Functional studies on DNA double-strand break repair proteins (MmRad51, Ku80) in mice

RecA in Escherichia coli and it's homologue, ScRad51 in Saccharomyces cerevisiae, play important roles in recombinational repair. ScRad51 homologues have been discovered in a wide range of organisms including Schizosaccharomyces pombe, lily, chicken, mouse and human. To date there is no direct evidence to describe that mouse Rad51(MmRad51) is involved in DNA double-strand break repair. In order to elucidate the role of MmRad51 in vivo, it was mutated by the embryonic stem (ES) cell/gene targeting technology in mice. The mutant embryos arrested in development shortly after implantation. There was a decrease in cell proliferation followed by programmed cell death, and trophectoderm-derived cells were sensitive to $\gamma$-radiation. Severe chromosome loss was observed in most mitotically dividing cells. The mutant embryos lived longer and developed further in a p53 mutant background; however, double-mutant embryonic fibroblasts failed to proliferate in tissue culture, reflecting the embryos limited life span. Based on these data, MmRad51 repairs DNA damage induced by $\gamma$-radiation, is needed to maintain euplody, and plays an important role in proliferating cells.^ Ku is a heterodimer of 70 and 80 kDs subunit, which binds to DNA ends and other altered DNA structures such as hairpins, nicks, and gaps. In addition, Ku is required for DNA-PK activity through a direct association. Although the biochemical properties of Ku and DNA-PKcs have been characterized in cells, their physiological functions are not clear. In order to understand the function of Ku in vivo, we generated mice homozygous for a mutation of the Ku80 gene. Ku80-deficient mice, like scid mice, showed severe immunodeficiency due to a impairment of V(D)J recombination. Mutant mice were semiviable and runted, cells derived from mutant embryos displayed hypersensitivity to $\gamma$-radiation, a decreased growth rate, a slow entry into S phase, altered colony size distributions, and a short life span. Based on these results, mutant cells and mice appeared to prematurely age. ^

Information bias and lifetime mortality risks of radiation-induced cancer

Additive and multiplicative models of relative risk were used to measure the effect of cancer misclassification and DS86 random errors on lifetime risk projections in the Life Span Study (LSS) of Hiroshima and Nagasaki atomic bomb survivors. The true number of cancer deaths in each stratum of the cancer mortality cross-classification was estimated using sufficient statistics from the EM algorithm. Average survivor doses in the strata were corrected for DS86 random error ($\sigma$ = 0.45) by use of reduction factors. Poisson regression was used to model the corrected and uncorrected mortality rates with covariates for age at-time-of-bombing, age at-time-of-death and gender. Excess risks were in good agreement with risks in RERF Report 11 (Part 2) and the BEIR-V report. Bias due to DS86 random error typically ranged from $-$15% to $-$30% for both sexes, and all sites and models. The total bias, including diagnostic misclassification, of excess risk of nonleukemia for exposure to 1 Sv from age 18 to 65 under the non-constant relative projection model was $-$37.1% for males and $-$23.3% for females. Total excess risks of leukemia under the relative projection model were biased $-$27.1% for males and $-$43.4% for females. Thus, nonleukemia risks for 1 Sv from ages 18 to 85 (DRREF = 2) increased from 1.91%/Sv to 2.68%/Sv among males and from 3.23%/Sv to 4.02%/Sv among females. Leukemia excess risks increased from 0.87%/Sv to 1.10%/Sv among males and from 0.73%/Sv to 1.04%/Sv among females. Bias was dependent on the gender, site, correction method, exposure profile and projection model considered. Future studies that use LSS data for U.S. nuclear workers may be downwardly biased if lifetime risk projections are not adjusted for random and systematic errors. (Supported by U.S. NRC Grant NRC-04-091-02.) ^

