Detection and sequence analysis of a nickel-induced mutation in a retroviral model system

We have developed a novel way to assess the mutagenicity of environmentally important metal carcinogens, such as nickel, by creating a positive selection system based upon the conditional expression of a retroviral transforming gene. The target gene is the v-mos gene in MuSVts110, a murine retrovirus possessing a growth temperature dependent defect in expression of the transforming gene due to viral RNA splicing. In normal rat kidney cells infected with MuSVts110 (6m2 cells), splicing of the MuSVts110 RNA to form the mRNA from which the transforming protein, p85$\sp{\rm gag-mos}$, is translated is growth-temperature dependent, occurring at 33 C and below but not at 39 C and above. This splicing "defect" is mediated by cis-acting viral sequences. Nickel chloride treatment of 6m2 cells followed by growth at 39 C, allowed the selection of "revertant" cells which constitutively express p85$\sp{\rm gag-mos}$ due to stable changes in the viral RNA splicing phenotype, suggesting that nickel, a carcinogen whose mutagenicity has not been well established, could induce mutations in mammalian genes. We also show by direct sequencing of PCR-amplified integrated MuSVts110 DNA from a 6m2 nickel-revertant cell line that the nickel-induced mutation affecting the splicing phenotype is a cis-acting 70-base duplication of a region of the viral DNA surrounding the 3$\sp\prime$ splice site. These findings provide the first example of the molecular basis for a nickel-induced DNA lesion and establish the mutagenicity of this potent carcinogen. ^

Analysis of the Soehner-Dmochowski strain of Moloney murine sarcoma virus

The Soehner-Dmochowski strain of murine sarcoma virus (MuSV-SD) was derived from a bone tumor of a New Zealand Black (NZB) rat infected with the Moloney strain of MuSV, which carries the gene encoding the v-mos protein. Serial passage of cell-free tumor extracts both decreased the latent period and resulted in osteosarcomas. Cells from a late passage tumor were established in culture, cell-free extracts frozen, and later inoculated into newborn NZB rats. One of the resulting bone tumors was established in culture and clonal cell lines derived, of which S4 was selected for the present study. The objectives of the study were two-fold: an examination of the genetic organization of MuSV-SD, and an examination of the biochemical characteristics of the viral proteins, since this is an acutely transforming virus which may yield insights into the mechanism of transformation caused by the v-mos protein. Blot hybridization of digested S4 genomic DNA reveals three candidate MuSV-SD integrated viral DNAs. The largest of these, MuSV-SD-6.5, was cloned from an S4 cosmid library, and the complete MuSV-SD-mos sequence was determined. The predicted amino acid sequence of the v-mos protein was compared to that of MuSV-124 and Ht-1, which show a 96.5% and 97.1% similarity, respectively. To characterize the MuSV-SD-mos protein further, immunochemical assays were performed using anti-mos antisera. The immunoblot analysis and immunoprecipitation assays demonstrated that similar levels of the v-mos protein were present in cells chronically infected with either MuSV-SD or MuSV-124; however, the immune complex kinase assay revealed greatly reduced in vitro serine kinase activity of the MuSV-SD-mos protein compared to that of MuSV-124. Sequence analysis demonstrated that the serine at amino acid residue 358 of the MuSV-SD-mos protein, like that of MuSV-Ht-1, had been mutated to a glycine. Mutations of this serine residue have been shown to affect the detectable in vitro kinase activity, however, v-mos proteins containing this mutation still retain transforming properties. Therefore, although the characteristic in vitro kinase activity of the MuSV-SD-mos protein has not been demonstrated, it is clear that this virus is a potent transforming agent. ^

Patient classification system: An integrated method for measuring nursing intensity and optimizing resource allocation

