An inter-machine comparison of tobacco smoke particle deposition in vitro from six independent smoke exposure systems.

There are several whole smoke exposure systems used to assess the biological and toxicological impact of tobacco smoke in vitro. One such system is the Vitrocell® VC 10 Smoking Robot and exposure module. Using quartz crystal microbalances (QCMs) installed into the module, we were able to assess tobacco smoke particle deposition in real-time. We compared regional deposition across the module positions and doses delivered by six VC 10s in four independent laboratories: two in the UK, one in Germany and one in China. Gauge R&r analysis was applied to the total data package from the six VC 10s. As a percentage of the total, reproducibility (between all six VC 10s) and repeatability (error within an individual VC 10) accounted for 0.3% and 7.4% respectively. Thus Gauge R&r was 7.7%, less than 10% overall and considered statistically fit for purpose. The dose-responses obtained from the six machines across the four different locations demonstrated excellent agreement. There were little to no positional differences across the module at all airflows as determined by ANOVA (except for one machine and at three airflows only). These results support the on-going characterisation of the VC 10 exposure system and suitability for tobacco smoke exposure in vitro.

Th2 but not Th1 immune bias results in altered lung functions in a murine model of pulmonary Cryptococcus neoformans infection.

Changes in airway dynamics have been reported in the rat model of pulmonary cryptococcosis. However, it is not known if Cryptococcus neoformans-induced changes in lung functions are related to the immunophenotype that develops in response to cryptococcal infection in the lungs. In this study we performed a parallel analysis of the immunophenotype and airway resistance (standard resistance of the airways [SRAW]) in BALB/c mice infected with highly virulent C. neoformans strain H99 and moderately virulent strain 52D. H99 infection evoked a Th2 response and was associated with increased SRAW, while the SRAW for 52D infection, which resulted in a predominantly Th1-skewed response, did not differ from the SRAW for uninfected mice. We found that an altered SRAW in mice did not positively or negatively correlate with the pulmonary fungal burden, the magnitude of inflammatory response, the numbers of T cells, eosinophils or eosinophil subsets, neutrophils, or monocytes/macrophages, or the levels of cytokines (interleukin-4 [IL-4], IL-10, gamma interferon, or IL-13) produced by lung leukocytes. However, the level of a systemic Th2 marker, serum immunoglobulin E (IgE), correlated significantly with SRAW, indicating that the changes in lung functions were proportional to the level of Th2 skewing in this model. These data also imply that IgE may contribute to the altered SRAW observed in H99-infected mice. Lung histological analysis revealed severe allergic bronchopulmonary mycosis pathology in H99-infected mice and evidence of protective responses in 52D-infected mice with well-marginalized lesions. Taken together, the data show that C. neoformans can significantly affect airflow physiology, particularly in the context of a Th2 immune response with possible involvement of IgE as an important factor.

Pneumocystis murina infection and cigarette smoke exposure interact to cause increased organism burden, development of airspace enlargement, and pulmonary inflammation in mice.

Chronic obstructive pulmonary disease (COPD) is characterized by the presence of airflow obstruction and lung destruction with airspace enlargement. In addition to cigarette smoking, respiratory pathogens play a role in pathogenesis, but specific organisms are not always identified. Recent reports demonstrate associations between the detection of Pneumocystis jirovecii DNA in lung specimens or respiratory secretions and the presence of emphysema in COPD patients. Additionally, human immunodeficiency virus-infected individuals who smoke cigarettes develop early emphysema, but a role for P. jirovecii in pathogenesis remains speculative. We developed a new experimental model using immunocompetent mice to test the interaction of cigarette smoke exposure and environmentally acquired Pneumocystis murina infection in vivo. We hypothesized that cigarette smoke and P. murina would interact to cause increases in total lung capacity, airspace enlargement, and pulmonary inflammation. We found that exposure to cigarette smoke significantly increases the lung organism burden of P. murina. Pulmonary infection with P. murina, combined with cigarette smoke exposure, results in changes in pulmonary function and airspace enlargement characteristic of pulmonary emphysema. P. murina and cigarette smoke exposure interact to cause increased lung inflammatory cell accumulation. These findings establish a novel animal model system to explore the role of Pneumocystis species in the pathogenesis of COPD.

Exacerbation of established pulmonary fibrosis in a murine model by gammaherpesvirus.