EXPOSURE OF PHARMACY PERSONNEL TO ANTINEOPLASTIC DRUGS

This dissertation consists of two parts: (1) Exposure of pharmacy personnel to antineoplastic drugs. The Salmonella reversion test was used to measure the mutagenic activities of urine concentrates from individuals preparing antineoplastic drugs for intravenous administration. Longitudinal studies were performed in which the total urine produced in 24-hour periods was collected, starting on a Sunday at 7 P.M. after a duty-free weekend and extending over an eight-day period. There was no detectable increase in mutagenic activity in the urine concentrates of three pharmacy administrators who had no contact with these drugs. All six individuals admixing drugs in open-faced, horizontal laminar flow hoods displayed a two-fold increase in mutagenesis by the fourth day with peak values of 2.7 to 24-fold occurring on days five and six, reduced values by day seven with a return to the spontaneous level by day eight. When four of the six positive individuals in the preceding experiment admixed comparable amounts of antineoplastic drugs in a closed-faced, vertical laminar flow hood, no increase in mutagenic activity was detected in their urine concentrates over the eight-day period. (2) Estimate of potential carcinogenic risks of antineoplastic drugs. Excision repair is the major repair system that is involved with the elimination of chemically induced DNA (deoxyribonucleic acid) lesions. This DNA excision repair capability increases in mammalian species with longer life span such as humans. In this study, the effect of functional DNA excision repair on the mutagenesis invoked by 17 antineoplastic drugs was determined by using a Salmonella/Microsome assay which was expanded to include some uvr('+) counterparts of the excisionless (uvrB) tester strains routinely employed. Although extrapolation cannot be made from bacteria to humans, one should be able to make a qualitative comparison as to which antineoplastic drugs are more potentially carcinogenic to humans based on the effects of excision repair on their mutagenesis in bacteria. The tested antineoplastic drugs were divided into three classes: those requiring excision repair for mutagenesis; those producing nonrepairable genetic damage; and those producing mostly repairable premutational DNA lesions. ^

Modulation of replicative senescence of skeletal muscle satellite cells by insulin -like growth factor-I (IGF-I)

Growth and regeneration of postnatal skeletal muscle requires a population of mononuclear myogenic cells, called satellite cells to add/replace myonuclei, which are postmitotic. Wedged between the sarcolemma and the basal lamina of the skeletal muscle fiber, these cells function as the stem cells of mature muscle fibers. Like other normal diploid cells, satellite cells undergo cellular senescence. Investigations of aging in both rodents and humans have shown that satellite cell self-renewal capacity decreases with advanced age. As a consequence, this could be a potential reason for the characteristically observed age-associated loss in skeletal muscle mass (sarcopenia). This provided the rationale that any intervention that can further increase the proliferative capacity of these cells should potentially be able to either delay, or even prevent sarcopenia. ^ Using clonogenicity assays to determine a cell's proliferation potential, these studies have shown that IGF-I enhances the doubling potential of satellite cells from aged rodents. Using a transgenic model, where the mice express the IGF-I transgene specifically in their striated muscles, some of the underlying biochemical mechanisms for the observed increase in replicative life span were delineated. These studies have revealed that IGF-I activates the PI3/Akt pathway to mediate downregulation of p27KIP1, which consequently is associated with an increase in cyclin E-cdk2 kinase activity, phosphorylation of pRb, and upregulation of cyclin A protein. However, the beneficial effects of IGF-I on satellite cell proliferative potential appears to be limited as chronic overexpression of IGF-I in skeletal muscles did not protect against sarcopenia in 18-mo old mice, and was associated with an exhaustion of satellite cell replicative reserves. ^ These results have shown that replicative senescence can be modulated by environmental factors using skeletal muscle satellite cells as a model system. A better understanding of the molecular basis for enhancement of proliferative capacity by IGF-I will provide a rational basis for developing more effective counter-measures against physical frailty. However, the implications of these studies are that these beneficial effects of enhanced proliferative potential by IGF-I may only be over a short-term period, and other alternative approaches may need to be considered. ^

Health-related quality of life and survival in liver transplant candidates.

