Biomedical subjects

L M Sullivan

Publications and source records attributed to L M Sullivan.

At least 73 records · Page 4Linked to original sources

PubMed1991

Factors influencing mutation at the hprt locus in T-lymphocytes: women treated for breast cancer.

Forty-nine women with breast cancer were enrolled in a prospective, longitudinal study of the genetic damage caused by treatment. Assays of mutant frequency at the hprt locus in peripheral blood lymphocytes were performed at approximately 6-month intervals for 2 years. Treatment consisted of surgery alone or additional tamoxifen, radiotherapy, or chemotherapy in various combinations. At 6 months, there was an elevation of mean mutant frequency compared to initial values (P = 0.004) which persisted for as many as 2 years. A significant elevation at 6 months occurred only in the group of women who received combination chemotherapy (P = 0.005). Within this group, 5 of 15 patients had striking elevations of mutant frequency following chemotherapy (greater than 3 SD). Three of these 5 women had serum folate levels in the deficient range, while only one of 9 patients with lesser responses to chemotherapy were folate deficient. The change in mutant frequency after chemotherapy was inversely related to serum folate levels (P = 0.05) and to the number of years of smoking cigarettes (P = 0.01). We conclude that of the various modalities used to treat breast cancer, only chemotherapy was accompanied by a high risk of somatic mutation. A subset of patients manifested substantial increases in mutant frequency, often in association with low serum folate levels.

Antineoplastic Agents↗

PubMed1991

Cytosine arabinoside enhancement of gamma irradiation induced mutations in human T-lymphocytes.

The frequency of 6-thioguanine resistant (TGr) mutants induced in human G0 phase T-lymphocytes by 200 cGy of gamma irradiation is greatly enhanced by incubation with cytosine arabinoside (ara-C) after irradiation. The mutant frequency increased with increasing incubation time in ara-C for up to 2 hr. This mutation induction required a phenotypic expression time of 5-8 days mass culture growth, similar to that found with mutants induced by 300 cGy of irradiation alone. Southern blot analysis of 40 isolated mutant clones revealed 8 independent mutations by T-cell receptor (TCR) gene rearrangement patterns. Four of these eight showed hprt gene structural alterations (0.50). An alternative method to allow phenotypic expression was developed to minimize the isolation of hprt/TCR sibling mutants. The use of in situ expression in the microtiter dish wells resulted in the isolation of 17 independent mutations in 19 mutant clones. Ten of these 17 mutations showed hprt structural alterations (0.59). The high fraction of mutations involving structural alterations detected by Southern blot analysis is consistent with the known induction of chromosome aberrations by irradiation plus ara-C treatment. We propose that both the increase in Mf and the increase in the incidence of hprt gene structural alterations are due to the accumulation of strand breaks in repairing regions of DNA under these conditions of ara-C induced inhibition of repair. We further propose that upon release of the ara-C inhibition, these repairing regions can interact to yield both gene mutations and chromosome aberrations.

Cells, Cultured↗

PubMed1990

In vivo hprt mutant frequencies in T-cells of normal human newborns.

Mutation at the hypoxanthine-guanine phosphoribosyl transferase locus (hprt; HPRT enzyme) in the human fetus was studied by clonal assay of placental cord blood samples from full-term newborns. Conditions for determining hprt mutant frequencies, as defined for adults, were also optimal for studies in newborns. The mean mutant frequency for 45 normal human newborns (37 male, 8 female) was 0.64 X 10(-6) (SD = 0.41 X 10(-6); median value = 0.58 X 10(-6). These values are approx. 10-fold lower than corresponding adult hprt mutant frequency values. Factors such as limiting-dilution cloning efficiencies, delay prior to study of sample, sex, cryopreservation or technician performing the assay did not significantly affect assay results. Maternal smoking did not result in elevated mutant frequency values. Most wild-type and mutant clones studied were CD4 surface antigen positive (helper/inducer). All hprt mutants analyzed lacked HPRT activity.

