Robert DeMars

Mutant enzymatic and cytological phenotypes in cultured human fibroblasts.

Fibroblasts were cultured from the cells of two children who shared some characteristics of Hurler syndrome, but they did not show corneal clouding and excessive excretion of mucopolysaccharides. The fibroblasts differ from those of controls and of patients with typical Hurler syndrome or other mucopolysaccharidoses in that they have abundant cytoplasmic inclusions, striking diminutions in beta-glucuronidase, and elevations in acid phosphatase.

Resistance of cultured human fibroblasts and other cells to purine and pyrimidine analogues in relation to mutagenesis detection.

Abstract In vitro enumeration of diploid human cell variants that are resistant to purine analogues is a possible method of detecting mutagenesis. Their incidences can be increased by the known mutagens, X-rays and N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG). Usefulness of this method depends on the kinds of hereditary changes that confer analogue-resistance on somatic cells. If resistance usually results from changes in genetic material, in vitro studies could be useful indicators of mutagenic effects on somatic cells and germ cells in vivo . If epigenetic changes are primarily responsible for analogue-resistant variants, their enumeration might not provide information relevant to germinal mutations but would still be a useful way to detect induction of general kinds of stable phenotypic changes that could cause cancer. This article outlines hypothetical epigenetic and genetic causes of somatic cell variation and a prospective genetic analysis of human cell variants that are resistant to 8-azaguanine (AG) or 2,6-diaminopurine ( (DAP). Recent evidences and arguments favoring epigenetic origins of resistance to base-analogues are inconclusive. The often cited high rate of changes causing impermeability to BUdR in hamster cells is based on one improperly executed determination. Comparisons of rates of variation conferring BUdR-resistance on cultured haploid and diploid frog cells included diploid variants that did not behave as mutants and ignored major sources of error in estimating mutation rates. AG-resistance could result from recessive mutations in X-chromosomal genes but comparisons of rates of mutation in hamster cells of different ploidies did not provide information about the numbers of X-chromosomes in the variants. Reports that normal rodent HGPRT reappeared in hybrids of enzyme-deficient rodent cells and HGPRT-containing cells of other species or in the rodent cells alone in response to the conditions of cell hybridization did not include adequate controls for reversions in mutant genes of the rodent cells. Questions about the epigenetic and genetic origins of analogue-resistance are mostly unanswered. It remains possible that some kinds of abnormal epigenetic changes cause somatic disease. Specific methods for detecting their occurrence and responsiveness to environmental factors should be devised by focusing efforts on traits that are normally subject to epigenetic regulation. Derepression of genes on the inactive X-chromosome and of liver phenylalanine hydroxylase production are presented as possible examples of abnormal epigenetic changes that could be quantitatively studied by direct selection in vitro .

Somatic cell mutation detection and quantification of X-ray-induced mutation in cultured, diploid human fibroblasts

Abstract We describe a system for detecting somatic cell mutation to 8-azaguanine (8AG) resistance in cultured, diploid human fibroblasts. Hypoxanthine-guanine phosphoribosyltransferase (HG-PRT)-deficient, AG-resistant fibroblasts from boys with the X-chromosomal, Lesch-Nyhan (L-N) mutation served as one type of prototype mutant cells. Both spontaneous and X-ray-induced mutation were studied. Recovery of L-N cells was a function both of density of normal cells and of the AG concentration used for selection. Optimum recovery was achieved at an initial inoculum of 2·10 4 normal cells per 60 mm diameter culture dish and an AG concentration of 8·10 −6 M . Efficiency of recovery was between 39 and 90% and controls to determine this efficiency were included in mutagenesis experiments. Attempts to free normal cell populations of pre-existing AG-resistant mutant cells by pregrowth in HAT medium failed because, unlike L-N mutants, most spontaneous AG-resistant mutants can grow in HAT medium. Although pre-existing mutants probably caused overestimation, the average spontaneous mutation rate derived from our experiments was 4.5·10 −6 per cell generation. Eliminating one large-yieldv experiment reduced this estimate to 1.9·10 −6 . Clonal survival of cultured human fibroblasts as a function of X-ray dose was studied. X-Irradiation increased the mutation rate above spontaneous background. Minimum estimates of the increases were 1.13·10 −9 per R per cell at 75 R, 7.49·10 −8 per R per cell at 125 R, 6.87·10 −8 per R per cell at 150 R and 2.16·10 −7 per R per cell at 250 R. The total mutagenic effect and the induced mutation rate appeared to be dose-dependent. Normal parental cell strains and their derived AG-resistant mutants had similar X-ray sensitivities indicating that X-rays induced mutations rather than selected for pre-existing mutants. Because of the realism of the cultured diploid, human fibroblast model vis-a-vis in vivo human cellular events, the mutation detection system described herein is proposed as being potentially useful for environmental monitoring.