Health-related quality of life (HRQOL) is an important measure of the effects of chronic liver disease in affected patients that helps guide interventions to improve well-being. However, the relationship between HRQOL and survival in liver transplant candidates remains unclear. We examined whether the Physical Component Summary (PCS) and Mental Component Summary (MCS) scores from the Short Form 36 (SF-36) Health Survey were associated with survival in liver transplant candidates. We administered the SF-36 questionnaire (version 2.0) to patients in the Pulmonary Vascular Complications of Liver Disease study, a multicenter prospective cohort of patients evaluated for liver transplantation in 7 academic centers in the United States between 2003 and 2006. Cox proportional hazards models were used with death as the primary outcome and adjustment for liver transplantation as a time-varying covariate. The mean age of the 252 participants was 54 +/- 10 years, 64% were male, and 94% were white. During the 422 person years of follow-up, 147 patients (58%) were listed, 75 patients (30%) underwent transplantation, 49 patients (19%) died, and 3 patients were lost to follow-up. Lower baseline PCS scores were associated with an increased mortality rate despite adjustments for age, gender, Model for End-Stage Liver Disease score, and liver transplantation (P for the trend = 0.0001). The MCS score was not associated with mortality (P for the trend = 0.53). In conclusion, PCS significantly predicts survival in liver transplant candidates, and interventions directed toward improving the physical status may be helpful in improving outcomes in liver transplant candidates.

Evaluating minimal important differences for the FACT-Melanoma quality of life questionnaire

Objectives. Minimal Important Differences (MIDs) establish benchmarks for interpreting mean differences in clinical trials involving quality of life outcomes and inform discussions of clinically meaningful change in patient status. As such, the purpose of this study was to assess MIDs for the Functional Assessment of Cancer Therapy–Melanoma (FACT-M). ^ Methods. A prospective validation study of the FACT-M was performed with 273 patients with stage I to IV melanoma. FACT-M, Karnofsky Performance Status (KPS), and Eastern Cooperative Oncology Group Performance Status (ECOG-PS) scores were obtained at baseline and 3 months following enrollment. Anchor- and distribution-based methods were used to assess MIDs, and the correspondence between MID ranges derived from each method was evaluated. ^ Results. This study indicates that an approximate range for MIDs of the FACT-M subscales is between 5 to 8 points for the Trial Outcome Index, 4 to 5 points for the Melanoma Combined Subscale, 2 to 4 points for the Melanoma Subscale, and 1 to 2 points for the Melanoma Surgery Subscale. Each method produced similar but not identical ranges of MIDs. ^ Conclusions. The properties of the anchor instrument employed to derive MIDs directly affect resulting MID ranges and point values. When MIDs are offered as supportive evidence of a clinically meaningful change, the anchor instrument used to derive thresholds should be clearly stated along with evidence supporting the choice of anchor instrument as the most appropriate for the domain of interest. In this analysis, the KPS was a more appropriate measure than the ECOG-PS for assessing MIDs. ^

Intensive care noise and mean arterial blood pressure in ELBW neonates

Objective. Loud noises in neonatal intensive care units (NICUs) may impede growth and development for extremely low birthweight (ELBW, < 1000 grams) newborns. The objective of this study was to measure the association between NICU sound levels and ELBW neonates' arterial blood pressure to determine whether these newborns experience noise-induced stress. ^ Methods. Noise and arterial blood pressure recordings were collected for 9 ELBW neonates during the first week of life. Sound levels were measured inside the incubator, and each subject's arterial blood pressures were simultaneously recorded for 15 minutes (at 1 sec intervals). Time series cross-correlation functions were calculated for NICU noise and mean arterial blood pressure (MABP) recordings for each subject. The grand mean noise-MABP cross-correlation was calculated for all subjects and for lower and higher birthweight groups for comparison. ^ Results. The grand mean noise-MABP cross-correlation for all subjects was mostly negative (through 300 sec lag time) and nearly reached significance at the 95% level at 111 sec lag (mean r = -0.062). Lower birthweight newborns (454-709 g) experienced significant decreases in blood pressure with increasing NICU noise after 145 sec lag (peak r = -0.074). Higher birthweight newborns had an immediate negative correlation with NICU sound levels (at 3 sec lag, r = -0.071), but arterial blood pressures increased to a positive correlation with noise levels at 197 sec lag (r = 0.075). ^ Conclusions. ELBW newborns' arterial blood pressure was influenced by NICU noise levels during the first week of life. Lower birthweight newborns may have experienced an orienting reflex to NICU sounds. Higher birthweight newborns experienced an immediate orienting reflex to increasing sound levels, but arterial blood pressure increased approximately 3 minutes after increases in noise levels. Increases in arterial blood pressure following increased NICU sound levels may result from a stress response to noise. ^

Improving the Quality of Life of spousal caregivers of stroke survivors: A cost effectiveness analysis