Adolescent↗

PubMed1990

In vitro induction, expression and selection of thioguanine-resistant mutants with human T-lymphocytes.

Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes by a cell cloning assay. The in vitro growth of mass cultures as well as cell cloning is accomplished by the use of crude T-cell growth factor (TCGF) and irradiated human lymphoblastoid feeder cells. These initial studies employed irradiation of G0 phase peripheral blood mononuclear cells from a single individual. After exposure to gamma-irradiation from a 137Cs source, the cells were stimulated with the mitogen phytohemagglutinin (PHA) and maintained in exponential growth with exogenous TCGF to allow phenotypic expression of the 6-thioguanine-resistant (TGr) mutants. The mutant frequency was determined by measuring cell cloning efficiency in microtiter dishes in the absence and presence of TG, with an optimal selection density of 1 X 10(4) cells/well. The development of this in vitro assay should allow direct study of susceptibility to gamma-irradiation in the human population in terms of both cytotoxicity and mutation induction.

Cells, Cultured↗

PubMed1990

Southern-blot analyses of human T-lymphocyte mutants induced in vitro by gamma-irradiation.

G0 phase cultures of human peripheral blood T-lymphocytes from a single individual were exposed to 300 rad of gamma-irradiation from a 137Cs source and cultured in vitro for 8 days to allow phenotypic expression. Thioguanine-resistant (TGr) mutants were isolated by a cell cloning assay in microtiter plates. These mutants were studied by Southern blot analysis to define the gross structural alterations in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene by use of an hprt cDNA probe. A similar analysis of the T-cell receptor (TCR) gene rearrangement patterns was employed to define the independent nature of each mutant colony by use of TCR beta and gamma cDNA probes. 74 mutants were isolated in 5 separate experiments. TCR gene rearrangement analysis showed these to represent 24 independent mutations, of which 18 contained hprt structural alterations. These alterations included simple deletions (10/18) as well as more complex rearrangements resulting in molecular weight changes of restriction fragments representing both the 5' and 3' regions of the hprt gene (4/18 and 4/18, respectively). These results demonstrate that gamma-irradiation primarily induces TGr mutations through gross structural alterations in the hprt gene and that these alterations are randomly distributed across the gene. This approach to mutation analysis will provide information on the types of alterations induced by this irradiation, especially the extent of deletions involving the hprt gene. These results also demonstrate the feasibility of employing in vitro exposure of human T-lymphocytes to a single mutagenic agent as an aid to understanding the mechanisms of mutations occurring in vivo in humans.

Blotting, Southern↗

PubMed1990

How important is functional status as a predictor of service use by older people?

In studies of older people, it is often assumed that biophysical, or functional, status is the primary determinant of formal service use. This article reports baseline data from a longitudinal study of a community-based, linked random sample of frail elders (n = 635) and their informal caregivers (n = 429) to investigate the relative contribution of social circumstances to the use of community-based formal services. Elder respondents were categorized into three groups defined by their primary source of care: (a) informal only, (b) mixed help with predominantly informal care, (c) mixed help with predominantly formal services. Of the respondents, 79% received most of their help from informal caregivers, whereas 21% relied on formal services for most of their assistance. A series of logistic regression models were developed to identify variables that discriminated between major sources of care. The social factor of living alone is the consistent predictor of reliance on formal services. Only for those elders living alone does the physical factor of level of frailty predict reliance on formal services. Elders who live with a caregiver, particularly a spouse, are likely not to use any formal services regardless of their level of frailty. Finally, elders reliant on formal services receive much less care overall.

Activities of Daily Living↗

PubMed1989

Longitudinal study of the in vivo hprt mutant frequency in human T-lymphocytes as determined by a cell cloning assay.

