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Biomedical subjects

R G Langlois

Publications and source records attributed to R G Langlois.

At least 19 recordsLinked to original sources
PubMed1995

Sensitivity of somatic mutations in human umbilical cord blood to maternal environments.

To assess the potential effect of maternal environments on human embryonic/fetal somatic mutation, we measured the frequencies of hypoxanthine-guanine phosphoribosyltransferase (HPRT, hprt gene), mutant T lymphocytes (Mf), and glycophorin A (GPA) variant erythrocytes (Vf) of both allele-loss (phi/N) and allele-loss-and-duplication (N/N) phenotypes in umbilical cord blood. The mean hprt Mf (1.40 +/- 1.11 x 10(-6), N = 66) and GPA Vf (phi/N 4.0 +/- 2.2 x 10(-6), N = 114; N/N 2.7 +/- 2.0 x 10(-6), N = 91) were significantly lower than those previously reported for adult populations. In addition, the hprt Mf was significantly higher than that of a published study of newborn cord blood samples from a geographically distant population (0.64 +/- 0.41 x 10(-6), N = 45, P < 0.01; t test, P < 0.01, Mann-Whitney U test). An examination of the demographic data from these two populations led to the sampling of 10 additional newborns specifically matched to the published study for maternal socioeconomic status. The hprt Mf (0.70 +/- 0.49 x 10(-6)) of this selected population was consistent with the published report and significantly lower than that of our initial population (P < 0.03, t test; P < 0.01, Mann-Whitney U test). These results indicate that there is an environmental effect related to maternal socioeconomic status on the frequency of embryonic/fetal somatic mutations. Molecular analyses of hprt mutants from this cohort with elevated Mf revealed a significant decrease in the relative contribution of gross structural mutations to the overall Mf (25 of 38, 66% vs. 34 of 41, 83%, P = 0.024, chi 2 test), suggesting that the higher Mf resulted from an elevated level of "point" mutations. No individual maternal demographic or environmental factor was identified as contributing more significantly than other any factor to the observed variability in hprt Mf or GPA Vf.

Adult↗
PubMed1992

Comparison of strategies to detect and quantitate uniquely marked cells in intra- and inter-species hemopoietic chimeras.

Evaluation of the outcome of successful bone marrow transplantation and indepth studies of transplantation biology rely increasingly upon detection and enumeration of donor hemopoietic cells in the transplanted recipients. The ability to detect and enumerate low levels of donor engraftment in interphase cell subpopulations in hemopoietic chimeras is particularly important for studies of mixed lineage chimerism, early relapse manifestations, and engraftment of subpopulations present at low frequency. We describe and compare the sensitivity and specificity of DNA-based detection strategies (fluorescence in situ hybridization, in vitro DNA amplification using the polymerase chain reaction) and flow cytometric analysis of cell surface markers to detect cells carrying marker DNA or proteins in syngeneic (mouse-to-mouse) and xenogeneic (mouse-to-human, monkey, sheep) backgrounds. DNA-based detection strategies offer advantages of rapid analysis and enumeration of target cell frequencies with detection sensitivities approximating 10(-4). The sensitivity of immunofluorescence-linked flow cytometric-based detection of nucleated leukocytes approached 10(-3), whereas flow cytometric-based detection of fixed human erythrocytes was feasible at cell frequencies of 10(-5). Data described in this manuscript should facilitate selection of appropriate methodologies for assessment of hemopoietic chimerism following transplantation.

Animals↗
PubMed1992

Biodosimetry for a radiation worker using multiple assays.

Four state-of-the-art biodosimeters--GPA mutations, chromosome translocations, micronuclei, and dicentrics--were used to evaluate a radiation worker who believed that the official dosimetry records substantially underestimated his actual dose. Dosimetry records indicated that the worker received 0.56 Sv during a 36-y employment history, always within the dose limits. In contrast, the worker believed that his dose equivalent may have been more than 2.5 Sv because much of the exposure was received during the early days of health physics when dosimetry capabilities and practices were not as good as they are today. Because there are no biodosimetric assays that have been fully validated for the long-term low-level exposures received by the worker, we did not expect to obtain particularly useful point-estimates of dose. However, because the discrepancy between the dosimetry records and the worker's belief was so large, we believed that biodosimetry using multiple assays together with probabilistic assessment of the uncertainties would provide useful insight. Results showed that the frequencies of chromosome translocations and GPA mutations (stable biodosimeters) were significantly elevated when compared with those for unexposed controls. Our analysis suggests that dose-equivalent estimates in the approximately 0.4 to approximately 2 Sv range (which include the value in the dosimetry records) cannot be confidently excluded at this time based on biodosimetry; however, a value greater than 2.5 Sv appears unlikely. Important new information on the temporal stability of chromosome translocations is also presented.