The spontaneous azaguanine-resistant mutants of diploid human fibroblasts

SummaryThe range of incidences of azaguanine-resistant colonies in cultures of fibroblasts from 16 unrelated humans was 0.4×10-6 to 19×10-6 and the mean value was 4.1×10-6. A fluctuation test showed that most or all of the mutant colonies derived from mutations that occurred during in vitro proliferation of the fibroblasts and before exposure to azaguanine. The estimated rate of spontaneous mutation was 0.45×10-6 to 1.8×10-6 per cell generation. At least ten independent mutants, comprising two general classes, were studied. Class I mutants were a minority and resembled cells from boys having the Lesch-Nyhan syndrome: they had very little HG-PRT activity, showed maximum resistance to azaguanine and could not utilize hypoxanthine for growth. At least 90% of the mutants were in Class II: their apparent HG-PRT activities ranged between normal and Lesch-Nyhan amounts, they were partially sensitive to azaguanine and they could utilize hypoxanthine. Some Class II mutants resembled cells cultured from a family having an X-chromosomal mutant gene that does not cause the Lesch-Nyhan syndrome but does confer resistance to azaguanine, although the quantity of HG-PRT activity is apparently normal and hypoxanthine can be utilized. Electrophoretic differences between the HG-PRT activities of normal and mutant strains were not detected but other qualitative alterations were observed in some mutants.

The D1 trisomy syndrome

This paper further defines the phenotype which results from trisomy for a particular autosome of the D group (13–15) in the cells of the developing embryo. From our 7 patients plus 7 additional cases reported by others, the principal pattern of anomalies in these grossly malformed babies is presented. Though there is variability in the expression of the D 1 trisomy from patient to patient, the total pattern of anomalies is specific enough to allow for clinical recognition in the nursery for newborn infants. As with the other autosomal trisomy syndromes (Downs syndrome and the 18 trisomy syndrome), this condition occurs more commonly at older maternal age.

Diploid azaguanine-resistant mutants of cultured human fibroblasts.

Two azaguanine-resistant clones of cultured, human fibroblasts were isolated from unrelated strains of karyotypically normal, male cells. The most resistant mutant has little hypoxanthine-guanine phosphoribosyltransferase activity, is virtually unable to incorporate hypoxanthine (a normal substrate of the enzyme), and resembles fibroblasts cultured from boys with the Lesch-Nyhan syndrome. The less resistant mutant has about one-third as much enzyme activity as its parent strain and is less able to utilize hypoxanthine. Both mutants are morphologically and karyotypically normal. These mutations may have occurred at the X-chromosomal, hypoxanthine-guanine phosphoribosyltransferase locus and may provide a realistic experimental model for studying mutation in human genetic material.

Specificity of monoclonal antibodies directed against human and murine class II histocompatibility antigens as analyzed by binding to HLA-deletion mutant cell lines

The specificity of 70 monoclonal anti-Ia monoclonal antibodies (MoAbs) (18 mouse allo-induced and 52 rodent anti-human) was studied with a panel of 17 HLA-deletion mutants that were derived from a single parent line and vary in expression of Ia antigens due to deletion of different subregions of HLA. MoAb binding was analyzed both by ELISA and flow microfluorometry. Characterization of the MoAbs with respect to specificity for products of subregions of a DR1-DC1-SB2 haplotype revealed great complexity. Many antibodies were quite specific for DR-linked determinants (26 MoAbs), DC-linked determinants (5 MoAbs), or determinants indistinguishable from SB (4 MoAbs). However, many MoAbs bound to products of more than one subregion: DR + SB (+/- weak DC) (22 MoAbs); DR + DC (3 MoAbs); or DR + DC + SB (1 MoAb). Furthermore, a number of the MoAbs bound unequally to products of the two HLA haplotypes analyzed, particularly among those recognizing DC1-linked determinants and the murine alloinduced MoAbs. Finally, despite strong structural homologies of murine I-A to human DC and murine I-E to human DR, the intraspecies cross-reactions of MoAbs do not closely follow that pattern. These data: (1) illustrate the usefulness of HLA-deletion mutant cell lines for analysis of the specificity of MoAbs and for delineation of HLA subregions; (2) demonstrate the great diversity of MoAbs specific for class II molecules and the high frequency of MoAbs that bind to products of more than one Ia subregion, particularly DR and SB. In view of such complexity, many (perhaps most) MoAbs cannot be relied on to unambiguously identify products of a particular Ia subregion, without extensive characterization.

