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Biomedical subjects

R A Phillips

Publications and source records attributed to R A Phillips.

At least 145 records · Page 8Linked to original sources
PubMed1988

Infrequent genomic rearrangement and normal expression of the putative RB1 gene in retinoblastoma tumors.

Retinoblastoma (RB) tumors develop when both alleles of a gene (RB1) are mutated and unable to function normally. Recently, Friend et al. [S. H. Friend, R. Bernards, S. Rogelj, R. A. Weinberg, J. M. Rapaport, D. M. Albert, and T. P. Dryja, Nature (London) 32:643-646, 1986] reported the cloning of a gene, 4.7R, with some properties expected for the RB1 gene, namely, a high frequency (30%) of genomic rearrangements in tumors and absence of message in all RB tumors examined. To extend the characterization of this gene, we used 4.7R probes to search for genomic rearrangements of DNA and to study the expression of the 4.7R gene in RB tumors, osteosarcoma (OS) tumors arising in RB patients, and other normal and malignant tissues. In 34 previously unreported RB and OS tumors arising in RB patients, we observed only four (12%) with genomic abnormalities. Transcripts of 4.7R were present in 12 of 17 RB tumors, 2 of 2 OS tumors, and all non-RB tumors and normal tissues tested. We were unable to confirm the high frequency of truncated messages of 4.7R in RB tumors reported by Lee et al. (W. H. Lee, R. Bookstein, F. Hong, L. J. Young, J. Y. Shaw, and E. Y. Lee, Science 235:1394-1399, 1987) and Fung et al. (Y. K. Fung, A. L. Murphree, A. Tang, J. Qian, S. H. Hinrichs, and W. F. Benedict, Science 236:1657-1661, 1987) but did confirm the presence of a truncated transcript in the RB cell line Y79. Of the RB and RB-related OS tumors which appeared normal on Southern blots, 2 of 26 or 12% had abnormal transcripts, giving a combined frequency of 22% abnormalities in the 4.7R gene detectable by Southern and Northern (RNA) blot analyses.

Gene Expression Regulation↗
PubMed1988

Determinants of atrial natriuretic factor in the adult respiratory distress syndrome.

We measured plasma atrial natriuretic factor over the clinical course of 12 patients with the adult respiratory distress syndrome (ARDS). A total of 33 sequential measurements were correlated with 23 hemodynamic, pulmonary, and renal parameters. Atrial natriuretic factor was found to be eight times higher than age-matched healthy control subjects (p = 0.001). Although atrial natriuretic factor correlated well with pulmonary capillary wedge pressure (r = 0.592), there was no significant relationship between right atrial pressure and plasma atrial natriuretic factor (r = 0.258). Atrial natriuretic factor was best related to pulmonary artery systolic pressure (r = 0.751). We conclude that in ARDS, plasma atrial natriuretic factor is markedly elevated and arterial levels are best determined by left atrial pressure and pulmonary hemodynamics, and not by right sided cardiac pressures. The role of increased circulating atrial natriuretic factor in the pathophysiology of ARDS is to be clarified in future studies.

Aged↗
PubMed1988

Effects of cyclosporine on systemic MHC expression. Evidence that non-T cells produce interferon-gamma in vivo and are inhibitable by cyclosporine.

