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Biomedical subjects

R A Phillips

Publications and source records attributed to R A Phillips.

At least 163 records · Page 9Linked to original sources
PubMed1985

Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice.

We have used the random chromosomal integration sites of retrovirus vectors as unique clonal markers to analyze cell lineage relationships within the hemopoietic stem cell hierarchy. Using a high efficiency protocol for retrovirus-mediated gene transfer, anemic W/Wv mutant mice were reconstituted with bone marrow cells infected with a NEO vector. Analysis of the DNA from bone marrow, thymus, and spleen of these reconstituted W/Wv mice indicated insertion of the vector into primitive pluripotent stem cells capable of producing both myeloid and lymphoid progeny as well as into more committed stem cells apparently restricted to either the myeloid or lymphoid lineages. The neo gene was also expressed in these mice, as they contained a variety of G418 resistant in vitro colony-forming cells. These results demonstrate high-efficiency gene transfer and expression in primitive hemopoietic stem cells and provide a direct approach for analyzing the hemopoietic stem cell hierarchy.

Animals↗
PubMed1985

Changes in collateral channel filling immediately after controlled coronary artery occlusion by an angioplasty balloon in human subjects.

Transluminal coronary angioplasty can serve as a model for controlled coronary artery occlusion and reperfusion which enables assessment of short-term changes in collateral vessel filling in patients with severe atherosclerotic coronary artery disease. In 16 patients with isolated left anterior descending or right coronary artery disease (greater than or equal to 75% stenosis) and normal left ventricular function, collateral filling to the artery being dilated was visualized by contrast injection into the contralateral artery using a second arterial catheter. During balloon inflation, contralateral dye injection was performed as soon as the patient developed angina or ST-T changes or at 90 seconds in those patients without symptoms or signs of ischemia. Grades of collateral filling from the contralateral vessel were: 0 = none; 1 = filling of side branches of the artery to be dilated via collateral channels without visualization of the epicardial segment; 2 = partial filling of the epicardial segment via collateral channels; 3 = complete filling of the epicardial segment of the artery being dilated via collateral channels. At baseline angiography, nine patients had grade 0 collateral filling, seven had grade 1 and none had grade 2 or 3. During coronary occlusion by balloon inflation, collateral filling improved by one grade in eight patients, two grades in five patients, three grades in two patients and remained the same in one patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1985

The survival of parasite eggs throughout the soil profile.

An experiment using lysimeters suggested that the eggs of Taenia saginata and Ascaris lumbricoides survive for only a short time when applied to pasture in sewage sludge. However, a subsequent experiment which followed the survival of eggs throughout the soil profile demonstrated that some T. saginata eggs could still be found at 200 days on the soil surface, and that survival increased down the profile. Rainfall is shown to be able to wash eggs into the soil where they may be afforded protection from radiation and desiccation; this may have little epidemiological significance.

Animals↗
PubMed1985

Osteosarcoma and retinoblastoma: a shared chromosomal mechanism revealing recessive predisposition.

Survivors of the heritable form of retinoblastoma subsequently develop second primary osteosarcomas at substantially greater frequency than either the general population or survivors of nonheritable retinoblastoma. Here we present molecular genetic evidence that the development of these two disparate tumor types involves specific somatic loss of constitutional heterozygosity for the region of human chromosome 13 that includes the RB1 locus. Similar events occur during the genesis of nonheritable osteosarcoma but not in several other embryonal tumors or sarcomas. These findings suggest that a conceptual approach toward defining the number of genes whose recessive mutant forms predispose to cancer is the molecular genetic analysis of clinically associated tumor types. They also suggest that the molecular basis of mixed cancer families may be the differential expression of a single pleiotropic recessive mutation by tissue specific mitotic segregation abnormalities.

Alleles↗
PubMed1985

The function of antigen-presenting cells in mice with severe combined immunodeficiency.

We have examined the antigen-presenting function of spleen cells in the C.B-17 scid mouse, a mutation that severely impairs the development of T and B lymphocytes. We show that antigen-presenting cells (APC) of SCID mice function normally in antigen-specific proliferative responses of primed T cells and in the antigen-specific activation of IL 2-producing T cell hybridomas. In both quantitative and qualitative terms, APC of SCID mice are equivalent to those of normal mice. These results indicate that the development and differentiation of APC function in vivo is independent of signals from mature, functional T or B lymphocytes.

Animals↗
PubMed1985

Monoclonal antibodies can precipitate low-density lipoprotein. I. Characterization and use in determining apolipoprotein B.