A patient classification system was developed integrating a patient acuity instrument with a computerized nursing distribution method based on a linear programming model. The system was designed for real-time measurement of patient acuity (workload) and allocation of nursing personnel to optimize the utilization of resources.^ The acuity instrument was a prototype tool with eight categories of patients defined by patient severity and nursing intensity parameters. From this tool, the demand for nursing care was defined in patient points with one point equal to one hour of RN time. Validity and reliability of the instrument was determined as follows: (1) Content validity by a panel of expert nurses; (2) predictive validity through a paired t-test analysis of preshift and postshift categorization of patients; (3) initial reliability by a one month pilot of the instrument in a practice setting; and (4) interrater reliability by the Kappa statistic.^ The nursing distribution system was a linear programming model using a branch and bound technique for obtaining integer solutions. The objective function was to minimize the total number of nursing personnel used by optimally assigning the staff to meet the acuity needs of the units. A penalty weight was used as a coefficient of the objective function variables to define priorities for allocation of staff.^ The demand constraints were requirements to meet the total acuity points needed for each unit and to have a minimum number of RNs on each unit. Supply constraints were: (1) total availability of each type of staff and the value of that staff member (value was determined relative to that type of staff's ability to perform the job function of an RN (i.e., value for eight hours RN = 8 points, LVN = 6 points); (2) number of personnel available for floating between units.^ The capability of the model to assign staff quantitatively and qualitatively equal to the manual method was established by a thirty day comparison. Sensitivity testing demonstrated appropriate adjustment of the optimal solution to changes in penalty coefficients in the objective function and to acuity totals in the demand constraints.^ Further investigation of the model documented: correct adjustment of assignments in response to staff value changes; and cost minimization by an addition of a dollar coefficient to the objective function. ^

Biologic and molecular analysis of tumor suppressor loci in human glioblastoma multiforme

Molecular and cytogenetic analyses of human glioblastomas have revealed frequent genetic alterations, including major deletions in chromosomes 9, 10, and 17, suggesting the presence of glioma-associated tumor suppressor genes on these chromosomes. To examine this hypothesis, copies of chromosomes 2, 4, and 10 derived from a human fibroblast cell line were independently introduced into a human glioma cell line, U251, by microcell-mediated chromosomal transfer. Successful transfer of chromosomes in each case was confirmed by resistance to the drug G418, indicating the presence of the neomycin-resistance gene previously integrated into each transferred chromosome. The presence of novel chromosomes and or chromosomal fragments was also demonstrated by molecular and karyotypic analyses. The hybrid clones containing either a novel chromosome 4 or chromosome 10 displayed suppression of the tumorigenic phenotype in vivo and suppression of the transformed phenotype in vitro, while cells containing a transferred chromosome 2 failed to alter their tumorigenic phenotype. The hybrid cells containing chromosome 4 or 10 exhibited a significant decrease in their saturation density, altered cellular morphology at high cell density, but only a slight decrease in their exponential growth rate. A dramatic decrease was observed in growth of cells with chromosome 4 or 10 in soft agarose, with the number and size of the colonies being greatly reduced, compared to the parental or chromosome 2 containing cells. The introduction of chromosome 4 or 10 also completely suppressed tumor formation in nude mice. These studies indicate that chromosome 10, as hypothesized, and chromosome 4, a novel finding for gliomas, harbor tumor suppressor loci that may be directly involved in the initiation or progression of normal glial precursors to human glioblastoma multiforme. ^

Analysis of prognostic factors for the development of metastases and survival in renal cell carcinoma patients