Chromosome Aberrations↗
PubMed1992

Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero.

Hemopoietic stem cells from human fetal liver were transplanted in utero into preimmune fetal sheep (48-54 days of gestation). The fate of donor cells was followed using karyotype analysis, by immunofluorescence labeling with anti-CD antibodies, and by fluorescent in situ hybridization using human-specific DNA probes. Engraftment occurred in 13 of 33 recipients. Of five live born sheep that exhibited chimerism, all expressed human cells in the marrow, whereas three expressed them in blood as well. Engraftment was multilineage (erythroid, myeloid, and lymphoid) and human hemopoietic progenitors (multipotent colony-forming units, colony-forming units-granulocyte, macrophage, and erythroid burst-forming units) capable of forming colonies in vitro were detected in all five lambs for greater than 2 yr. These progenitors responded to human-specific growth factors both in vitro and in vivo. Thus the administration of recombinant human IL-3 and granulocyte macrophage-colony-stimulating factor to chimeric sheep resulted in a 2.1-3.4-fold increase in the relative expression of donor (human) cells. These results demonstrate that the permissive environment of the preimmune fetal sheep provides suitable conditions for the engraftment and long-term multilineage expression of human hemopoietic stem cells in a large animal model. In this model, donor human cells appear to retain certain phenotypic and functional characteristics that can be used to manipulate the size of donor cell pool.

Animals↗
PubMed1991

Novel biodosimetry methods applied to victims of the Goiânia accident.

Two biodosimetric methods under development at the Lawrence Livermore National Laboratory were applied to five persons accidentally exposed to a 137Cs source in Goiânia, Brazil. The methods used were somatic "null" mutations at the glycophorin A locus detected as missing proteins on the surface of blood erythrocytes and chromosome translocations in blood lymphocytes detected using fluorescence in-situ hybridization. Biodosimetric results obtained approximately 1 y after the accident using these new and largely unvalidated methods are in general agreement with results obtained immediately after the accident using dicentric chromosome aberrations. Additional follow-up of Goiânia accident victims will 1) help provide the information needed to validate these new methods for use in biodosimetry and 2) provide independent estimates of dose.

Accidents↗
PubMed1990

Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).

Antibodies, Monoclonal↗
PubMed1990

An improved flow cytometric assay for somatic mutations at the glycophorin A locus in humans.

An improved method has been developed for the glycophorin A assay for somatic cell mutations in humans. The new assay, named the "BR6" assay, can be performed on a commercially available, single-beam flow cytometer, in contrast to the previously described 1W1 assay that required a dual-beam flow sorter. A modified cell labeling method developed for the BR6 assay results in improved separation of normal and mutant phenotype cells, as well as eliminating some cellular artifacts that affected the 1W1 assay. Parallel measurements on samples from 17 normal donors showed that the BR6 assay yields comparable variant cell frequencies and improved measurement precision compared with the 1W1 assay. A detailed analysis of three individuals who showed large differences in background variant frequency with the 1W1 assay confirmed that these differences could also be detected with the BR6 assay. A dramatically elevated variant cell frequency was seen with the BR6 assay of an individual exposed to a high level of ionizing radiation in an accident at Goiania, Brazil.

Adult↗
PubMed1990

The effect of chemotherapy on the in vivo frequency of glycophorin A 'null' variant erythrocytes.