The in vivo frequency of mutants resulting from mutation at the hprt locus in human T-lymphocytes can be determined by a cloning assay. This assay quantifies the frequency of 6-thioguanine-resistant (TGr) T-cells through growth of colonies in 96-well microtiter dishes. The reproducibility of the TGr mutant frequency values has now been assessed in a longitudinal study of six individuals (three male, three female, aged 22-33 years) employing 4-5 blood samples over a 26-37 week time period. Cloning assays were performed with both fresh and cryopreserved cell samples. No significant differences were found among the mutant frequency values for multiple samples from each individual with both fresh and cryopreserved cell samples. These results demonstrate the reproducibility of this cloning assay for in vivo mutant frequency determinations in human T-lymphocytes.

Adult↗

PubMed1989

Informal care for frail elders: the role of secondary caregivers.

Although the majority of informal care is provided by one person, most elders receive help from other caregivers as well. This paper describes the identity and activities of these secondary caregivers, who are often the spouse and children of the primary caregiver. They provide a wide variety of help, but much less than the primary caregiver. Further, this help is provided in a pattern supplementary rather than complementary to that of the primary caregiver.

Aged↗

PubMed1988

Functional role of specific secreted and cell surface molecules in tumour cell invasion and metastasis.

The invasion and metastatic spread of tumour cells are complex biological processes involving an array of cell surface and secreted protein molecules which function as specific recognition and catalytic agents in each step of the malignant cascade. In order to begin to identify these molecules and implicate them as true functioning agents, our laboratory has developed selected panels of monoclonal antibodies that have been screened for their ability to inhibit specific manifestations of the malignant phenotype in various model systems. Since monoclonal antibodies recognize only single and unique determinants, it is hypothesized that any monoclonal antibody which is specifically antifunctional will allow for the identification and isolation of relevant protein molecules.

Animals↗

PubMed1988

Molecular analyses of in vivo hypoxanthine-guanine phosphoribosyltransferase mutations in human T-lymphocytes: II. Demonstration of a clonal amplification of hprt mutant T-lymphocytes in vivo.

Recent molecular analysis of in vivo-derived hprt mutant T-lymphocytes cloned from human blood show that mutants occurring at the normal frequency (approximately 5 X 10(-6) in healthy young individuals generally represent independent hprt mutations. Here we report that in an individual with a high mutant frequency (86-620 X 10(-6],92% (61/66) of the mutant clones are descendents of an original mature T-cell precursor that has undergone in vivo clonal expansion. Therefore, these mutants could represent as few as one original hprt mutation. If so, correcting for the clonal expansion yields a revised calculated mutant frequency (Mf) value for this individual that is near the normal range. These hprt mutant clones all showed identically rearranged T-cell receptor (TCR) beta and gamma gene patterns by Southern blot analysis. All the clones were surface marker CD4+, showed no obvious chromosomal aberration, and had no detectable hprt gene structural alteration. This TCR-defined T-cell clone appears to have expanded in the blood of the individual over a 6-month period and persists at high levels after nearly 4 years. This finding illustrates the need to analyze mutants from individuals with high mutant frequencies at the molecular level in order to estimate hprt mutation frequency from the calculated hprt mutant frequency. The possibility that spontaneous hprt mutants might arise in vivo preferentially in dividing cells, and implications of this, are discussed.

Adult↗

PubMed1988

Mutagenicity monitoring in humans by autoradiographic assay for mutant T lymphocytes.

Somatic cell mutation which occurs in vivo in humans can be determined by measurement of the frequency of the 6-thioguanine-resistant (TGr) T lymphocytes in samples of peripheral blood. This frequency can be determined by either a short-term autoradiographic methodology or a longer cell-cloning methodology. The advantage of the former is the relative simplicity of the assay, while the latter allows recovery of mutant clones for further characterization. This report presents results of a longitudinal study of cancer chemotherapy nurses and other health care personnel by use of the autoradiography assay. The use of this assay in human mutagenicity monitoring and the analysis of the TGr cell frequencies are discussed in terms of age effects and validation of 'elevated' frequencies by use of the clonal assay. This report then presents evidence that both assays yield similar TGr cell frequencies in two groups of 'normal adults'. The mean variant frequency (+/- S.D.) for 82 autoradiographic assays was 8.7 (+/- 6.1) X 10(-6), while the mean mutant frequency (+/- S.D.) for 115 clonal assays was 6.5 (+/- 4.8) X 10(-6). In addition, concurrent autoradiographic and clonal assays on 33 individuals yielded mean values (+/- S.D.) of 8.4 (+/- 8.5) X 10(-6) and 10.5 (+/- 6.3) X 10(-6), respectively.