Back ground and Purpose. There is a growing consensus among health care researchers that Quality of Life (QoL) is an important outcome and, within the field of family caregiving, cost effectiveness research is needed to determine which programs have the greatest benefit for family members. This study uses a multidimensional approach to measure the cost effectiveness of a multicomponent intervention designed to improve the quality of life of spousal caregivers of stroke survivors. Methods. The CAReS study (Committed to Assisting with Recovery after Stroke) was a 5-year prospective, longitudinal intervention study for 159 stroke survivors and their spousal caregivers upon discharge of the stroke survivor from inpatient rehabilitation to their home. CAReS cost data were analyzed to determine the incremental cost of the intervention per caregiver. The mean values of the quality-of-life predictor variables of the intervention group of caregivers were compared to the mean values of usual care groups found in the literature. Significant differences were then divided into the cost of the intervention per caregiver to calculate the incremental cost effectiveness ratio for each predictor variable. Results. The cost of the intervention per caregiver was approximately $2,500. Statistically significant differences were found between the mean scores for the Perceived Stress and Satisfaction with Life scales. Statistically significant differences were not found between the mean scores for the Self Reported Health Status, Mutuality, and Preparedness scales. Conclusions. This study provides a prototype cost effectiveness analysis on which researchers can build. Using a multidimensional approach to measure QoL, as used in this analysis, incorporates both the subjective and objective components of QoL. Some of the QoL predictor variable scores were significantly different between the intervention and comparison groups, indicating a significant impact of the intervention. The estimated cost of the impact was also examined. In future studies, a scale that takes into account both the dimensions and the weighting each person places on the dimensions of QoL should be used to provide a single QoL score per participant. With participant level cost and outcome data, uncertainty around each cost-effectiveness ratio can be calculated using the bias-corrected percentile bootstrapping method and plotted to calculate the cost-effectiveness acceptability curves.^

Cardiovascular diseases risk factors during the first year of life

The pattern of change in cardiovascular risk factors, blood pressure (SBP and DBP) and plasma total cholesterol (TC), over time, their tracking and their relation to anthropometric measurements during the first year of life were investigated. Also, the effect of breast feeding on TC and the relationship of blood pressure measurements and family history of CVD risk factors were examined. One hundred five newborn term, healthy infants who were seen at a pediatric clinic in The Woodlands, Texas were followed longitudinally from 2 weeks to 1 year of age. TC, blood pressure, weight and length of the infants were measured at age 2 weeks, and again at 2, 4, 6, 9 and 12 months. In addition, family history, maternal and paternal, of CVD risk factors was obtained. Data analyses included only 40 infants who completed one year of follow up.^ At 2 weeks of age, the median value for TC was 23 mg/dl higher for females than for males. This difference disappeared as infants got older. For males, most of the increase in TC median levels, from 114 to 137 mg/dl, occurred between the ages of 2 weeks and 2 months, whereas for the female group, TC levels increased moderately, about 10 mg/dl, between 9 and 12 months of age. Tracking of TC was examined by using Spearman's correlation analysis. There were strong correlations between measurements taken as early as 2 weeks of age with later measurements. These correlations were stronger and more significant for males than for females (for males, r varied between 0.51 to 0.70, whereas for females, r varied between 0.11 to 0.70). The association of body measurements with TC is no more than modest and is closer for female infants than for male infants. Analysis, also, showed that infants who received breast milk had a TC mean value 47 mg/dl higher than that for infants who received formula milk only during the period of breast feeding and this difference disappeared by age 12 months.^ In both genders, most of the increase in blood pressure (about 10-15 mmHg in both SBP and DBP) occurred during the first 4 months of life. Most of the increase for male infants occurred during the first 2 months of life, while for females, the increase in SBP and DBP was between the age of 2 and 4 months. Neither SBP nor DBP track well during the first year of life and most of the correlations between measurements at different ages were not significant for either gender. The cross-sectional relationship of blood pressure measurements and selected body measurements was assessed. For females, only at age of 12 months did DBP have positive and significant correlations with weight, length and Quetelet index (r = 0.57, 0.60 and 0.57, respectively). There were no significant correlations between blood pressure and body measurements for males. Finally, analysis showed that maternal history of CV risk factors was significantly related to SBP in the female infant group, but not for males. For DBP, neither maternal nor paternal history was related. ^