A human in vivo somatic cell assay based on the enumeration of variant erythrocytes lacking expression of an allelic form of the cell-surface sialoglycoprotein, glycophorin A, was applied to the study of blood samples from patients obtained prior to, during, and following chemotherapy for malignant disease in order to determine the effect of mutagenic chemical agents on the frequency of variant cells. In 22 patients assayed prior to therapy, the mean variant cell frequency was 11.9 per million, which was not significantly different from that observed in healthy controls. In an initial cross-sectional survey, blood samples were obtained at various times during and after therapy from 30 patients diagnosed with a variety of malignancies who were treated with one or more known mutagenic agents including adriamycin, bleomycin, cis-platinum, cyclophosphamide, dacarbazine, etoposide, lomustine, mechlorethamine, melphalan, mitomycin C, and procarbazine. Significant elevations in the mean frequency of variant cells over pre-therapy and normal levels were observed in samples obtained during and after therapy. In a time-series study, 14 breast cancer patients treated with CAF (cyclophosphamide, adriamycin, 5-fluorouracil), CMF (cyclophosphamide, methotrexate, 5-fluorouracil), or VMF (vinblastine, methotrexate, 5-fluorouracil) adjuvant chemotherapy were sampled repeatedly during and after therapy. For the CAF and CMF patients an increase in the frequency of variant cells was observed with a lag in the appearance of induced variants after initiation of therapy; variant frequencies gradually increased during therapy reaching a maximum at or shortly after the end of therapy, then declined to near pre-therapy levels within 6 months. The maximum level of induced variants ranged from 2- to 7-fold over pre-therapy or normal levels depending on the combination of agents used. The breast cancer patients treated with both adriamycin and cyclophosphamide showed consistent elevations in the frequency of variant cells; patients treated only with cyclophosphamide showed lower and more variable elevations. The data demonstrate that mutagenic chemotherapy agents induce elevated levels of glycophorin A variant erythrocytes consistent with the hypothesis that variant cells result from somatic mutation. The elevations in variant cells were transient, suggesting that these agents primarily affect the rapidly cycling committed erythroid cell population.

Adolescent↗
PubMed1990

Determination of guanine-plus-cytosine content of bacterial DNA by dual-laser flow cytometry.

A dual-laser flow cytometer was used to analyse different species of bacteria for the molar percentage of guanine-plus-cytosine (% G + C) without the need for DNA extraction or purification. Ethanol-fixed bacterial cells were stained with a combination of DNA-specific fluorochromes, Hoechst 33258 and chromomycin A3, which bind to AT- and GC-rich regions of DNA, respectively. A linear relationship (r = 0.99) was demonstrated between the log of the ratio of chromomycin A3 to Hoechst 33258 fluorescence and the log of the % G + C as determined by thermal denaturation (Tm) or buoyant density centrifugation (Bd) methods. Linearity was maintained for all bacterial species tested over the range of 28-67% G + C. A standard curve was constructed using five strains whose % G + C had been determined by other methods. From the equation describing this line, the % G + C values of nine other strains with known DNA base composition, together with the five strains used to construct the curve, were calculated using the chromomycin A3 to Hoechst 33258 ratio and were in agreement with values obtained by Tm, Bd or HPLC. The reproducibility of flow cytometric analysis (mean error 0.7% G + C) compared well with the reproducibility of other methods. Mixtures containing two species were also analysed. Two cell populations could be discerned in mixtures containing two species which differed in base composition by as little as 4% G + C.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosine↗
PubMed1990

Multiple endpoints for somatic mutations in humans provide complementary views for biodosimetry, genotoxicity and health risks.

While monitoring people with multi-endpoint analysis is only just beginning, the indications of complementarity of these assays are already evident. Since two different tissue types are analyzed, effects of uneven exposure or tissue-specific sensitivities or development kinetics and persistence of each cell type can be addressed. The differing analytical techniques in determining variant cells lead to particular advantages for each assay procedure. The GPA assay can be performed rather quickly on small blood volumes (as little as 0.1 ml). A similar potential exists for the Hb assay, although sophisticated instrumentation development is still required to make this assay easily performable. Both the HPRT and HLA assays require long term tissue growth and many-fold larger (10-30 ml) blood samples. However, both the HPRT and HLA assays have the advantage that they select variant cells that contain nuclei, so that detailed characterization of their mutagenesis is possible using Southern blots, hybridization analysis, and gene sequencing. Since erythrocytes have no nucleic acids, so these effects cannot be measured using the GPA or Hb assay. Other differences between the assays are the target size, chromosomal location, and control signals. Each gene locus is on a different chromosome, with GPA, HLA and Hb genes on autosomes and HPRT on the X chromosome. The target size for GPA and HPRT are about 40kb, whereas HLA A locus is 5kb and the Hb target is one base. These differences should result in much different sensitivity to different mutagenic phenomena, such as radiation effects or different chemical effects. The multiple differences between assays that are designed to detect similar kinds of events occurring in vivo in humans leads us to conclude that all these assays could well be performed on the same blood samples, and the information gained would be much more than the separate assays could yield. Biodosimetry and estimation of cancer initiation could become realistic goals instead of simply significant desires.

Aging↗
PubMed1989

Evidence for increased in vivo mutation and somatic recombination in Bloom's syndrome.

