Faculty Bibliography

We have examined the exposure and conservation of antigenic epitopes on the surface envelope glycoproteins (gp120 and gp41) of 26 intact, native, primary human immunodeficiency virus type 1 (HIV-1) group M virions of clades A to H. For this, 47 monoclonal antibodies (MAbs) derived from HIV-1-infected patients were used which were directed at epitopes of gp120 (specifically V2, C2, V3, the CD4-binding domain [CD4bd], and C5) and epitopes of gp41 (clusters I and II). Of the five regions within gp120 examined, MAbs bound best to epitopes in the V3 and C5 regions. Only moderate to weak binding was observed by most MAbs to epitopes in the V2, C2, and CD4bd regions. Two anti-gp41 cluster I MAbs targeted to a region near the tip of the hydrophilic immunodominant domain bound strongly to >90% of isolates tested. On the other hand, binding of anti-gp41 cluster II MAbs was poor to moderate at best. Binding was dependent on conformational as well as linear structures on the envelope proteins of the virions. Further studies of neutralization demonstrated that MAbs that bound to virions did not always neutralize but all MAbs that neutralized bound to the homologous virus. This study demonstrates that epitopes in the V3 and C5 regions of gp120 and in the cluster I region of gp41 are well exposed on the surface of intact, native, primary HIV-1 isolates and that cross-reactive epitopes in these regions are shared by many viruses from clades A to H. However, only a limited number of MAbs to these epitopes on the surface of HIV-1 isolates can neutralize primary isolates

Because immunologic classification of human immunodeficiency virus type 1 (HIV) might be more relevant than genotypic classification for designing polyvalent vaccines, studies were undertaken to determine whether immunologically defined groups of HIV ('immunotypes') could be identified. For these experiments, the V3 region of the 120-kDa envelope glycoprotein (gp120) was chosen for study. Although antibodies (Abs) to V3 may not play a major protective role in preventing HIV infection, identification of a limited number of immunologically defined structures in this extremely variable region would set a precedent supporting the hypothesis that, despite its diversity, the HIV family, like the V3 region, might be divisible into immunotypes. Consequently, the immunochemical reactivities of 1,176 combinations of human anti-V3 monoclonal Abs (MAbs) and V3 peptides, derived from viruses of several clades, were studied. Extensive cross-clade reactivity was observed. The patterns of reactivities of 21 MAbs with 50 peptides from clades A through H were then analyzed by a multivariate statistical technique. To test the validity of the mathematical approach, a cluster analysis of the 21 MAbs was performed. Five groups were identified, and these MAb clusters corresponded to classifications of these same MAbs based on the epitopes which they recognize. The concordance between the MAb clusters identified by mathematical analysis and by their specificities supports the validity of the mathematical approach. Therefore, the same mathematical technique was used to identify clusters within the 50 peptides. Seven groups of peptides, each containing peptides from more than one clade, were defined. Inspection of the amino acid sequences of the peptides in each of the mathematically defined peptide clusters revealed unique 'signature sequences' that suggest structural motifs characteristic of each V3-based immunotype. The results suggest that cluster analysis of immunologic data can define immunotypes of HIV. These immunotypes are distinct from genotypic classifications. The methods described pave the way for identification of immunotypes defined by immunochemical and neutralization data generated with anti-HIV Env MAbs and intact, viable HIV virions

Methods are needed for retrospective estimation of long-term ozone exposures in epidemiologic studies. The overall objective of this study was to evaluate whether data from available U.S. ozone monitoring sites are useful for estimating lifetime ozone exposures of young adults (for example, college students). Several aspects of this question were evaluated. First, we applied and (compared several spatial interpolation methods to a set of long-term average ozone data from all U.S. monitoring sites in operation from 1981 through 1990. Interpolation methods included simple and weighted averages, linear regression, and, in an exploratory way, kriging. The comparison of methods was carried out for five different metrics of ozone concentration: the daily one-hour maximum (MAX1) and eight-hour maximum (MAX8), the average ozone concentrations between 10 a.m. and 6 p.m. (MID8) and between 10 a.m. and 10 p.m. (MID12), and the sum of all hourly ozone concentrations greater than or equal to 60 parts per billion (ppb) (SUM06). We also tested whether interpolations were improved by modeling the influence of covariates such as population density, elevation, and weather on ozone concentrations. We analyzed the reliability of a set of newly developed questions about past activity levels among a group of 52 freshmen students at Yale University. This was done by analyzing the agreement between answers to the same questionnaire administered two times, one month apart (test and retest), to the same students. Finally, we combined the interpolation models with residential history information obtained by questionnaire to derive long-term ozone exposure estimates for a group of 200 Yale freshmen. Results of our study showed that the density of available monitoring sites appears to be adequate for estimating spatial patterns of long-term average ambient ozone concentrations. A simple regression-based interpolation on the three nearest sites produced consistently good results. Including covariates in the interpolation models did not substantially improve the estimates. The largest estimation errors occurred for areas where ozone concentrations were highest. The newly developed activity history questions exhibited fair to moderate reliability, The results of this work imply that reasonably precise estimates of long-term ambient ozone concentrations for use in large-scale epidemiologic studies can be achieved by interpolating ozone concentrations between available U.S. monitoring sites. This study did not address the issues of whether and how retrospective data on factors that modify exposure or dose (e.g., indoor/outdoor penetration of ozone and time outdoors) can be used to derive estimates of long-term personal ozone exposures and contribute to the assessment of received dose