Age Factors↗

PubMed1988

Minimal antigenicity of intron A in human recipients demonstrated by three analytical methods.

Antibodies to recombinant human interferon alpha 2b (Intron A) were detected in only a small number of 101 Intron A recipients. This group of cancer patients received Intron A for a mean treatment time of 4.3 months and were selected from disease categories in which subjects were expected to be immunocompetent. Three methods for the detection of antibodies were employed: (a) a bioassay measuring the neutralizing activity of the sera for the antiviral action of interferon alpha 2b, (b) a radioimmunological assay measuring the ability of the sera to prevent the detection of interferon alpha 2b by an immunoradiometric assay (IRMA), and (c) an enzyme-linked immunosorbent assay (ELISA) that measures the binding of immunoglobulins to interferon alpha 2b attached to a solid support. Three of the 101 patients developed neutralizing activity during treatment. Two of these exhibited low neutralizing titers of 1:6-1:9 and were unreactive in the IRMA and ELISA, while only one was positive by bioassay, IRMA, and ELISA. An additional seven patients were positive only in the ELISA. Six of these were borderline positive, i.e., the posttreatment:pretreatment ratio was less than or equal to 5. The results of this study confirm that Intron A is minimally antigenic in human subjects.

Antibody Formation↗

PubMed1987

Limited cleavage of cellular fibronectin by plasminogen activator purified from transformed cells.

The substrate specificity and direct catalytic activity of plasminogen activator (PA) was examined under conditions where its natural substrate, plasminogen, was missing or inhibited. PA, purified from cultures of transformed chicken fibroblasts, was incubated with purified preparations of potential substrates. The adhesive glycoprotein fibronectin, isolated from normal chicken fibroblast extracellular matrix, underwent limited but specific cleavage by PA in the absence of plasminogen. Analysis of the cleavage products by polyacrylamide gels under both reducing and nonreducing conditions indicated that PA-mediated cleavage occurred near the carboxyl terminus of fibronectin but on the amino-terminal side of the interchain disulfide bridge, thus disrupting the native dimeric fibronectin molecule. Under the identical conditions, chicken ovalbumin was not cleaved while the established substrate, chicken plasminogen, was extensively converted to plasmin. A monoclonal antibody, directed against avian PA and shown to inhibit plasminogen-free, cell-mediated matrix degradation, specifically inhibited the fibronectin cleavage. A human PA, urokinase, also cleaved fibronectin under plasminogen-free conditions yielding a limited number of high molecular weight cleavage products.

Animals↗

PubMed1987

Refinement of a T-lymphocyte cloning assay to quantify the in vivo thioguanine-resistant mutant frequency in humans.

Cell cloning by limiting dilution in 96-well microtiter plates has been employed to isolate colonies of human T-lymphocytes resistant to the purine analogue, 6-thioguanine (TG). These colonies show stability of the TG-resistant (TG1) phenotype, lack hypoxanthine guanine phosphoribosyl transferase (HPRT) activity and thus appear to be the result of in vivo somatic cell mutation events. In order to employ this T-lymphocyte cloning assay for quantitative determination of the in vivo TGr mutant frequency in humans, we have defined the optimal conditions for T-cell colony formation with both nonselected and TG-selected cells. The parameters investigated include medium, serum, amount of the mitogen phytohaemagglutinin, amount of T-cell growth factor (TCGF) and the number of irradiated feeder or accessory cells. Under the optimal conditions, the fraction of positive wells is proportional to the number of cells plated per well with both nonselection and TG selection conditions. T-cell cloning efficiencies therefore are independent of inoculum size. There was some evidence for a decline in TGr mutant cell cloning at densities greater than 2 x 10(4) cells per round-bottom well, possibly due to metabolic cooperation between wild-type and mutant cells. The conditions defined in this study appear to provide a quantitative measurement of the in vivo TGr mutant frequency in human T-lymphocytes.

