PubMed Health⌕ Search

Biomedical subjects

B W Grant

Publications and source records attributed to B W Grant.

13 recordsLinked to original sources
PubMed1999

HPRT mutations in vivo in human CD 34+ hematopoietic stem cells.

The HPRT mutations in T lymphocytes are widely utilized as biomarkers of environmental exposure and effect. The HPRT gene detects a wide variety of mutation types, many of which are similar at the molecular level to those found in oncogenes in cancers. However, it remains to be determined whether the assay for mutations in T lymphocytes is reflective of mutagenic events in tissues or cells which have high frequencies of malignancy in humans. We now demonstrate that the HPRT gene can be utilized to detect mutations in myeloid stem cells, which are frequent progenitor cells of leukemias. This myeloid stem cell assay shows an age related increase in mutation at HPRT and also detects increases in mutant frequency (M-MF) in patients who have undergone chemotherapy. The myeloid mutants are confirmed to have mutations in the HPRT gene by DNA sequence analysis. Increases in M-MF are seen as expected in the clonally unstable myeloid stem cells of patients with myelodysplastic syndromes; however, unexpectedly these patients also have elevated T-lymphocyte mutant frequencies (T-MF). A good correlation is shown between M-MFs and T-MFs in the same patients. Thus, it appears that the T-lymphocyte assay, which is technically much less demanding than the myeloid assay, appears to faithfully represent the frequency of mutagenic events in the myeloid lineage.

Adult↗
PubMed1992

Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC).

Osteonectin is an adhesive, cell, and extracellular matrix-binding glycoprotein found primarily in the matrix of bone and in blood platelets in vivo. Osteonectins isolated from these two sources differ with respect to the complexity of their constituent N-linked oligosaccharide. In this study, osteonectin synthesized by bone-forming cells (osteoblasts) and platelet-producing cells (megakaryocytes) in vitro was analyzed to determine if the proteins produced were analogous in terms of glycosylation to those isolated from bone and platelets, respectively. Immunoblot analyses of osteonectin produced by the osteoblast-like cell lines, SaOS-2 and MG-63, indicated that secreted and intracellular forms of the molecule are structurally distinct. Endoglycosidase treatment and immunoblotting of osteonectin secreted from SaOS-2 and MG-63 cells, under serum-deprived conditions, suggested that the molecule possessed a complex type oligosaccharide unlike the high-mannose moiety found on bone matrix-derived osteonectin. Biosynthetic labeling of SaOS-2 cells and human megakaryocytes indicated that both cell types synthesize osteonectin de novo. Electrophoretic and glycosidase sensitivity analyses of [35S]-osteonectin isolated from lysates of metabolically labeled SaOS-2 cells and megakaryocytes indicated that these two cell types synthesize osteonectin molecules that are identical in oligosaccharide structure to the isolated bone and platelet proteins. These data suggest that the intracellular form of the osteonectin molecule is glycosylated differently in SaOS-2 cells and megakaryocytes but that the extracellular form which is secreted from platelets in vivo and osteoblasts in vitro is characterized by the presence of a complex type N-linked oligosaccharide.

Adult↗
PubMed1991

Demonstration of osteonectin mRNA in megakaryocytes: the use of the polymerase chain reaction.

Platelets have been shown to release osteonectin on thrombin stimulation. The origin of platelet osteonectin was unclear as it may have been synthesized by megakaryocytes or it may have been endocytosed from plasma as other platelet alpha-granule constituents are. Platelet osteonectin has a larger apparent molecular size than the bone species, although the molecular basis for this difference has not been elucidated. These two issues have been addressed here by (1) examining the potential for osteonectin biosynthesis in human megakaryocytes by demonstrating the presence of osteonectin mRNA in purified megakaryocytes, and (2) comparing the coding portion of osteonectin transcript in megakaryocytes to the size of its bone counterpart. Because of the limitations of cell population purity and in obtaining sufficient numbers of megakaryocyte cells for Northern analysis, we have used the polymerase chain reaction (PCR) to detect the presence of human osteonectin mRNA in megakaryocyte and megakaryocyte-depleted bone marrow cells. Isolation of RNA, cDNA synthesis, and PCR were performed on human osteosarcoma SaOS-2 cells, enriched megakaryocytes, and megakaryocyte-depleted cells. Restriction enzyme analysis of PCR DNA products confirmed the identity of the products as those encoding osteonectin for all three cell populations studied. In addition, the sizes of DNA indicate that osteonectin genomic DNA, nuclear RNA, or altered transcript were not amplified, and that the transcript from megakaryocytes is the same size as that from bone cells. These data suggest that the difference in protein size between platelet and bone osteonectin is due to posttranslational modification. To overcome the possibility that megakaryocyte signal originated from contaminating cells (less than 5% by cell count), all three cell populations were diluted to less than one cell per tube and PCR amplification was performed. Limiting dilution analyses demonstrated the presence of osteonectin mRNA in single megakaryocytes as well as in single cells from the cell population depleted of megakaryocytes, suggesting the capacity for osteonectin biosynthesis in all cells studied. The procedure we describe in this report can be used to examine specific characteristics of mRNA molecules in heterogeneous cell populations and in situations where only small quantities of cells can be obtained.

