Biomedical subjects

William B Slayton

Publications and source records attributed to William B Slayton.

13 recordsLinked to original sources

PubMed2006

Differential effects of recombinant thrombopoietin and bone marrow stromal-conditioned media on neonatal versus adult megakaryocytes.

Umbilical cord blood (CB) is a valuable source of stem cells for transplantation, but CB transplantations are frequently complicated by delayed platelet engraftment. The reasons underlying this are unclear. We hypothesized that CB- and peripheral-blood (PB)-derived megakaryocytes (MKs) respond differently to the adult hematopoietic microenvironment and to thrombopoietin (Tpo). To test this, we cultured CB- and PB-CD34(+) cells in adult bone marrow stromal conditioned media (CM) or unconditioned media (UCM) with increasing concentrations of recombinant Tpo and compared the effects of these conditions on CB-versus PB-MKs. PB-MKs reached highest ploidy in response to UCM + 100 ng/mL rTpo, and the addition of CM inhibited their maturation. In contrast, CB-MKs reached highest ploidy in CM without rTpo, and high rTpo concentrations (> 0.1 ng/mL) inhibited their maturation. This is the first evidence that human neonatal and adult MKs have substantially different biologic responses to Tpo and potentially to other cytokines.

Adult↗

PubMed2006

Stem cell research.

One of the most active areas of research in medicine today is stem cell biology. This review introduces the reader to the field of stem cell biology and its therapeutic potential. More importantly, the potential application of stem cell therapy in acute lung injury will be explored.

Animals↗

PubMed2006

Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5.

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.

Animals↗

PubMed2005

Developmental differences in megakaryocyte maturation are determined by the microenvironment.

Historically, physicians have attributed delayed platelet engraftment following umbilical cord blood transplant to decreased numbers of stem cells in cord blood compared with adult bone marrow. However, recent studies suggest that delayed platelet engraftment may be caused by an intrinsic inability of neonatal stem cells to produce mature, polyploid megakaryocytes. We tested this hypothesis by transplanting adult bone marrow and newborn liver hematopoietic stem and progenitor cells from transgenic mice expressing green fluorescent protein into myeloablated wild-type recipients and comparing the size and ploidy levels of megakaryocytes that developed in adult transplant recipients. Transplanted stem and progenitor cells, regardless of their source, gave rise to megakaryocytes that were larger than normal adult megakaryocytes as early as 7 days post-transplant. However, megakaryocytes that developed after transplant of neonatal stem and progenitor cells were significantly smaller than those derived from adult stem and progenitor cells. Furthermore, megakaryocytes derived from neonatal cells had lower ploidy values than megakaryocytes derived from adult cells at 18 days post-transplant, when ploidy could first be reliably measured in the bone marrow. These differences in size and ploidy disappeared by 1 month post-transplant. The largest megakaryocytes developed in the spleen. These results suggest that, in the mouse, the microenvironment is responsible for some of the maturational differences in size and ploidy between neonatal and adult megakaryocytes. Furthermore, neonatal and adult megakaryocyte progenitors also have cell-intrinsic differences in the way they engraft and respond to thrombocytopenic stress. These differences may contribute to the delay in platelet engraftment that frequently complicates cord blood transplants.

Age Factors↗

PubMed2004

PU.1 supports proliferation of immature erythroid progenitors.

Despite normal levels of erythropoiesis in PU.1(-/-) embryos, PU.1(-/-) fetal hematopoietic progenitors are unable to establish sustained erythropoiesis in the adult bone marrow. This study demonstrates that PU.1(-/-) fetal erythroid progenitors are synergistically expanded by TPO plus SCF, but not combinations of EPO plus SCF, IL-3 or GM-CSF. The EPO defect is not corrected by a constitutively active variant of EPOR. Microarray analysis identified several candidate PU.1 target genes known to affect cytokine signaling and gene regulation in the erythroid lineage. These data suggest that PU.1 plays an important role in regulating the proliferation of immature erythroid progenitors.

Animals↗

PubMed2004

A neonate with severe thrombocytopenia and radio-ulnar synostosis.

