Caspase-8 dependent apoptosis induction in malignant myeloid cells by TLR stimulation in the presence of IFN-alpha.

Pro-apoptotic signalling upon toll-like receptor (TLR) stimulation in myeloid cells is normally antagonized by the simultaneous activation of anti-apoptotic pathways. We have previously reported that IFN-alpha can sensitize human monocytes to apoptosis induction by lipopolysaccharide (LPS). Based on these results we investigated whether similarly apoptosis can be cooperatively induced in myeloid tumor cells. When testing established acute myeloid leukemia (AML) cell lines we found the monocytic cell line THP-1 to be sensitive to IFN-alpha plus LPS induced apoptosis, which was partially dependent on caspase-8 and was associated with an enhanced expression of Fas/CD95. We extended our study to 29 short term blast lines from patients with AML and observed additive effects of IFN-alpha and LPS on cell death only with few samples indicating that sensitivity to IFN-alpha plus LPS inducible apoptosis is present in a fraction of AML samples only with no obvious correlation with certain FAB phenotypes.

Use of highly sensitive mitochondrial probes to detect microchimerism in xenotransplantation models.

Chimerism, defined as the co-existence of cells of different origin within the same organism, has received much attention in hematopoietic cell and organ transplantation because of the strict relationship between its establishment and the induction of specific tolerance. Traditional methods applied for chimerism detection, such as immunohistochemistry, cytogenetics, fluorescent-activated cell sorter analysis, and serological and biochemical testing, are limited by their sensitivity. We have established a highly sensitive molecular approach based on the amplification of the mitochondrial cytochrome B gene and tested its specificity and sensitivity level in six different mammalian species, including human, pig, mouse, rat, sheep and rabbit. Increased sensitivity of detection of specific amplification products was obtained by the non-radioactive Southern blot technique. This novel approach allows the detection of one cell against the background of 1 to 4 x 10(6) xenogenec cells and will be helpful for high-sensitivity analysis of donor cell engraftment after xenotransplantation procedures in these animal models.

Conditioning of neonatal pigs using low-dose chemotherapy and murine fetal tissue before murine hybridoma transplantation.

Immunosuppression, myeloablation, and the use of immunologically immature tissue can overcome major histocompatibility complex barriers by inducing tolerance. With the goal of inducing tolerance to BALB/c-derived murine hybridoma cells producing the 4C6 monoclonal antibody (mAb), we transplanted BALB/c fetal tissue into neonatal pigs during a regimen of low-dose conditioning with busulfan and cyclophosphamide. After the tolerance induction phase, animals received intraperitoneal injections of 4C6 mAb hybridoma cells. Evidence of persistence of injected cells over time was sought by molecular analysis of peripheral blood for the presence of mouse genomic sequences and circulating 4C6 mAb. Persistence of donor polymerase chain reaction signal during the entire duration of the study, detectable mAb titer for 6 weeks, and a twofold increase of mAb concentration after a boost hybridoma infusion was observed in one animal receiving six consecutive administrations of the conditioning regimen. Our model has the distinctive advantage of allowing functional monitoring of engrafted cells for studying tolerance induction strategies. In addition, this model could be the basis for approaches aimed at producing mAbs in tolerized large animals.

Engraftment potential of human amnion and chorion cells derived from term placenta.

BACKGROUND: Fetal membranes are tissues of particular interest for several reasons, including their role in preventing rejection of the fetus and their early embryologic origin. which may entail progenitor potential. The immunologic reactivity and the transplantation potential of amnion and chorion cells, however, remain to be elucidated. METHODS: Amnion and chorion cells were isolated from human term placenta and characterized by immunohistochemistry, flow cytometric analysis, and expression profile of relevant genes. The immunomodulatory characteristics of these cells were studied in allogeneic and xenogeneic mixed lymphocyte reactions and their engraftment potential analyzed by transplantation into neonatal swine and rats. Posttransplant chimerism was determined by polymerase chain reaction analysis with probes specific for human DNA. RESULTS: Phenotypic and gene expression studies indicated mesenchymal stem cell-like profiles in both amnion and chorion cells that were positive for neuronal, pulmonary, adhesion, and migration markers. In addition, cells isolated both from amnion and chorion did not induce allogeneic nor xenogeneic lymphocyte proliferation responses and were able to actively suppress lymphocyte responsiveness. Transplantation in neonatal swine and rats resulted in human microchimerism in various organs and tissues. CONCLUSIONS: Human amnion and chorion cells from term placenta can successfully engraft neonatal swine and rats. These results may be explained by the peculiar immunologic characteristics and mesenchymal stem cell-like phenotype of these cells. These findings suggest that amnion and chorion cells may represent an advantageous source of progenitor cells with potential applications in a variety of cell therapy and transplantation procedures.