The ability of lipopolysaccharide to induce major histocompatibility complex hyperexpression in vivo in a variety of mouse tissues--particularly kidney--and the effect of cyclosporine on this process were studied. MHC expression was measured by a radiolabeled antibody-binding assay using tissue homogenates, as well as by assessment of tissue sections by indirect immunoperoxidase staining. LPS administered to mice in two doses, 4 days apart, induced an increase in class I expression in several tissues but also induced an increase in class II expression in kidney. A similar increase in class II expression in kidney was not elicited with polyinosinic acid/polycytidylic acid, an agent that induces release of IFN-alpha/beta and increases class I MHC product expression. Thus we reasoned that LPS in vivo may release IFN-gamma, which then induces increased expression of MHC products. We validated this hypothesis by demonstrating that monoclonal antibody against IFN-gamma inhibited the induction of renal MHC products by LPS. However, the LPS effects did not require the participation of T cells, being demonstrable in nude mice and in mice with severe combined immunodeficiency. Moreover, the effect of LPS on MHC expression in normal and nude mice was inhibited by in vivo administration of monoclonal antibody against IFN-gamma just as it was in normal mice. Thus the class II hyperexpression that follows LPS is apparently mediated by non T cells and is due to the systemic release of IFN-gamma. This mechanism was inhibited by high doses of CsA in vivo, both in normal and in nude mice. The results indicate that there is a non T cell pathway for IFN-gamma release (and MHC induction) in vivo that is sensitive to CsA. This observation raises the possibility that some of the immunosuppressive effects of CsA may be due to inhibition of mediator release from non T cells.

Animals↗
PubMed1987

High efficiency gene transfer and expression in normal murine B lymphocytes.

The introduction of new genetic information into hematopoietic cells offers a new approach for investigating the molecular events controlling differentiation. Retrovirus vectors have been used to transfer new genes with high efficiency into murine hematopoietic cells, primarily of the myeloid lineage. In this report, we show that vectors carrying the dominant, selectable gene for neomycin resistance (neo gene) can successfully infect normal murine B lymphocytes (CFU-B). The infected CFU-B formed colonies in vitro in high concentrations (750 micrograms/ml) of G418, a neomycin analogue. That B lymphocytes contained the neo gene was confirmed by the findings that the putative B cell colonies growing in G418 contained antibody-producing cells and that the cells responding to the B cell mitogen, LPS, were resistant to G418. Infection of normal spleen cells with different vectors containing a variety of transcriptional regulatory sequences resulted in 7-40% of the CFU-B becoming G418 resistant. Introduction of the immunoglobulin heavy chain enhancer into NEO vectors appeared to augment the expression of the neo gene, since the level of G418 resistance was higher in B cells infected with a NEO vector containing the enhancer than in cells infected with a vector lacking the enhancer.

Animals↗
PubMed1987

Doppler echocardiographic analysis of left ventricular filling in treated hypertensive patients.

Early detection and prevention of cardiac dysfunction is an important goal in the management of hypertensive patients. In this study, Doppler echocardiography was used to evaluate the pattern of left ventricular diastolic filling in 38 subjects: 18 treated hypertensive patients (blood pressure 141 +/- 17/83 +/- 10 mm Hg, mean +/- SD) without other coronary risk factors and 20 risk-free normotensive subjects of similar age (47 +/- 10 and 49 +/- 13 years, respectively). Peak velocity of late left ventricular filling due to the atrial contraction was greater in hypertensive compared with normotensive subjects (69 +/- 14 versus 52 +/- 13 cm/s; p less than 0.001). Peak velocity of late filling was significantly greater in hypertensive versus normotensive subjects in those aged 50 years or younger and those older than age 50 (65 +/- 12 versus 50 +/- 11; p less than 0.01 and 75 +/- 15 versus 56 +/- 15 cm/s; p less than 0.05, respectively). In hypertensive subjects, peak velocity of late filling did not correlate with routine indexes of hypertensive heart disease (including posterior wall thickness and left ventricular mass), systolic and diastolic blood pressure or duration of hypertension. These results indicate that increased velocity of late left ventricular filling may be independent of left ventricular hypertrophy and persist despite effective blood pressure control.

Adult↗
PubMed1987

Modulation of gene expression in multiple hematopoietic cell lineages following retroviral vector gene transfer.