Bone marrow failure syndromes can be associated with abnormalities of the forearms. We observed a neonate with congenital thrombocytopenia who had bilateral radio-ulnar synostosis and fifth finger clinodactly. We performed an evaluation of the mechanism causing the thrombocytopenia using a combination of direct and indirect measures of thrombopoiesis. These tests indicated decreased platelet production. This entity of congenital hyporegenerative thrombocytopenia with bilateral radio-ulnar synostosis and fifth-finger clinodactly is an uncommon but easily recognizable form of congenital amegakaryocytic thrombocytopenia (CAMT). This entity can be distinguished from the TAR syndrome (thrombocytopenia and absent radii) by the distinctive orthopedic issues, different underlying genetic mutations, and a more worrisome prognosis for CAMT than for TAR.

Diagnosis, Differential↗

PubMed2003

Characterization of thymic progenitors in adult mouse bone marrow.

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1(-)) (Thy1.1(-) progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1(-) progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1(-) population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.

Aging↗

PubMed2003

Dapsone therapy for children with immune thrombocytopenic purpura.

Dapsone has been shown to be effective in treating adults with immune thrombocytopenic purpura (ITP). This retrospective review describes the authors' experience using dapsone in children with refractory, symptomatic ITP. Seven children were treated with dapsone. Dapsone was discontinued in two patients because of methemoglobinemia. In the remaining five patients, three achieved platelet counts of more than 100 x 10(3)/microL. Discontinuation resulted in a rapid decline in platelet counts in all three patients. Two of the three responded to a second round of treatment. Additional study of dapsone in children is warranted. Children receiving dapsone should be monitored for methemoglobinemia.

Adolescent↗

PubMed2003

Early stages of hematopoietic differentiation.

Mouse bone marrow contains hematopoietic stem cells as well as progenitor cells, which are partially differentiated offspring of stem cells. We have utilized several approaches to separate progenitors from stem cells in order to characterize essential differences between these two stages of development. As a first approach, we utilized the supravital fluorescent dye rhodamine-123 (Rh-123) to distinguish quiescent stem cells (Rh-123(low)) from metabolically active progenitor cells (Rh-123(hi)). Analysis of megakaryocyte potential in a tissue culture assay demonstrated that Rh-123(hi) progenitor cells were capable of robust megakaryocyte differentiation, while Rh-123(low) stem cells produced fewer colonies containing megakaryocytes. Transplantation of the two cell populations into irradiated recipients revealed the opposite outcome, suggesting that the tissue culture assay failed to predict behavior in a transplant setting. We also evaluated functional potential of lymphoid progenitors isolated by selecting for differential expression of Thy-1.1 and c-kit. The potential of defined cell populations to differentiate as T or B lymphocytes in vivo was dependent upon the time post transplant at which animals were evaluated. These studies underscore the need for caution in the interpretation of lineage potentials evaluated by both in vitro and in vivo assays.

Animals↗

PubMed2003

Response to intravenous immunoglobulin predicts splenectomy response in children with immune thrombocytopenic purpura.

OBJECTIVE: Response to intravenous immunoglobulin (IVIG) has been shown to predict response to splenectomy in adults with immune thrombocytopenic purpura (ITP). However, reports in children have been inconsistent. We sought to determine whether response to IVIG is predictive of response to splenectomy in children. METHODS: Thirty-two assessable children were identified by a retrospective chart review. Response was graded according to previously published criteria as follows: "excellent" (platelets >150 000 within 1 week), "good" (platelets between 50 000 and 150 000), and "poor" (platelets <50 000). "Response" refers to both splenectomy and IVIG, and response to splenectomy was counted only when it was durable. RESULTS: Twenty-one of 23 patients who had a good or excellent response to IVIG also had an excellent response to splenectomy. Six of 9 patients who had a poor response to IVIG also had a poor response to splenectomy. Response to IVIG was a sensitive predictor of response to splenectomy in 88% of patients. Response to IVIG had a specificity of 75%, a positive predictive value of 91%, and a negative predictive value of 67%. Response to prednisone and length of time to splenectomy were not correlated with splenectomy response. CONCLUSIONS: These results suggest that response to IVIG is predictive of response to splenectomy in children with chronic ITP. This correlation may be of value in deciding whether a splenectomy should be performed in children with chronic ITP.