Blotting, Southern↗
PubMed1990

Phase I trial of intraperitoneal recombinant interleukin-2/lymphokine-activated killer cells in patients with ovarian cancer.

Ten patients with ovarian cancer refractory to conventional therapy were treated with intraperitoneal (i.p.) recombinant interleukin-2 (rIL-2) and lymphokine-activated killer cells (LAK). The 28-day protocol consisted of 6 priming i.p. rIL-2 infusions on days 0, 4, 6, 8, 10, and 12. Leukapheresis was performed for mononuclear cell collection on days 15, 16, 17, and 18 and lymphokine-activated killer cells were given i.p. with the rIL-2 on days 19 and 21. Three additional i.p. rIL-2 infusions were given on days 23, 25, and 27. Three dose levels of rIL-2 were tested: 5 X 10(5), 2 X 10(6), and 8 X 10(6) units/m2 body surface area. The dose-limiting toxicity was abdominal pain secondary to ascites accumulation with significant weight gain. Other toxic effects included decreased performance status, fever, nausea and vomiting, diarrhea, and anemia. Peripheral lymphocytosis and eosinophilia were seen at all dose levels. The maximum tolerated dose is 8 X 10(6) units/m2/dose. Peripheral and peritoneal IL-2 levels were measured with a bioassay using an IL-2-dependent cell line. At the highest dose level, serum IL-2 was greater than 10 units/ml for 18 h. After the first infusion, a 2-log dilution of the i.p. IL-2 was measured in the serum. In the postleukapheresis i.p. IL-2-dosing period less IL-2 was detected in the serum than in the earlier i.p. IL-2-priming period. The induction and persistence of LAK activity were studied. Peritoneal LAK activity was detected as early as 4 days after the first i.p. infusion, by day 11 in all evaluable patients, and persisted for the 6-day interval between priming IL-2 and LAK/IL-2 infusion. Peritoneal lytic activity persisted until day 28 in 5 tested patients. These peritoneal cells retained lytic activity 48 h in culture medium without rIL-2 present. Peritoneal LAK activity correlated with the percentage of mononuclear cells and the percentage of CD56-positive mononuclear cells in the peritoneum. The yield of peripheral lymphocytes after the six i.p. priming doses of rIL-2 correlated with the dose level of rIL-2 infused. Peripheral blood LAK activity showed a minimal, however progressive, increase during the treatment protocol. LAK activity could be enhanced if rIL-2 was present during the 4-h assay. These studies indicate that i.p. rIL-2 infusion induced durable regional LAK activity and primes peripheral blood cells for LAK activity if exposed briefly to additional IL-2.

Adult↗
PubMed1990

Effects of recombinant interferons on human megakaryocyte growth.

Interferons (IFNs) have been shown to suppress the proliferation of human pluripotent and single-lineage hematopoietic progenitor cells. Treatments with IFNs have reduced platelet counts in patients with myeloproliferative disorders (MPD) but have not altered platelet counts in patients with healthy marrows. We assessed recombinant alpha and gamma IFN (rIFN-alpha and rIFN-gamma) preparations for their effect on the growth of marrow megakaryocytes (MKs) from normal donors and from patients with MPD. In addition, the interactions of recombinant interleukin 3 (rIL-3) recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) with the suppressive effects of rIFNs were examined. The addition of rIFN-alpha to liquid cultures resulted in a dose-dependent inhibition of normal marrow and MPD marrow MK growth. Inhibition with rIFN-gamma was only observed in normal marrows at 2500 U/ml and 12,500 U/ml; and rIFN-gamma unexpectedly stimulated MK growth in some culture conditions. Thirty units per milliliter rIL-3 overcame the inhibitory effect of rIFN-alpha on MK growth, but rGM-CSF or 3 U/ml rIL-3 did not. These studies with rIFN-alpha demonstrate that MPD marrow MKs and their precursor cells are no more sensitive to rIFN-alpha than are normal marrow MKs. Clinically, rIFN-gamma would be expected to be less effective than rIFN-alpha at controlling thrombocytosis in patients with MPD.

Adult↗
PubMed1988

Effect of commercial peritoneal dialysis fluids on the lytic function of lymphokine-activated killer cells.

Intraperitoneal (ip) immunotherapies are often administered in saline or peritoneal dialysis solutions. In preparation for a Phase I i.p. interleukin-2/lymphokine-activated killer cell (IL-2/LAK) trial in patients with recurrent ovarian cancer, we evaluated the effect of six solutions, including two 1.5% dextrose peritoneal dialysis solutions (PDS), on the lytic function of LAK. In vitro IL-2-activated LAK cells were exposed to solutions, washed with Hanks' balanced salt solution, and lytic activity was measured in a standard 4-h chromium release assay using Daudi as a tumor target. LAK function was abrogated after 2 h of exposure to Inpersol and after only 20 min of exposure to Dianeal PD-2. Five percent dextrose also significantly decreased lytic capabilities of LAK, whereas 0.9% sodium chloride or lactated Ringers had no deleterious effect on function. Adjustment of PDS to pH greater than 6.5 decreased the damaging effect on LAK function, suggesting that the low pH of PDS and 5% dextrose results in a loss of LAK cell viability and therefore lytic function. Based on these data, we have chosen to administer IL-2/LAK i.p. in lactated Ringers.