Retrovirus vectors offer a simple and highly efficient method for introducing new genes into mammalian cells. Here, we have examined the efficiency of gene transfer into hematopoietic cells with retrovirus vectors carrying the neomycin (neo) resistance gene expressed from different transcriptional regulatory regions. Direct infection of mouse bone marrow cells resulted in high efficiencies of gene transfer into a variety of myeloid progenitor cells, including pluripotent, erythroid, and granulocyte-macrophage colony-forming cells with all the vectors examined. However, the progeny derived from individual pluripotent progenitor cells infected with different vectors differed markedly in the proportion of G418-resistant progenitor cells, as judged by their ability to survive selection in the drug G418. This biological assay suggests that the highest level of expression was observed when the neo gene was expressed from constructs that contained the herpes thymidine kinase promoter rather than the viral long terminal repeat or the simian virus 40 early region promoter. In contrast, neo gene expression was highest in fibroblasts infected with the vector containing the simian virus 40 early region promoter. These results show that high and sustainable levels of gene expression in hematopoietic cells can be obtained with retrovirus vectors containing appropriate transcriptional regulatory regions.

Animals↗
PubMed1987

Re-evaluation of the sublocalization of esterase D and its relation to the retinoblastoma locus by in situ hybridization.

In situ hybridization of a cDNA probe for the esterase D gene (ESD) was carried out on human chromosomes. The probe hybridized most strongly to 13q14.2 and 13q14.3. This observation raises doubts concerning the most recently published assignment of ESD to 13q14.1. A deletion in an individual with retinoblastoma was reported to separate the closely linked ESD and retinoblastoma (RB1) loci, placing ESD proximal to RB1. Quantitative in situ hybridization studies of this deletion do not confirm this interpretation. Rather, they suggest that ESD is missing from the deleted chromosome 13 and duplicated on the normal homolog. From these findings, we conclude that the deletion in this individual cannot be used to determine the orientation nor the sublocalization of ESD and RB1 within the 13q14 region.

Carboxylesterase↗
PubMed1986

An expanded population of natural killer cells in mice with severe combined immunodeficiency (SCID) lack rearrangement and expression of T cell receptor genes.

Mice with severe combined immunodeficiency syndrome (SCID) exhibit an impairment in both T and B cell maturation, whereas myelopoiesis remains unaffected. We report here that spleens from SCID mice have undergone phenotypic expansion of cells bearing the NK-2 and asialo GM1 markers (70-80%) characteristic of NK cells and this expansion is accompanied by a 3-4-fold enrichment in NK cytolytic activity over their normal C.B-17 littermates. Furthermore, the NK cells from SCID mice do not rearrange or express T cell receptor alpha or beta genes, or a third T cell rearranging gene, gamma. These findings suggest that (a) T cell receptors are not necessary for NK-mediated cytolysis, and (b) either NK cells constitute an entirely distinct lineage or NK cell function is acquired in pre-T cells prior to the expression of T cell receptor genes.

Animals↗
PubMed1986

Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency.

A process unique to lymphocyte differentiation is the rearrangement of genes encoding antigen-specific receptors on B and T cells. A mouse mutant (C.B-17scid) with severe combined immune deficiency, i.e., that lacks functional B and T cells, shows no evidence of such gene rearrangements. However, rearrangements were detected in Abelson murine leukemia virus-transformed bone marrow cells and in spontaneous thymic lymphomas from C.B-17scid mice. Most of these rearrangements were abnormal: approximately 80% of Igh rearrangements deleted the entire Jh region, and approximately 60% of TCR beta rearrangements deleted the entire J beta 2 region. The deletions appeared to result from faulty D-to-J recombination. No such abnormal rearrangements were detected in transformed tissues from control mice. The scid mutation may adversely affect the recombinase system catalyzing the assembly of antigen receptor genes in developing B and T lymphocytes.

Abelson murine leukemia virus↗
PubMed1986

Full reconstitution of the immune deficiency in scid mice with normal stem cells requires low-dose irradiation of the recipients.

