Biomedical subjects

Robert W Georgantas

Publications and source records attributed to Robert W Georgantas.

5 recordsLinked to original sources

PubMed2006

Ex vivo soluble fas ligand treatment of donor cells to selectively reduce murine acute graft versus host disease.

BACKGROUND: Allogeneic bone marrow transplantation (BMT) and donor lymphocyte infusion (DLI) provide valuable treatments for a range of diseases. However, the therapeutic utility of BMT and DLI is reduced by the high incidence of graft-versus-host disease (GvHD) mediated by activated donor T lymphocytes directed against recipient alloantigens. METHODS: Using mouse models, we developed and evaluated a strategy to selectively enhance activation-induced cell death (AICD) of anti-recipient T cells within transplant donor cell populations, with the goal of reducing GvHD. Responder T lymphocytes were incubated ex vivo with irradiated allogenic stimulator cells in a mixed lymphocyte reaction (MLR) in the presence of soluble Fas ligand (sFasL) to induce AICD in alloreactive cells. RESULTS: This ex vivo sFasL treatment reduced proliferation to the allogeneic stimulator cells in vitro and abrogated acute GvHD capacity in vivo. In contrast, the secondary immune responsiveness of the ex vivo sFasL-treated responder T cells to an unrelated model antigen was preserved. Furthermore, upon adoptive transfer in a DLI model, ex vivo sFasL-treated T cells were able to reject a model tumor. Finally, ex vivo sFasL treatment of bone marrow cells did not reduce their hematopoietic engraftment capacity. CONCLUSIONS: Thus, ex vivo treatment with sFasL appears to have potential for translation to clinical cell processing of BMT allografts and DLI infusions.

Acute Disease↗

PubMed2005

TAGmapper: a web-based tool for mapping SAGE tags.

Serial Analysis of Gene Expression (SAGE) is an important means of obtaining quantitative information about expression of genes in different samples. Short SAGE tags are 10 nucleotides long and often contain enough information to uniquely identify the gene(s) corresponding to the tag. We have observed, however, that the currently available resources are not adequate for accurate mapping of all SAGE tags to genes. Here, we describe development of a web-based tool called TAGmapper (http://tagmapper.ibioinformatics.org), which provides a comprehensive and accurate mapping of SAGE tags to genes. We were able to map SAGE tags accurately in several instances where two other popular resources, SAGEmap (http://www.ncbi.nlm.nih.gov/projects/SAGE/) and SAGE Genie (http://cgap.nci.nih.gov/SAGE), provided incorrect or no assignment of tags to genes. Finally, we experimentally determined the expression of a subset of genes assigned by TAGmapper using DNA microarrays and/or quantitative PCR to confirm the reliability of the gene mappings. We anticipate that TAGmapper will be a useful tool in functional genomic approaches by providing accurate identification of genes in SAGE experiments.

Base Sequence↗

PubMed2004

Microarray and serial analysis of gene expression analyses identify known and novel transcripts overexpressed in hematopoietic stem cells.

The human CD34(+)/CD38(-)/Lin(-) cell subset, comprising approximately 1-10% of the CD34(+) cell population, contains few of the less primitive hematopoietic (lineage-committed) progenitor cells (HPCs) but most of the primitive in vivo engrafting (lympho-)hematopoietic stem cells (HSCs). We analyzed gene expression in CD34(+)/CD38(-)/Lin(-) cell populations isolated from normal human adult donor bone marrow, neonatal placental/umbilical cord blood, and mobilized adult donor peripheral blood stem-progenitor cells. As measured by Affymetrix microarrays, 4746 genes were expressed in CD34(+)/CD38(-)/Lin(-) cells from all three tissues. We also determined the transcriptomes of the stem cell-depleted, HPC-enriched CD34(+)/[CD38/Lin](++) cell population from each tissue. Comparison of CD34(+)/CD38(-)/Lin(-) (HSC-enriched) versus CD34(+)/[CD38/Lin](++) (HPC-enriched, HSC-depleted) cells from each tissue yielded 81 genes overrepresented and 90 genes underrepresented, common to all three of the CD34(+)/CD38(-)/Lin(-) cell populations. These transcripts, which are selectively expressed in HSCs from all three tissues, include a number of known genes (e.g., transcription factors, receptors, and signaling molecules) that might play roles in key functions (e.g., survival, self-renewal, differentiation, and/or migration/adhesion) of human HSCs. Many genes/transcripts of unknown function were also detected by microarray analysis. Serial analysis of gene expression of the bone marrow HSC and HPC populations confirmed expression of most of the overrepresented transcripts for which reliable serial analysis of gene expression tags were detected and additionally suggested that current microarrays do not detect as many as 30% of the transcripts expressed in HSCs, including a number of previously unknown transcripts. This work is a step toward full definition of the transcriptome of normal human HSCs and may identify new genes involved in leukemogenesis and cancer stem cells.

ADP-ribosyl Cyclase↗

PubMed2003

Kinetics of CCR7 expression differ between primary activation and effector memory states of T(H)1 and T(H)2 cells.

We explored the kinetics of CCR7 expression on T(H)1 and T(H)2 polarized cells as well as on antigen-specific T cell lines at various stages of differentiation. A striking pattern of early (days 7-14) inducible CCR7 expression was seen preferentially on primary T(H)1 cell lines, as compared to T(H)2 cells, and was dependent on the strength and duration of the T cell receptor signal. Upon repeated restimulation (days 21-28) and differentiation, a switch occurred in which T(H)2 cells had high CCR7 expression, whereas T(H)1 cells lost CCR7 expression. Chronic (8 weeks and later) effector memory cell lines were 95% CCR7 negative. These data demonstrate an ordered pattern of CCR7 expression that suggest more rapid priming of T(H)1 cells in the lymph node, and delayed priming with prolonged CCR7 expression during T(H)2 responses, and may have implications for tracking T(H)1 effector T cells ex vivo in autoimmune diseases.

Antigen Presentation↗

PubMed2002

Human CD34+ hematopoietic stem/progenitor cells express high levels of FLIP and are resistant to Fas-mediated apoptosis.

We sought to determine whether lympho-hematopoietic stem-progenitor cells (HSC) from human placental/umbilical cord blood (CB) or adult mobilized blood (PBSC) are sensitive to Fas-induced apoptosis. Human CD34+ cells from CB or PBSC were cultured in serum-free medium, with or without hematopoietic growth factors (FKT: FLT-3 ligand [FL], KIT ligand [KL], and thrombopoietin [TPO]), and with or without soluble Fas ligand (sFasL) or agonistic anti-Fas antibody. After 5-48 hours of culture, cells were assessed for viability and stained with Annexin V and 7-Aminoactinomycin D for apoptosis analysis by fluorescence-activated cell sorting. Cultured cells were also assessed by in vitro hematopoietic colony-forming cell (CFC) and in vivo nonobese diabetic/severe combined immunodeficient mouse engraftment potential (SEP) assays. Levels of Fas, FLICE inhibitory protein (FLIP), and Caspase 8 mRNA in CD34+ cells were determined by real-time quantitative polymerase chain reaction. Expression of FLIP was confirmed by Western blotting. No decrease in viability, CFC, or SEP was observed in CB or PBSC CD34+ cells cultured in the presence of sFasL or agonistic anti-Fas antibody. Human CB and mobilized PBSC CD34+ cells expressed high levels of FLIP, low ratios of Caspase 8:FLIP, and low levels of Fas. Thus, human CB and PBSC CD34+ HSC were resistant to Fas pathway agonists. High-level expression of FLIP likely provides one level of protection of CD34+ cells from Fas-mediated apoptosis.

Animals↗