Adolescent↗

PubMed2002

High incidence of epithelial cancers in mice deficient for DNA polymerase delta proofreading.

Mutations are a hallmark of cancer. Normal cells minimize spontaneous mutations through the combined actions of polymerase base selectivity, 3' --> 5' exonucleolytic proofreading, mismatch correction, and DNA damage repair. To determine the consequences of defective proofreading in mammals, we created mice with a point mutation (D400A) in the proofreading domain of DNA polymerase delta (poldelta, encoded by the Pold1 gene). We show that this mutation inactivates the 3' --> 5' exonuclease of poldelta and causes a mutator and cancer phenotype in a recessive manner. By 18 months of age, 94% of homozygous Pold1(D400A/D400A) mice developed cancer and died (median survival = 10 months). In contrast, only 3-4% of Pold1(+/D400A) and Pold1(+/+) mice developed cancer in this time frame. Of the 66 tumors arising in 49 Pold1(D400A/D400A) mice, 40 were epithelial in origin (carcinomas), 24 were mesenchymal (lymphomas and sarcomas), and two were composite (teratomas); one-third of these animals developed tumors in more than one tissue. Skin squamous cell carcinoma was the most common tumor type, occurring in 60% of all Pold1(D400A/D400A) mice and in 90% of those surviving beyond 8 months of age. These data show that poldelta proofreading suppresses spontaneous tumor development and strongly suggest that unrepaired DNA polymerase errors contribute to carcinogenesis. Mice deficient in poldelta proofreading provide a tractable model to study mechanisms of epithelial tumorigenesis initiated by a mutator phenotype.

Amino Acid Sequence↗

PubMed2002

The spleen is a major site of megakaryopoiesis following transplantation of murine hematopoietic stem cells.

The stem cell pool can be fractionated by using the mitochondrial dye, rhodamine-123, into Rho(low) hematopoietic stem cells and Rho(high) progenitors. Rho(low) stem cells permanently engraft all lineages, whereas Rho(high) progenitors transiently produce erythrocytes, without substantial platelet or granulocyte production. We hypothesized that the inability of the Rho(high) cells to produce platelets in vivo was due to the fact that these cells preferentially engraft in the spleen and lack marrow engraftment. Initially, we demonstrated that Rho(high) progenitors produced more megakaryocytes in vitro than Rho(low) stem cells did. To study the activity of the Rho(low) and Rho(high) subsets in vivo, we used mice allelic at the hemoglobin and glucose phosphate isomerase loci to track donor-derived erythropoiesis and thrombopoiesis. Rho(low) stem cells contributed to robust and long-term erythroid and platelet engraftment, whereas Rho(high) progenitors contributed only to transient erythroid engraftment and produced very low numbers of platelets in vivo. Donor-derived megakaryopoiesis occurred at higher densities in the spleen than in the bone marrow in animals receiving Rho(low) stem cells and peaked around day 28. Blockade of splenic engraftment using pertussis toxin did not affect the peak of splenic megakaryopoiesis, supporting the hypothesis that these megakaryocytes were derived from progenitors that originated in the bone marrow. These data emphasize that in vitro behavior of hematopoietic progenitor cell subsets does not always predict their behavior following transplantation. This study supports a major role for the spleen in thrombopoiesis following engraftment of transplanted stem cells in irradiated mice.

Animals↗

PubMedDate not supplied

Lineage-specific trisomy 21 in a neonate with resolving transient myeloproliferative syndrome.

The cellular events that lead to transient myeloproliferative syndrome (TMS) in patients with trisomy 21 mosaicism confined to the hematopoietic system are poorly understood. The authors attempt to define the event that led to the development of TMS in a single patient with clonal trisomy 21. A phenotypically normal neonate with clonal trisomy 21 is described. At the time when his TMS was resolving, fluorescent in situ hybridization analysis was performed on cell populations sorted by flow cytometry to determine what cell populations contained trisomic cells. Trisomy 21 was found in cells of the erythrocytic and monocytic lineages, but not in the stem cells, progenitor compartment, megakaryocytes, lymphocytes, or neutrophils. These results support the hypothesis that, in this neonate, trisomy 21 occurred in a multipotent hematopoietic progenitor, and a subsequent event led to the appearance of the blast population.

Adult↗