We produced 20 mouse monoclonal antibodies against human plasma low-density lipoprotein (LDL). Individually they failed to precipitate LDL in agarose gel by the double-immunodiffusion technique; collectively they did, or as few as two combined monoclonal antibodies could do so. To mimic polyclonal antibodies in determination of apolipoprotein B (apo B) by radial immunodiffusion, a combination of four particular monoclonal antibodies (clones A, B, C, and D) was necessary. We characterized these four clones with respect to temperature dependency, affinity, total binding to 125I-labeled LDL, and specificity to the different species of apolipoprotein B. Two monoclonal antibodies (B and C) bound 100% of 125I-labeled LDL; clones A and D bound 80% and 87%, respectively. All four clones bound maximally to LDL at 4 degrees C. The affinity constants for clones A, B, C, and D were 0.6, 2.1, 3.8, and 2.3 X 10(9) L/mol, respectively. By the Western blotting technique, the four monoclonal antibodies all reacted with the species B-100 and B-74 of apolipoprotein B, and to various degrees with B-48 and B-26. Radial immunodiffusion (chi) and direct enzyme-linked immunosorbent assay (y) with a mixture of the four monoclonal antibodies gave almost identical results for 70 patients: y = 0.921 chi-2.58; r = 0.933.

1-Propanol↗
PubMed1985

Natural killer (NK) cells are present in mice with severe combined immunodeficiency (scid).

Spleen cells from C.B- 17 scid mice with severe combined immunodeficiency disease exhibit natural killer cell (NK) activity against YAC lymphoma targets in a standard 4-hr 51Cr release assay. The cytolytic activity is demonstrable only at high effector to target ratios but can be augmented at least sevenfold by the interferon inducer poly I:C. The pattern of target lysis is specific, because splenocytes from poly I:C-primed C.B-17 scid mice lyse NK-sensitive YAC cells and not the insensitive P815 mastocytoma. The presence of several NK-associated antigens on C.B-17 scid splenocytes was tested by pretreating cells with the appropriate antiserum plus complement before testing for NK activity. The results indicate that a proportion of NK effectors in C.B-17 scid mice bear surface NK 2.1 and Asialo GM1 but are negative for Thy-1.

Animals↗
PubMed1984

Clonal generation of multipotent and unipotent hemopoietic blast cell colonies in vitro.

Colonies with a unique compact morphology and displaying high replating potential develop in methycellulose cultures when bone marrow cells from hydroxyurea- or 5-fluorouracil-treated mice are cultured in the presence of syngeneic thymocytes. These compact colonies are clonal, originating from a single unit, the compact-colony-forming unit (CFU). Based on replating analysis, at least three groups of compact colonies exist: (1) those that do not replate, (2) those that replate and give only GM colonies, and (3) those that give rise to granulocyte-macrophage (GM), erythroid (E), and mixed-erythroid (MIX/E) colonies. Colonies in the last group have a mean size of 2,060 cells and generate an average of 244 colonies in secondary cultures. Velocity sedimentation studies showed the compact-CFU to be similar in size to the MIX/E-CFU, sedimenting at rates between 3.5 and 7 mm/hour. In addition to giving rise to large numbers of in vitro CFU, compact-CFU are also able to generate day 8 spleen colony-forming units (CFU-S).

Animals↗
PubMed1984

Isochromosome 6p, a unique chromosomal abnormality in retinoblastoma: verification by standard staining techniques, new densitometric methods, and somatic cell hybridization.

Study of chromosome rearrangements in retinoblastoma tumors revealed that all tumors contained either an unusual isochromosome and/or extra copies of chromosome 1q. Extra copies of chromosome 1q occur in many malignancies. The pattern of G-bands suggested that the isochromosome was derived from either the short arm of chromosome 6, i(6p), or the long arm of chromosome 17, i(17q). Standard staining techniques using G-, C-, Q-, and R-banding; high resolution G-banding; and density profile analysis were consistent with the characteristic isochromosome of retinoblastoma being i(6p), rather than i(17q). This conclusion was substantiated by the analysis of segregants derived from retinoblastoma X mouse hybrid cells which had been grown in bromodeoxyuridine to select for loss of chromosome 17. The unique isochromosome was not lost under these conditions confirming that it is an i(6p) rather than an i(17q). The i(6p) abnormality has not been observed frequently in other tumors, but occurs in 60% of retinoblastoma tumors. Thus, although the mutation predisposing to retinoblastoma is known to map at 13q14, somatic amplification of genes on 1q and 6p may play a role in the pathogenesis of this tumor.

Animals↗
PubMed1984

Characterization of a stable, anchorage-dependent clone obtained from a spontaneously transformed mouse cell line.

A variant nontransformed clone, I21, was selected from the spontaneously transformed mouse fibroblast line, IT22. Selection was done by plating IT22 in methylcellulose and picking single cells after 2 d. Cultures derived from these single cells were selected again and one clone, I21, derived from the second round of selection was characterized extensively. I21 and IT22 have the same plating efficiency (PE) on plastic, but in agarose they differ by 1000-fold. In comparison to IT22, I21 has a normal morphological appearance, a lower saturation density, a higher viability in stationary phase, an increased doubling time, an increased chromosome content, and is unable to form tumors in nude mice. I21 has remained remarkably stable in culture and has not reverted to the transformed phenotype for at least 300 generations in culture. Over 100 clones of I21, expanded to 10(6) cells, failed to show an increased PE in agarose. Even expansion of the rare colonies of I21 that grow in agarose failed to produce clones similar to IT22.