Introduction and objective. A number of prognostic factors have been reported for predicting survival in patients with renal cell carcinoma. Yet few studies have analyzed the effects of those factors at different stages of the disease process. In this study, different stages of disease progression starting from nephrectomy to metastasis, from metastasis to death, and from evaluation to death were evaluated. ^ Methods. In this retrospective follow-up study, records of 97 deceased renal cell carcinoma (RCC) patients were reviewed between September 2006 to October 2006. Patients with TNM Stage IV disease before nephrectomy or with cancer diagnoses other than RCC were excluded leaving 64 records for analysis. Patient TNM staging, Furhman Grade, age, tumor size, tumor volume, histology and patient gender were analyzed in relation to time to metastases. Time from nephrectomy to metastasis, TNM staging, Furhman Grade, age, tumor size, tumor volume, histology and patient gender were tested for significance in relation to time from metastases to death. Finally, analysis of laboratory values at time of evaluation, Eastern Cooperative Oncology Group performance status (ECOG), UCLA Integrated Staging System (UISS), time from nephrectomy to metastasis, TNM staging, Furhman Grade, age, tumor size, tumor volume, histology and patient gender were tested for significance in relation to time from evaluation to death. Linear regression and Cox Proportional Hazard (univariate and multivariate) was used for testing significance. Kaplan-Meier Log-Rank test was used to detect any significance between groups at various endpoints. ^ Results. Compared to negative lymph nodes at time of nephrectomy, a single positive lymph node had significantly shorter time to metastasis (p<0.0001). Compared to other histological types, clear cell histology had significant metastasis free survival (p=0.003). Clear cell histology compared to other types (p=0.0002 univariate, p=0.038 multivariate) and time to metastasis with log conversion (p=0.028) significantly affected time from metastasis to death. A greater than one year and greater than two year metastasis free interval, compared to patients that had metastasis before one and two years, had statistically significant survival benefit (p=0.004 and p=0.0318). Time from evaluation to death was affected by greater than one year metastasis free interval (p=0.0459), alcohol consumption (p=0.044), LDH (p=0.006), ECOG performance status (p<0.001), and hemoglobin level (p=0.0092). The UISS risk stratified the patient population in a statistically significant manner for survival (p=0.001). No other factors were found to be significant. ^ Conclusion. Clear cell histology is predictive for both time to metastasis and metastasis to death. Nodal status at time of nephrectomy may predict risk of metastasis. The time interval to metastasis significantly predicts time from metastasis to death and time from evaluation to death. ECOG performance status, and hemoglobin levels predicts survival outcome at evaluation. Finally, UISS appropriately stratifies risk in our population. ^

Has implementation of integrated mental health services in primary care programs positively impacted health outcomes among the mentally ill in developing countries?

Objective. The World Health Organization (WHO) estimates that nearly 450 million people suffer from a mental disorder in the world. Developing countries do not have the health system structure in place to support the demand of mental health services. This study will conduct a review of mental health integration in primary care research that is carried out in low-income countries identified as such from the World Bank economic analysis. The research follows the standard of care that WHO has labeled appropriate in treatment of mental health populations. Methods. This study will use the WHO 10 principles of mental health integration into primary care as the global health standard of care for mental health. Low-income countries that used these principles in their national programs will be analyzed for effectiveness of mental health integration in primary care. Results. This study showed that mental health service integration in primary care did have an effect on health outcomes of low-income countries. However, information did not lead to significant quantitative results that determined how positive the effect was. Conclusion. More ethnographic research is needed in low-income countries to truly assess how effective the program is in integrating with the health system currently in place.^ ^

THE APPLICATION OF PATH ANALYSIS TO ASSESSMENT OF IRON NUTRITIONAL STATUS IN MAN

Path analysis has been applied to components of the iron metabolic system with the intent of suggesting an integrated procedure for better evaluating iron nutritional status at the community level. The primary variables of interest in this study were (1) iron stores, (2) total iron-binding capacity, (3) serum ferritin, (4) serum iron, (5) transferrin saturation, and (6) hemoglobin concentration. Correlation coefficients for relationships among these variables were obtained from published literature and postulated in a series of models using measures of those variables that are feasible to include in a community nutritional survey. Models were built upon known information about the metabolism of iron and were limited by what had been reported in the literature in terms of correlation coefficients or quantitative relationships. Data were pooled from various studies and correlations of the same bivariate relationships were averaged after z- transformations. Correlation matrices were then constructed by transforming the average values back into correlation coefficients. The results of path analysis in this study indicate that hemoglobin is not a good indicator of early iron deficiency. It does not account for variance in iron stores. On the other hand, 91% of the variance in iron stores is explained by serum ferritin and total iron-binding capacity. In addition, the magnitude of the path coefficient (.78) of the serum ferritin-iron stores relationship signifies that serum ferritin is the most important predictor of iron stores in the proposed model. Finally, drawing upon known relations among variables and the amount of variance explained in path models, it is suggested that the following blood measures should be made in assessing community iron deficiency: (1) serum ferritin, (2) total iron-binding capacity, (3) serum iron, (4) transferrin saturation, and (5) hemoglobin concentration. These measures (with acceptable ranges and cut-off points) could make possible the complete evaluation of all three stages of iron deficiency in those persons surveyed at the community level. ^

DEVELOPMENT AND APPLICATION OF SHORT-TERM MUTAGENICITY AND CYTOTOXICITY BIOASSAYS FOR INTEGRATED HEALTH ASSESSMENT STUDIES OF COAL FLY ASH EMISSIONS