Methods are needed for estimating long-term ozone concentrations in epidemiology studies. The objective of this study was to apply and compare several alternative spatial interpolation models in a set of long-term average ozone data from 1112 U.S. monitoring sites in operation between 1981 and 1990. The comparison of methods was carried out for five different metrics of ozone concentration: the daily one-hour and eight-hour maxima, the average concentrations between 10AM and 6PM, between 10AM and 10PM, and the sum of all hourly ozone concentrations greater than or equal to 60 ppb. We also tested whether estimates were more precise when covariates (population density, elevation, meteorology) were added to the interpolation models. Results of our study showed that the ozone monitoring network is useful for estimating spatial patterns in long-term average ambient ozone concentrations. Reasonably good interpolations were possible using a variety of models; optimal results were obtained using a regression-based interpolation on three nearest sites. Inclusion of covariates in the interpolation models did not substantially improve the estimates. The largest estimation errors occurred where ozone concentrations were highest. The results of this work imply that reasonably precise long-term ozone concentration estimates can be achieved throughout the U.S. by interpolation of ozone concentrations between available U.S. monitoring sites

The functions of metallothioneins (MTs) have been debated for at least a decade. Because it seems unlikely that they evolved only to protect cells against exogenous heavy metals, it has been suggested that MTs have roles in scavenging reactive intermediates, controlling zinc and copper homeostasis, and controlling transfer of zinc to transcription factors and other proteins. Previously, we demonstrated that Chinese hamster G12 cells which overexpress MT have greatly reduced spontaneous mutation rates, suggesting that MT evolved to prevent spontaneous mutagenesis induced by free nuclear zinc ions. We have now isolated G12 transfectants which express antisense RNA to MT. Immunofluorescent staining reveals MT protein in both the nucleus and the cytoplasm in parental cells. A clone expressing high levels of antisense RNA (AMT30) shows reduced basal and induced levels of MT protein. AMT30 cells are hypersensitive to cadmium, zinc, copper and mercury chlorides as well as to menadione. Glutathione levels in AMT30 and G12 cells do not differ. AMT30 cells are spontaneous mutators, showing a spontaneous mutation rate 5-10 times that of G12 cells or G12 cells transfected with vector alone. Only transfectants which show a high level of MT antisense expression (i.e., AMT30) had greatly elevated spontaneous mutation rates. These results support our hypothesis that a major role of MT is to act as an endogenous antimutagen probably via scavenging of reactive intermediates in the nucleus. AMT30 cells should be useful in delineating the sources of spontaneous mutagenesis

In addition to being the single greatest known environmental cause of cancer, cigarette smoke (CS) is also a major contributor to heart disease. We reported previously that 1) inhalation of either mainstream or sidestream CS promotes aortic arteriosclerotic plaque development; 2) 1,3 butadiene, a vapor-phase component of CS, promotes plaque development at 20 ppm, which at the time was only 2 times higher than the threshold limit value; and 3) individual tar fraction carcinogens in CS, including polynuclear aromatic hydrocarbons (PAHs) and nitrosamines, either do not promote plaque development or do so only at high concentrations. These results suggested that the tar fraction is not the primary source of plaque-promoting agents in CS. We asked whether repeated exposure to the tar fraction of CS, collected in a cold trap (TAR), promotes plaque development in an avian model of arteriosclerosis. Acetone extracts of mainstream CS tar from burning, unfiltered reference cigarettes were solubilized in dimethyl sulfoxide (DMSO) and injected weekly into cockerels for 16 weeks (25 mg/kg/week). Positive controls were injected weekly with the synthetic PAH carcinogen, 7,12 dimethylbenz(a)anthracene (DMBA) dissolved in DMSO and negative controls were injected with DMSO. Plaque location and prevalence did not differ from group to group. Morphometric analysis of plaque cross-sectional areas showed that plaque sizes, which are log-normally distributed, were significantly larger in the DMBA cockerels compared to both the TAR and DMSO groups. There were no significant differences in plaque size between DMSO and TAR cockerels. The results reported here, combined with other recent findings, support the conclusion that the primary arteriosclerotic plaque-promoting components of CS are in the vapor phase