Animals↗
PubMed1984

Retinoblastoma: a model of oncogenesis.

Early events in oncogenesis can be understood in retinoblastoma (RB) because of several unusual clinical features: intraocular location of the tumor results in early diagnosis and frequent cure by surgery; survivors demonstrate a dominantly transmitted hereditary predisposition to RB; heritable and nonheritable subgroups can usually be distinguished clinically. Mathematical analysis of the clinical data lead Knudson to formulate the hypothesis that two mutations are required for RB tumor production. The first mutation (M1) occurs in the germ line of hereditary RB patients and in a somatic retinal cell in nonhereditary RB patients; for all RB patients, the second mutation (M2) occurs in the somatic retinal cell that becomes malignant. The locus of M1 (the RB locus), suspected on the basis of deletion patients to be at 13q14, was confirmed by linkage to the esterase D (ESD) gene locus in hereditary families. Studies utilizing multiple polymorphic markers, (ESD isoenzymes, restriction fragment length polymorphisms and karyotypic heteromorphisms) have shown that a somatic change from heterozygosity in constitutional cells to homozygosity in RB tumors occurs frequently for chromosome 13q but not for other chromosomes. Thus, M2 produces malignancy by somatic loss of the normal allele on the homologous chromosome 13. The normal allele at the RB locus probably regulates differentiation. In its absence, uncontrolled proliferation occurs. The genetic mechanisms defined in RB for expression of a dominantly inherited mutation may well apply to other malignancies and other dominantly inherited diseases.

Cell Transformation, Neoplastic↗
PubMed1984

Maintenance of hemopoiesis in long-term bone marrow cultures from S1/S1d and W/Wv mice.

Although phenotypically similar, the cellular defects in congenitally anemic mice of genotype W/Wv and S1/S1d are quite different. W/Wv mice have defective hemopoietic stem cells; in contrast, S1/S1d mice have normal stem cells, but their hemopoietic microenvironment cannot support normal differentiation of the stem cells. We also observed defective hemopoiesis as measured by granulocyte-macrophage colony-forming units (GM-CFU) in long-term bone marrow cultures (LTBMC) established with marrow from these mutants, but in contrast to an earlier report we obtained long-term maintenance of hemopoiesis (up to 20 weeks) albeit at a lower level than the control (28% of control for S1/S1d and 23% for W/Wv). These levels probably reflect more accurately the in vivo effects of the mutations than the severe defect reported previously. However, this level of hemopoiesis and the high variability observed in replicate flasks in LTBMC make it difficult to study these mutants in tissue culture.

Animals↗
PubMed1984

Functional status of cells from lymphoid and myeloid tissues in mice with severe combined immunodeficiency disease.

Cells from mice with severe combined immunodeficiency disease (SCID) were tested in assays that measure myeloid and lymphoid function. Results showed that C.B-17 scid and their normal counterparts (C.B-17) have similar levels of spleen colony-forming units. The frequency of in vitro myeloid colony-forming units in C.B-17 scid spleen is elevated, but the absolute number of colony-forming units in C.B-17 scid and C.B-17 spleen is similar. The absolute number of bone marrow colony-forming units in C.B-17 scid and C.B-17 mice is comparable. Cells from C.B-17 scid spleen are consistently negative in all tests of B and T cell function. C.B-17 scid splenocytes fail to proliferate in response to T and B cell mitogens or to allogeneic lymphocytes in a one-way MLR; C.B-17 scid cells do serve as stimulators in MLR. B lymphocyte colony-forming units are absent, as are cytotoxic lymphocyte precursors and cells that can generate T cell colonies with cytotoxic progenitors. The microenvironment of the C.B-17 scid mouse is conducive to lymphocyte differentiation, because functional B and T cells are easily detectable in mice reconstituted with normal bone marrow cells. The results of this study indicate that scid specifically impairs the differentiation of stem cells into mature lymphocytes; myeloid cell differentiation is not affected.

Animals↗
PubMed1983

Hemopoiesis in spleen and bone marrow cultures.

Long-term cultures established from spleen cells were compared to those established from bone marrow cells for their ability to maintain hemopoiesis as measured by the presence of hemopoietic progenitor cells (in vitro CFU) and multipotent stem cells (CFU-S). The frequency of both in vitro CFU and CFU-S increased dramatically during the first 2 weeks in the spleen cultures. Following this early peak of activity, the number of progenitors and stem cells declined to undetectable levels by week 6 of culture. During this short phase of hemopoiesis, large amounts of GM-CSF could be detected in the supernatant of the spleen cultures. In contrast, bone marrow cultures did not share this early peak of hemopoiesis; however, they maintained activity for much longer periods of time than did the spleen cultures. When spleen stem cells were seeded onto functional bone marrow adherent cells, spleen-derived in vitro CFU were maintained well beyond week 6 of culture. Spleen cultures established from athymic nu/nu mice showed a greatly reduced ability to support hemopoiesis while those from S1/S1d mice maintained GM-CFU as well as cultures from normal mice.

Animals↗