RATIONALE: Idiopathic pulmonary fibrosis is a progressive disease with high mortality. Although most patients have a slow, progressive course, some patients will have an acute deterioration in function or acute exacerbation, which carries a poor prognosis. In some cases, acute deterioration is associated with infection. Herpesviruses have been associated with this disease. Fibrocytes have also been shown to be important in the pathogenesis of pulmonary fibrosis. OBJECTIVES: To develop a murine model for infectious exacerbation of preexisting fibrosis, and provide mechanistic insight into the role of herpesviruses in fibrotic disease. METHODS: We used a model of fluorescein isothiocyanate-induced pulmonary fibrosis in mice. Infection with a murine gammaherpesvirus was given at time of established lung fibrosis. Measurements were made at the time of peak lytic viral replication. MEASUREMENTS AND MAIN RESULTS: We demonstrate that infection with gammaherpesvirus can exacerbate established fluorescein isothiocyanate-induced fibrosis evidenced by increased total lung collagen, histologic changes of acute lung injury, and diminished lung function. Gammaherpesvirus can exacerbate preexisting fibrosis in a Th1 cytokine environment and in the absence of Th2 cytokines. Gammaherpesvirus increases fibrocyte recruitment to the lung in wild-type, but not CCR2(-/-) mice, in part because viral infection up-regulates production of CCL2 and CCL12, chemokines important for fibrocyte recruitment. In contrast, mouse adenovirus infection did not exacerbate collagen deposition. CONCLUSIONS: These data provide a new model for gammaherpesvirus exacerbation of established pulmonary fibrosis. The up-regulation of chemokines during viral infection and subsequent recruitment of fibrocytes to the lung likely contribute to augmentation of pulmonary fibrosis.

Emergency care in California: robust capacity or busted access?

Licensed emergency department (ED) capacity is a static measure that is inadequate to evaluate a system that the public and policymakers expect to respond dynamically to individual patients in a timely manner. Government mandates on hospital-based providers, undersupply of trained and willing personnel, and private market imperatives all curtail the functional capacity of the emergency care system. Although most Californians still live within a few miles of the closest hospital, many ambulance patients are diverted much further because of ED crowding. Many ambulatory patients are delayed so long in waiting rooms that they return home without ever being seen.

The Emergency Medical Treatment and Labor Act as a federal health care safety net program.

Despite the greatest economic expansion in history during the 1990s, the number of uninsured U.S. residents surpassed 44 million in 1998. Although this number declined for the first time in recent years in 1999, to 42.6 million, the current economic slow-down threatens once again to increase the ranks of the uninsured. Many uninsured patients use hospital emergency departments as a vital portal of entry into an access-impoverished health care system. In 1986, Congress mandated access to emergency care when it passed the Emergency Medical Treatment and Labor Act (EMTALA). The EMTALA statute has prevented the unethical denial of emergency care based on inability to pay; however, the financial implications of EMTALA have not yet been adequately appreciated or addressed by Congress or the American public. Cuts in payments from public and private payers, as well as increasing demands from a larger uninsured population, have placed unprecedented financial strains on safety net providers. This paper reviews the financial implications of EMTALA, illustrating how the statute has evolved into a federal health care safety net program. Future actions are proposed, including the pressing need for greater public safety net funding and additional actions to preserve health care access for vulnerable populations.

Lessons learned during 15 years of clinical information system experience.

This article describes lessons learned during an initial intensive care unit point-of-care clinical information system implementation and subsequent expansions to other units and hospitals in a multihospital healthcare delivery system. Although the implementation and expansions were primarily successful, lessons learned include developing a broad base of support, making decisions through consensus, addressing conflict when it occurs, keeping user expectations realistic, preparing for the change process, implementing the computer information system in stages, challenging existing work processes, viewing the implementation as a process, and choosing a project leader with outstanding communication and group process skills in addition to technical skills.

Quantification of changes in c-myc mRNA levels in normal human bronchial epithelial (NHBE) and lung adenocarcinoma (A549) cells following chemical treatment.

Lung tumors frequently exhibit altered expression of oncogenes and/or tumor suppressor genes. Although some of these alterations are believed to arise from chemical exposure, the ability of specific chemicals to cause distinct changes in gene expression is not well characterized. We previously reported the development of a quantitative reverse transcriptase/polymerase chain reaction (RT/PCR) method for measuring c-myc mRNA levels, and reported that c-myc proto-oncogene expression is significantly increased in small-cell lung carcinoma cells. In the present study, quantitative RT/PCR was used to assess the effect of model toxins cycloheximide (CHX), a protein synthesis inhibitor, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a DNA alkylating agent, on c-myc mRNA levels in normal human bronchial epithelial (NHBE) and lung adenocarcinoma (A549) cells. Expression of c-myc was evaluated at 1-100 microM CHX and MNNG and was compared to the cytotoxic response as measured by the neutral red assay. Cycloheximide elicited a dose-dependent increase in c-myc mRNA levels in NHBE and A549 cells, but did not alter expression of the housekeeping gene beta-actin. A maximum increase for c-myc expression (200% of control) was observed 5 h after treatment with noncytotoxic concentrations. In contrast, MNNG elicited a dose-dependent decrease in c-myc expression in A549 cells, but no significant change in c-myc was observed in NHBE cells. The results from this study suggest that the quantitative RT/PCR method may be an appropriate technique for monitoring gene expression changes following chemical exposure. Hence, these types of studies may assist in the identification of specific chemicals which may induce the genetic alterations involved in the development of lung cancer as well as provide information relevant to the interactive effects of chemicals within complex mixtures.