Cytotoxicity Tests, Immunologic↗
PubMed1987

Quantitation of human in vitro megakaryocytopoiesis by radioimmunoassay.

The isolation and characterization of human megakaryocyte growth factors has been hampered because evaluation of megakaryocyte growth in semisolid medium requires both lengthy incubation and visual quantitation. In addition, colony formation requires cell division, while most regulation of platelet production may involve individual, nonproliferating differentiating megakaryocytes. We have developed a radioimmunoassay (RIA) that makes use of an iodinated murine monoclonal antibody (MoAb) specific for platelet/megakaryocyte glycoprotein IIb/IIIa (GPIIb/IIIa) to measure megakaryocyte production in liquid marrow culture. This assay is sensitive to 3 X 10(3) platelets (roughly 30 megakaryocytes) and linear up to 1 X 10(6) platelets, and thus it provides a useful range for quantitating megakaryocyte production in in vitro marrow culture. Significant differences (threefold to fivefold) in megakaryocyte/platelet-specific GPIIb/IIIa complex are detected between stimulated and unstimulated marrow cultures by day 7, although antigen accrual in stimulated cultures continues through at least day 16. Conditions that promote megakaryocyte growth in semisolid medium (ie, aplastic plasma and PHA-LCM) have also been facilitory in liquid culture. This rapid and sensitive assay for cell-bound GPIIb/IIIa should facilitate recognition and isolation of megakaryocyte and platelet growth factors.

Antibodies, Monoclonal↗
PubMed1986

Lymphocyte populations and TAC-antigen in diffuse B-cell lymphomas.

Immune cell populations in 8 diffuse histology B-cell lymphomas were analysed in frozen tissue sections by indirect immunofluorescence to gain insight into their possible modulating influence in these tumors. Use of monoclonal antibodies to identify cellular and extracellular antigens combined with nuclear counterstaining allowed precise quantitation, localization and comparison of T- and B-lymphocyte populations. T lymphocytes clustered in non-random fashion. Areas of high T-lymphocyte density manifested higher T4:T8 ratios than locales with fewer T lymphocytes (p less than 0.05). Few cells had surface antigens (Leu 7, 73.1, OKM1) associated with natural killing. Cells strongly reactive with anti-TAC (Interleukin-2 receptor, associated with T-lymphocyte activation) were also T11 reactive and were usually helper (Leu 3) phenotype. In addition, B-lineage lymphoma cells in some tissues reacted with anti-TAC. The pattern of tumor cell reactivity with anti-TAC correlated with Rappaport histologic classification. These findings suggest that non-malignant T lymphocytes modulate B-lymphoma cell growth in situ, and that in some lymphomas the T-cell product IL-2 may be an important local growth factor.

Antibodies, Monoclonal↗
PubMed1983

Immune cell populations in cutaneous delayed-type hypersensitivity.

Delayed-type hypersensitivity (DTH) is a prototypic T lymphocyte-mediated response to antigenic challenge. In this study, mononuclear cells infiltrating the skin during cutaneous response to tuberculin in presensitized human subjects (responders) and nonimmune controls were identified using monoclonal antibodies by indirect immunofluorescence. In both responders and controls the infiltrate consisted mainly of T lymphocytes (T11+ and OKT3+) and monocytes (OKM1+, 63D3+, Mo2+) which initially accumulated in proximity to small blood vessels and later infiltrated the interstitial dermis and epidermis. More T lymphocytes reacted with OKT4 than with OKT8. 6 h after tuberculin the ratio of OKT4/OKT8 in tissue from responders exceeded that in blood, whereas in tissues studied at 15-48 h and in all control tissues those ratios in blood and tissue were similar. Evidence of T lymphocyte activation was sought using monoclonal antibodies anti-Tac, OKT9, and OKT10. In responders but not in controls the proportion of infiltrating cells reactive with these antibodies increased during the course of DTH. The presence of activated T lymphocytes in tissue was not associated with a comparable increase in peripheral blood cell populations identified by anti-Tac and OKT10. Studies using anti-B1, Leu-7, and anti-IgD/IgM revealed comparatively few reactive cells. Dual-labeling studies demonstrated that most Leu-7--reactive cells also bound T11 while fewer bound OKM1 or OKT8 and that cells reactive with OKIa1 and T11 constituted largely nonoverlapping populations. Specific patterns of reactivity were not observed when tissues were stained with anti-human C3, or poly C9-MA, a monoclonal antibody reactive with a neoantigen on polymerized C9 of the membrane attack complex of complement. The number of epidermal Langerhans cells identified by OKT6 was similar in responders and controls. Thus, the cutaneous response to tuberculin in sensitized individuals is characterized by early enrichment of the OKT4 subpopulation of T lymphocytes in tissue infiltrates and subsequent (15-48 h) evidence of T lymphocyte activation.

Animals↗