Two respirable coal fly ash samples ((LESSTHEQ) 3(mu)m), one from a pressurized fluidized-bed combustion miniplant and one from a conventional combustion power plant, were investigated for physical properties, chemical composition and biological activity. Electron microscopy illustrated irregularity in fluidized-bed combustion fly ash and sphericity in conventional combustion fly ash. Elemental analysis of these samples showed differences in trace elements. Both fly ash samples were toxic in rabbit alveolar macrophage and Chinese hamster ovary cell systems in vitro. The macrophages were more sensitive to toxicity of fly ash than the ovary cells. For measuring the cytotoxicity of fly ash, the most sensitive parameters were adenosine triphosphate in the alveolar macrophage system and viability index in the hamster ovary system. Intact fluidized-bed combustion fly-ash particles showed mutagenicity only in strains TA98 and TA1538 without metabolic activation in the Ames Salmonella assay. No mutagenicity was detected in bioassay of conventional combustion fly ash particles. Solvent extraction yielded more mass from fluidized-bed combustion fly ash than from conventional combustion fly ash. The extracts of fluidized-bed combustion fly ash showed higher mutagenic activity than conventional combustion fly ash. These samples contained direct-acting, frameshift mutagens.^ Fly ash samples collected from the same fluidized-bed source by cyclones, a fabric filter, and a electrostatic precipitator at various temperatures were compared for particle size, toxicity, and mutagenicity. Results demonstrated that the biological activity of coal fly ash were affected by the collection site, device, and temperature.^ Coal fly ash vapor-coated with 1-nitropyrene was developed as a model system to study the bioavailability and recovery of nitroaromatic compounds in fly ash. The effects of vapor deposition on toxicity and mutagenicity of fly ash were examined. The nitropyrene coating did not significantly alter the ash's cytotoxicity. Nitropyrene was bioavailable in the biological media, and a significant percentage was not recovered after the coated fly ash was cultured with alveolar macrophages. 1-Nitropyrene loss increased as the number of macrophages was increased, suggesting that the macrophages are capable of metabolizing or binding 1-nitropyrene present in coal fly ash. ^

Immune response to hepatitis B vaccination in drug using populations: A systematic review and meta-regression analysis

Hepatitis B virus (HBV) is a significant cause of liver diseases and related complications worldwide. Both injecting and non-injecting drug users are at increased risk of contracting HBV infection. Scientific evidence suggests that drug users have subnormal response to HBV vaccination and the seroprotection rates are lower than that in the general population; potentially due to vaccine factors, host factors, or both. The purpose of this systematic review is to examine the rates of seroprotection following HBV vaccination in drug using populations and to conduct a meta-analysis to identify the factors associated with varying seroprotection rates. Seroprotection is defined as developing an anti-HBs antibody level of ≥ 10 mIU/ml after receiving the HBV vaccine. Original research articles were searched using online databases and reference lists of shortlisted articles. HBV vaccine intervention studies reporting seroprotection rates in drug users and published in English language during or after 1989 were eligible. Out of 235 citations reviewed, 11 studies were included in this review. The reported seroprotection rates ranged from 54.5 – 97.1%. Combination vaccine (HAV and HBV) (Risk ratio 12.91, 95% CI 2.98-55.86, p = 0.003), measurement of anti-HBs with microparticle immunoassay (Risk ratio 3.46, 95% CI 1.11-10.81, p = 0.035) and anti-HBs antibody measurement at 2 months after the last HBV vaccine dose (RR 4.11, 95% CI 1.55-10.89, p = 0.009) were significantly associated with higher seroprotection rates. Although statistically nonsignificant, the variables mean age>30 years, higher prevalence of anti-HBc antibody and anti-HIV antibody in the sample population, and current drug use (not in drug rehabilitation treatment) were strongly associated with decreased seroprotection rates. Proportion of injecting drug users, vaccine dose and accelerated vaccine schedule were not predictors of heterogeneity across studies. Studies examined in this review were significantly heterogeneous (Q = 180.850, p = 0.000) and factors identified should be considered when comparing immune response across studies. The combination vaccine showed promising results; however, its effectiveness compared to standard HBV vaccine needs to be examined systematically. Immune response in DUs can possibly be improved by the use of bivalent vaccines, booster doses, and improving vaccine completion rates through integrated public programs and incentives.^