Antigen-Presenting Cells↗

PubMed1987

Molecular analyses of in vivo hprt mutations in human T-lymphocytes. I. Studies of low frequency 'spontaneous' mutants by Southern blots.

Fifty wild-type and 164 in vivo-derived hprt mutant T-cell clones obtained from eight non-mutagen-exposed adult males with mutant frequency values in the normal range (usually less than 10 X 10(-6) were studied by Southern blot analyses to determine frequency and extent of gross structural alterations in the hprt gene. Sixteen (9.8%) of the mutant clones showed hprt changes. No site or type of lesion predominated. Relative frequencies of gross structural alterations in the recovered hprt mutants did not differ among the eight individuals, within limits detectable by the study. DNA from 201 of these 214 clones was also studied with a T-cell receptor (TCR) beta gene probe as a marker for independence of in vivo-derived clones. Some clones were also studied with a TCR gamma gene probe. Ninety-four percent of wild-type and 89% of the hprt mutants were found to originate from independent in vivo precursors. Therefore, most of the recovered hprt mutants in the study were presumably derived from separate in vivo mutations. For non-mutagenized adults with normal mutant frequencies, in vivo mutant frequencies are thus reasonable approximations of in vivo mutation frequencies, although elsewhere we show that this is not necessarily true for individuals with grossly elevated mutant frequencies.

Adult↗

PubMed1986

An anticatalytic monoclonal antibody to avian plasminogen activator: its effect on behavior of RSV-transformed chick fibroblasts.

A monoclonal antibody has been raised against the serine protease, plasminogen activator (PA) produced by Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF), and selected for its ability to inhibit the catalytic activity of PA. The high specificity and anticatalytic nature of the antibody has allowed probing of the direct role of PA in cellular behavior. Microgram quantities of monoclonal IgG inhibit the overgrowth and the morphological changes associated with RSVCEF transformation and the degradation of extracellular matrix mediated by RSVCEF, indicating a catalytic role for PA in these cellular processes. Specific cleavage of extracellular matrix proteins by immunoaffinity-purified PA in the complete absence of plasminogen demonstrates a direct catalytic involvement of PA in matrix degradation.

Animals↗

PubMed1982

Eicosanoid production in rat blood as affected by fasting and dietary fat.

The effects of fasting and dietary linoleate on eicosanoid production in clotting rat blood incubated 10 minutes at 37 degrees C was studied. Fasting increased eicosanoid synthesis. Dietary manipulation of eicosanoid levels was best demonstrated in the fed rat model. TXB2, PGE2, F2 alpha, and E1 synthesis increased when dietary linoleate increased from 0.0 to 0.4% calories; from 0.4 to 29% linoleate calories there was no change in PG synthesis, but a decrease in TXB2 production was apparent.

Animals↗

PubMed1979

Serum amyloid A to monitor cancer dissemination.

The level of serum amyloid A, a protein previously found to behave as an acute-phase reactant, was measured by a radioimmunoassay in 621 patients with various neoplastic diseases free of inflammatory processes. In all but eight of the 289 patients with solid tumors with distant metastases, in all patients with myelocytic leukemia with high leukocyte counts, and in all patients with advanced lymphoma, the serum amyloid A level was above 400 ng/mL. It was below this value in all tested patients with lymphocytic leukemia and in 250 of 270 patients with solid malignant tumors with localized or regional disease. Among the 20 patients from this latter group with levels higher than 400 ng/mL, 16 developed distant metastases within 214 days from the initial measurement. The serum amyloid A level decreased significantly in patients with lymphoma and those with metastatic solid tumors who responded to chemotherapy. Thus the level of serum amyloid A can be used as a biochemical marker that discriminates between disseminated and localized or regional disease, and monitors the response to therapy.

Adult↗