Mice homozygous for an autosomal recessive mutation for the scid gene exhibit a defect that specifically impairs lymphoid differentiation but not myelopoiesis. Such mice can be cured of their lymphoid deficiency by grafts with normal bone marrow, although full reconstitution of lymphoid function is seldom obtained. Long-term bone marrow cultures (LTBMC) are devoid of all mature B and pre-B cells but contain lymphoid stem cells. We therefore reconstituted scid mice with LTBMC cells to study the kinetics of B lymphocyte reconstitution in normal and irradiated (4 Gy) scid recipients and in irradiated (9.5 Gy) co-isogenic C.B-17 mice. Detectable colony-forming B cells rapidly increased in the spleen and bone marrow of irradiated C.B-17 and irradiated scid recipients, reaching normal levels between 4 and 6 wk post-grafting. Unirradiated scid recipients showed limited reconstitution in spleen and very poor reconstitution in bone marrow. Unirradiated scid recipients also had relatively few surface Ig+ cells in spleen or bone marrow, whereas both groups of irradiated recipients had normal numbers between 4 and 6 wk post-reconstitution. Normal levels of cytotoxic T cell activity by 8 wk after reconstitution were observed only in the irradiated C.B-17 and irradiated scid recipients. Analysis of mice reconstituted with cells from LTBMC indicates that these cultures contain lymphoid stem cells with significant proliferative and self-renewal potential, and that full reconstitution of lymphoid function requires prior irradiation of the scid recipient.

Animals↗
PubMed1986

Generation of purified stromal cell cultures that support lymphoid and myeloid precursors.

Treatment of Dexter-type long-term bone marrow cultures with the antibiotic mycophenolic acid (MPA) eliminates all hemopoietic cells from the cultures, while a morphologically intact, adherent stromal cell layer is retained. The ability of these MPA treated stromal cell cultures to support long-term hemopoiesis was tested by seeding them with fresh bone marrow cells that had been passed through nylon wool. This procedure yields a relatively stromal cell depleted population of hemopoietic cells. An aliquot of 5 X 10(5) or 2.5 X 10(5) nylon wool passed bone marrow cells bearing the T6 chromosomal marker was seeded onto replicate MPA-treated stromal cell layers. The stromal cells stimulated the proliferation of the bone marrow cells, and nonadherent cells were present for up to 8 weeks of culture. Progenitors of granulocytes and macrophages (CFU-GM) were also present for this period of time despite weekly demi-depopulation, during culture feeding. Karyotypic analysis confirmed that the CFU-GM were derived from the reseeded population. Nylon wool-passed bone marrow cells seeded alone into empty flasks under identical conditions did not survive past 1 week. Cells from the reseeded cultures were also tested for early myeloid precursors (CFU-S) and injected into immunodeficient CBA/N mice to test for the presence of primitive B cell precursors. CFU-S were present in mice killed 11 days following injection of cells, and high levels of B cell colony-forming units (CFU-B) were present in mice 4 weeks post reconstitution. Further studies demonstrated that factors present in medium conditioned by the stromal cells could support the growth of CFU-GM. These data indicate that treatment of long-term bone marrow cultures with MPA results in a population of functional stromal cells.

Animals↗
PubMed1986

Cloning of the esterase D gene: a polymorphic gene probe closely linked to the retinoblastoma locus on chromosome 13.

The study of recessive oncogenes such as those responsible for retinoblastoma and Wilms tumor is difficult because the gene products involved are unknown and because the diseases are not associated with unique cellular or molecular phenotypes suitable for genetic manipulation. Since the gene for esterase D (ESD) is known to be tightly linked to the retinoblastoma locus (RB1) in the q14.1 band of chromosome 13, we have cloned the ESD gene from a human cDNA library by using oligonucleotides specific for a partial amino acid sequence of the purified enzyme to provide a genetic marker for further studies on retinoblastoma. The putative ESD gene codes for a message of 1.2 kilobases, which is present in all cell types examined, and maps to 13q14.1, thus confirming that it is the ESD gene. Restriction enzyme analysis reveals a restriction fragment length polymorphism with Apa I; this polymorphism results from the heterozygosity of 32% of the individuals tested and is shown to be useful in identifying carriers of the mutation responsible for retinoblastoma. A preliminary screen of 24 retinoblastoma tumors by Southern blot did not reveal any homologous deletions or rearrangements of the ESD locus.