The glycophorin A assay was used to estimate the frequency of mutations that accumulate in vivo in somatic cells of persons with Bloom's syndrome (BS). This assay measures the frequency in persons of blood type MN of variant erythrocytes that lack the expression of one allelic form of glycophorin A, presumably due to mutational or recombinational events in erythroid precursor cells. Samples of blood from persons with BS showed dramatic 50- to 100-fold increases in the frequency of variants of three types, those with a hemizygous phenotype, those with a homozygous phenotype, and those with what appears to be partial loss of the expression of one locus. The high frequency of homozygous variants, genetic evidence for altered allelic segregation of a specific biochemical locus, provides evidence for increased somatic crossing-over in vivo in BS. An increased generation of functional hemizygosity and homozygosity in their somatic cells may play an important role in the extreme cancer risk of persons with BS.

Adolescent↗
PubMed1989

Evidence for an elevated frequency of in vivo somatic cell mutations in ataxia telangiectasia.

Somatic cell mutation frequency in vivo was measured in individuals with high cancer risk who were from ataxia telangiectasia (A-T) families. The assay for somatic mutation measures the frequency of variant erythrocytes which are progeny of erythroid precursor cells with mutations that result in a loss of gene expression at the polymorphic glycophorin A (GPA) locus. Samples from 14 of 15 A-T homozygotes showed high frequencies of GPA gene expression-loss variant cells with normal expression of only one of the two alleles at the GPA locus (i.e., GPA hemizygous variant cells). The mean elevation of the frequency of hemizygous variant cells over those in normal controls and unaffected family members was 7-14-fold. A-T homozygotes also showed an increase in the frequency of cells in which one allele at the GPA locus had lost expression and in which the remaining allele was expressed at a homozygous level (i.e., GPA homozygous variant cells). Family members who are obligate A-T heterozygotes did not appear to have a significantly elevated frequency of GPA hemizygous or homozygous variant cells. These indications of elevated in vivo frequencies of variant erythrocytes in A-T homozygotes support a causal link between susceptibility to somatic mutation and susceptibility to cancer.

Adult↗
PubMed1987

Evidence for increased somatic cell mutations at the glycophorin A locus in atomic bomb survivors.

A recently developed assay for somatic cell mutations was used to study survivors of the atomic bomb at Hiroshima. This assay measures the frequency of variant erythrocytes produced by erythroid precursor cells with mutations that result in a loss of gene expression at the polymorphic glycophorin A (GPA) locus. Significant linear relations between variant frequency (VF) and radiation exposure were observed for three different variant cell phenotypes. The spontaneous and induced VFs agree with previous measurements of radiation-induced mutagenesis in other systems; this evidence supports a mutational origin for variant cells characterized by a loss of GPA expression and suggests that the GPA assay system may provide a cumulative dosimeter of past radiation exposures. VFs for some survivors differ dramatically from the calculated dose response, and these deviations appear to result primarily from statistical fluctuations in the number of mutations in the stem-cell pool. These fluctuations allow one to estimate the number of long-lived hemopoietic stem cells in humans.

Antibodies, Monoclonal↗
PubMed1987

Coordinate glycosylation and cell surface expression of glycophorin A during normal human erythropoiesis.

The expression of two epitopes on glycophorin A (GPA) during erythroid development was examined on normal human bone marrow using quantitative flow cytometry. The highly correlated binding of two monoclonal antibodies, one sensitive and the other insensitive to glycosylation, indicated that the two epitopes were coordinately expressed during erythroid development. Both antigens reached a maximum expression during the early normoblast stage and were maintained at a constant amount per cell throughout further maturation to erythrocytes. These data suggest that glycosylation of GPA, as detected by antibodies recognizing blood group (M) and (N) antigens, does not increase during erythroid maturation.

Antibodies, Monoclonal↗
PubMed1986

Measurements of the frequency of human erythrocytes with gene expression loss phenotypes at the glycophorin A locus.

An assay method is described for determining the frequency of human erythrocytes having a gene expression loss phenotype at the glycophorin A locus presumably due to in vivo somatic mutational events in erythroid precursor cells. Monoclonal antibodies specific for the M and N glycophorin A alleles are used to identify variant cells that lack the expression of one allele in blood samples from MN heterozygotes. Flow cytometry and sorting are used to enumerate and purify variant cells. Using three different antibody combinations which are sensitive to the loss of either the M or the N allele, we find that variant cells occur at a frequency of 1 X 10(-5) in normal donors. We also detect variant cells with an apparent homozygous phenotype suggesting that events leading to homozygosity may occur at similar frequencies to gene loss events. Significant increases in variant cell frequency are observed in cancer patients after exposure to mutagenic chemotherapy drugs.

Adult↗