When estimating a spontaneous mutation rate from either a single culture (C=1) or from the C parallel cultures (C>1) of a fluctuation experiment, the use of a large initial population size N0 to seed each culture will permit a gaussian approximation for the probability distribution of the number M of mutants at the time when the culture(s) has (have) grown to size N=N02g, i.e., experienced g doublings. Using this gaussian approximation we find that the maximum likelihood estimate mu of the expected number mu of mutants present in a culture in generation g is (exactly) (equation: see text) where r = 2g / g and M 2 is the average of the squares of the C mutant counts. The maximum likelihood estimate p of the unknown mutation rate p is p = 2 mu / gN assuming an 'ideal' experiment and that there were no mutants in the initial population. A well-behaved maximum likelihood estimate is known to be efficient in large samples and we illustrate by Monte Carlo simulation that indeed p is better (has smaller mean squared error) than our previous (Rossman et al., 1995) estimator (equation: see text) (M is the average mutant count) provided N0 is of the order 1/p or larger. This advantage exists even without a fluctuation experiment, i.e., for C = 1

Spontaneous mutagenesis is thought to play a crucial role in spontaneous carcinogenesis. We recently described a new mathematical model for estimation of the spontaneous mutation rate (mutation/gene/generations) based on the assumption that mutations are fixed in the S-phase of the cell cycle. With this definition, the spontaneous mutation rate should be independent of the growth rate. In the present study, we tested this hypothesis, using cell line G12, a transgenic Chinese hamster V79 derivative, which contains a single copy of the Escherichia coli gpt gene as a target for mutagenesis. The growth rate was modulated by varying the serum concentration or the seeding density, or by addition of suramin, transforming growth factor beta, or dichlorobenzimidazole riboside to the medium. Significant increases in the spontaneous mutation rate occurred when cell proliferation was blocked by serum deprivation. Density-dependent inhibition of growth and inhibition of growth by suramin, transforming growth factor beta, or dichlorobenzimidazole riboside did not result in significant increases in spontaneous mutation rates. The level of oxidants in cells cultivated in the presence of low concentrations of serum was higher compared to control cells, suggesting that the increases in the spontaneous mutation rates under low serum conditions may be partly a result of oxidative stress due to a lack of serum antioxidants. This was shown to be the case, because spontaneous mutation rates were significantly reduced in serum-depleted cells when antioxidants were added to the medium. We suggest that during carcinogenesis, when tumors are in a prevascularized state, the spontaneous mutation rate may be elevated, and this process may contribute to the genetic instability of the tumor cells

Certain mathematical artifacts which had been appended by others to Luria and Delbruck's [Genetics 28: 491-511, 1943] model of spontaneous mutagenesis in bacterial populations have added confusion to the modeling and measurement of spontaneous mutation rates. Additional confusion arises when models which had been tuned for experiments with bacterial cultures grown from a small inoculum are adapted for use with mammalian cell cultures grown from a large initial population. As one consequence, biologists still tend to grow the large number of parallel cultures required by the fluctuation test in order to avoid large errors due to the high variability in the number of mutants in a growing culture. By avoiding models with infinite mean values and certain mathematical approximations that lead to conceptual and practical difficulties, the large variance of the number of mutants can be avoided (and the precision of the estimated mutation rate controlled) through the use of sufficiently large initial cell populations. A direct consequence is that simpler experiments with fewer cultures may suffice. In this paper, after a discussion of the confusions, we extend our previous approach [Rossman et al.: Mutat Res 328:21-30, 1995] by giving improved formulas for the standard error of the estimated mutation rate. The improvement results from using a more inclusive model based on consideration of the variability due to both the biological phenomenon of the growing culture (growth and mutation) and the protocols used for selection (sampling and plating efficiency). Also included is the situation where the initial cell population is not assumed to be free of mutants but the initial mutant fraction is measured instead. These standard error formulas are useful in planning experiments that yield mutation rate estimates with planned precision and for comparing and testing hypotheses about mutation rates in two or more populations which are grown under different conditions