Mosaicism of peripheral blood lymphocyte populations in females heterozygous for the Lesch-Nyhan mutation

Phytohemagglutinin-stimulated thymidine-H3 incorporation into peripheral blood lymphocytes in vitro was inhibited by 8-azaguanine (AG) in cells from normal individuals, but not in cells from boys with the Lesch-Nyhan (L-N) syndrome. Artificial cell mixtures in which the AG-resistant minority L-N population comprised more than 1.0% of the total were clearly differentiated from normals by this technique. The method was used to search for blood cell mosaicism in females heterozygous for the X-chromosomal L-N mutation. In three adolescent girls, a minority population of AG-resistant lymphocytes comprising 5–10% of the total was found, while no such mosaicism was detected in a heterozygous woman of 40. Although blood cell mosaicism had not previously been found in female carriers of the L-N mutation, the sensitivity of the present method exceeds that of previous studies. Our findings suggest that in vivo selection against lymphocytes with the L-N mutation does occur but is not absolute and may be age related. The present method has applications in studying this phenomenon, and, with modification, may provide a direct assay system to detect the in vivo frequencies of AG-resistant lymphocytes.

Definition of Five New Simian Immunodeficiency Virus Cytotoxic T-Lymphocyte Epitopes and Their Restricting Major Histocompatibility Complex Class I Molecules: Evidence for an Influence on Disease Progression

ABSTRACT Simian immunodeficiency virus (SIV) infection of the rhesus macaque is currently the best animal model for AIDS vaccine development. One limitation of this model, however, has been the small number of cytotoxic T-lymphocyte (CTL) epitopes and restricting major histocompatibility complex (MHC) class I molecules available for investigating virus-specific CTL responses. To identify new MHC class I-restricted CTL epitopes, we infected five members of a family of MHC-defined rhesus macaques intravenously with SIV. Five new CTL epitopes bound by four different MHC class I molecules were defined. These included two Env epitopes bound by Mamu-A*11 and -B*03 and three Nef epitopes bound by Mamu-B*03, -B*04, and -B*17. All four restricting MHC class I molecules were encoded on only two haplotypes (b or c). Interestingly, resistance to disease progression within this family appeared to be associated with the inheritance of one or both of these MHC class I haplotypes. Two individuals that inherited haplotypes b and cseparately survived for 299 and 511 days, respectively, while another individual that inherited both haplotypes survived for 889 days. In contrast, two MHC class I-identical individuals that did not inherit either haplotype rapidly progressed to disease (survived <80 days). Since all five offspring were identical at their Mamu-DRBloci, MHC class II differences are unlikely to account for their patterns of disease progression. These results double the number of SIV CTL epitopes defined in rhesus macaques and provide evidence that allelic differences at the MHC class I loci may influence rates of disease progression among AIDS virus-infected individuals.

Direct Detection and Magnetic Isolation of Chlamydia trachomatis Major Outer Membrane Protein-Specific CD8+ CTLs with HLA Class I Tetramers

We recently identified HLA class I-presented epitopes in the major outer membrane protein (MOMP) of Chlamydia trachomatis that elicit CTL responses in human genital tract infections. T cells possessing cytolytic activities specific for these epitopes could be detected following in vitro stimulation of peripheral blood CD8+ T cells with peptides. In the present study we used HLA-A2 tetramers for detailed characterization of MOMP-specific CTL responses. Ex vivo tetramer analysis detected MOMP-specific T cells in the peripheral blood of infected individuals at significant frequencies (0.01–0.20% of CD8+ T cells). After in vitro stimulation with peptides, the frequencies of MOMP peptide-specific T cells increased up to 2.34% of CD8+ T cells in bulk cultures. In contrast, HLA-A2/MOMP tetramer-binding T cells were virtually undetectable in the peripheral blood from uninfected individuals, either ex vivo or after 3 wk of in vitro peptide stimulation of their T cells. Magnetically sorted, tetramer-bound T cells specifically lysed peptide-pulsed targets as well as C. trachomatis-infected epithelial cells with nearly 50-fold greater per cell efficiency than that of unsorted populations. This study provides conclusive evidence of in vivo induction of HLA class I-restricted CD8+ CTL responses to C. trachomatis MOMP. Direct detection of these cells with tetramers will allow their further characterization without prior manipulation and facilitate monitoring of CTL responses during infections and in immunization trials with MOMP-based vaccines.