Carboxylesterase↗
PubMed1985

Genetic origin of mutations predisposing to retinoblastoma.

Retinoblastoma is one of several human tumors to which predisposition can be inherited. Molecular genetic analysis of several nonheritable cases has led to the hypothesis that this tumor develops after the occurrence of specific mitotic events involving human chromosome 13. These events reveal initial predisposing recessive mutations. Evidence is presented that similar chromosomal events occur in tumors from heritable cases. The chromosome 13 found in the tumors was the one carrying the predisposing germline mutation and not the homolog containing the wild-type allele at the Rb-1 locus. These results suggest a new approach for identifying recessive mutant genes that lead to cancer and a conceptual basis for accurate prenatal predictions of cancer predisposition.

Alleles↗
PubMed1985

A detailed analysis of chromosomal changes in heritable and non-heritable retinoblastoma.

Full cytogenetic analysis of 27 different retinoblastoma tumors is presented. Gross aneuploidy of chromosome arms 6p and 1q were very common, being observed in 15/27 and 21/27 tumors, respectively. However, we found that chromosome 13 was rarely missing: only 3/27 had a detectable monosomy affecting 13q14. Monosomy of chromosome 13 by small deletion or rearrangement was also not observed in any of 12 retinoblastoma tumor lines analyzed detail at the 300-400 chromosome band level. A novel observation in retinoblastoma was the discovery of non-random translocations at three specific breakpoints, 14q32 (4/12), 17p12 (5/12), and 10q25 (3/12). Genomic rearrangements similar to those described involving C-myc in Burkitt lymphoma 14q+ cells could not be demonstrated in the four 14q+ retinoblastoma lines using molecular techniques, and a probe mapping to the site implicated to have an activating role in lymphoma. These data suggest that there is a target for rearrangement at 14q32 but it is not the same sequence used in some Burkitt lymphomas. Two other breakpoints (2p24 and 8q24) coincided with the mapped position of cellular oncogenes, but also failed to show a molecular rearrangement with the oncogene probes. The breakpoints, 10q25 and 17p12, are constitutional fragile sites which may predispose these regions to act as acceptors of translocations in malignant cells. One line had double minute chromosomes, and was the only one of 16 (6%) tested with the N-myc probe which had an amplification. Different tumors from single patients with multifocal heritable retinoblastoma showed independent karyotype evolution. Unilateral non-heritable tumors exhibited a high level of karyotype stability throughout both in vivo and in vitro growth. The various common patterns of aneuploidy and translocations probably confer an early selective advantage to malignant cells, rather than induce malignant transformation.

Aneuploidy↗
PubMed1985

Radiation sensitivity of fibroblasts of bilateral retinoblastoma patients as determined by micronucleus induction in vitro.

The radiation sensitivity of fibroblasts isolated from bilateral retinoblastoma (RB) patients was investigated using an in vitro micronucleus assay. Bilateral RB is an autosomal dominant disease associated with a single locus, RB-1; therefore, all cells in an affected individual carry the germ line mutation. The ability to identify gene carriers made it possible to study the effect of the RB-1 mutation in the heterozygous state on the sensitivity of the cells to chromosome breakage by gamma-rays. The micronucleus assay was chosen for this study since it is a quick and easy measure of chromosomal aberrations. The fibroblasts from bilateral RB patients did not differ systematically from the normal fibroblasts in either the spontaneous or the induced rates of micronucleus production. Thus, bilateral RB fibroblasts are not more sensitive to the clastogenic effects of gamma-radiation than the controls.

Adolescent↗