompanied by greater postischemic cerebral reflow (measured by laser Doppler) in the PS -/- mice. In addition, PS -/- mice demonstrated smaller infarct volumes (5-fold reduction, P<.05) and improved survival compared with PS +/+ mice (88% versus 44%, P<.05). Functional blockade of P-selectin in PS +/+ mice using a monoclonal antibody directed against murine P-selectin also improved early reflow and stroke outcome compared with control mice, with reduced cerebral infarction volumes noted even when the blocking antibody was administered after occlusion of the middle cerebral artery. These data are the first to demonstrate a pathophysiological role for P-selectin in stroke and suggest that P-selectin blockade may represent a new therapeutic target in the treatment of stroke.     

The role of oncostatin M in animal and human connective tissue collagen turnover and its localization within the rheumatoid joint

OBJECTIVE: To study the interaction of interleukin-1alpha (IL-1alpha) and oncostatin M (OSM) in promoting cartilage collagen destruction. METHODS: Bovine, porcine, and human cartilage and human chondrocytes were studied in culture. The levels of collagenase (matrix metalloproteinase 1 [MMP-1]) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by bioassay and enzyme-linked immunosorbent assay (ELISA). The levels of OSM in rheumatoid synovial fluid were measured by ELISA. RESULTS: When combined with OSM, IL-1alpha, IL-1beta, and tumor necrosis factor alpha released proteoglycan and collagen from cartilage. OSM was the only member of the IL-6 family to have this effect. Human tendon also responded to IL-1alpha and OSM. OSM increased the production of MMP-1 and TIMP-1 but when combined with IL-1alpha, synergistically promoted MMP-1 production in human chondrocytes and synovial fibroblasts. High levels of OSM were found in human rheumatoid synovial fluids, and confocal microscopy showed that OSM was produced by macrophages in rheumatoid synovial tissue. CONCLUSION: These results highlight an important new mechanism by which there is irreversible loss of collagen from cartilage.     

Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine

Plasmodium falciparum malaria parasites were transformed with plasmids containing P. falciparum or Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (dhfr-ts) coding sequences that confer resistance to pyrimethamine. Under pyrimethamine pressure, transformed parasites were obtained that maintained the transfected plasmids as unrearranged episomes for several weeks. These parasite populations were replaced after 2 to 3 months by parasites that had incorporated the transfected DNA into nuclear chromosomes. Depending upon the particular construct used for transformation, homologous integration was detected in the P. falciparum dhfr-ts locus (chromosome 4) or in hrp3 and hrp2 sequences that were used in the plasmid constructs as gene control regions (chromosomes 13 and 8, respectively). Transformation by homologous integration sets the stage for targeted gene alterations and knock-outs that will advance understanding of P. falciparum.