Gene expression profiling of cultured human bronchial epithelial and lung carcinoma cells.

Lung cancer is a complex collection of diseases that is thought to begin with single mutated progenitor cells and culminates in any of several clinically described pathologies. Our knowledge of the molecular events that lead to different lung cancer types--small cell carcinoma, squamous cell carcinoma, adenocarcinoma, and large cell carcinoma--is incomplete. Nonetheless, it is evident that genetic changes that impact multiple molecular networks are involved in the generation of each specific phenotype. Due to the obvious complexity of these processes, the simultaneous quantitative monitoring of changes in the expression of genes that define these networks can provide mechanistic information to increase our understanding of the molecular basis for human pulmonary carcinogenesis. To this end, we have employed a commercially available human cDNA array (Atlas Human Array, Clontech Laboratories) to systematically screen for alterations in the expression of 600 genes in normal human bronchial epithelial (NHBE) cells as well as in several lung carcinoma lines. Studies on the reproducibility and variability of array results indicate that a 2-fold or greater difference in the expression of a particular gene could be considered a real difference in transcript abundance. Accuracy of gene expression as measured in the array was verified by comparing mRNA levels of the proto-oncogene c-myc in the array with results obtained by traditional Northern blot analysis and by quantitative RT-PCR. Gene expression profiles were compared within and among cell types. The differential expression of 17 genes, including downregulation of MRP8 and MRP14 and upregulation of CYP1B1, was observed in all four carcinoma lines compared to NHBE cells. The direction of all 17 gene expression differences, either upregulation or downregulation relative to NHBE cells, was the same for all four carcinoma lines, underscoring their common molecular features. Each lung tumor line also exhibited a number of unique differences compared to both normal cells and the other tumor cell lines. These differences may be due to differences in the cellular origin and/or pathology of the cell lines studied.

Effect of treating isolated systolic hypertension on the risk of developing various types and subtypes of stroke: the Systolic Hypertension in the Elderly Program (SHEP).

CONTEXT: The Systolic Hypertension in the Elderly Program (SHEP) demonstrated that treating isolated systolic hypertension in older patients decreased incidence of total stroke, but whether all types of stroke were reduced was not evaluated. OBJECTIVE: To investigate antihypertensive drug treatment effects on incidence of stroke by type and subtype, timing of strokes, case-fatality rates, stroke residual effects, and relationship of attained systolic blood pressure to stroke incidence. DESIGN: The SHEP study, a randomized, double-blind, placebo-controlled trial began March 1, 1985, and had an average follow-up of 4.5 years. SETTING AND PARTICIPANTS: A total of 4736 men and women aged 60 years or older with isolated systolic hypertension at 16 clinical centers in the United States. INTERVENTIONS: Patients were randomly assigned to receive treatment with 12.5 mg/d of chlorthalidone (step 1); either 25 mg/d of atenolol or 0.05 mg/d of reserpine (step 2) could be added (n = 2365); or placebo (n = 2371). MAIN OUTCOME MEASURES: Occurrence, type and subtype, and timing of first strokes and stroke fatalities; and change in stroke incidence for participants (whether in active treatment or placebo groups) reaching study-specific systolic blood pressure goal (decrease of at least 20 mm Hg from baseline to below 160 mm Hg) compared with participants not reaching goal. RESULTS: A total of 85 and 132 participants in the active treatment and placebo groups, respectively, had ischemic strokes (adjusted relative risk [RR], 0.63; 95% confidence interval [CI], 0.48-0.82); 9 and 19 had hemorrhagic strokes (adjusted RR, 0.46; 95% CI, 0.21-1.02); and 9 and 8 had strokes of unknown type (adjusted RR, 1.05; 95% CI, 0.40-2. 73), respectively. Four subtypes of ischemic stroke were observed in active treatment and placebo group participants, respectively, as follows: for lacunar, n = 23 and n = 43 (adjusted RR, 0.53; 95% CI, 0.32-0.88); for embolic, n = 9 and n = 16 (adjusted RR, 0.56; 95% CI, 0.25-1.27); for atherosclerotic, n = 13 and n = 13 (adjusted RR, 0. 99; 95% CI, 0.46-2.15); and for unknown subtype, n = 40 and n = 60 (adjusted RR, 0.64; 95% CI, 0.43-0.96). Treatment effect was observed within 1 year for hemorrhagic strokes but was not seen until the second year for ischemic strokes. Stroke incidence significantly decreased in participants attaining study-specific systolic blood pressure goals. CONCLUSIONS: In this study, antihypertensive drug treatment reduced the incidence of both hemorrhagic and ischemic (including lacunar) strokes. Reduction in stroke incidence occurred when specific systolic blood pressure goals were attained. JAMA. 2000;284:465-471

A natural flavonoid present in unripe plantain banana pulp (Musa sapientum L. var. paradisiaca) protects the gastric mucosa from aspirin-induced erosions.

The active anti-ulcerogenic ingredient was extracted from unripe plantain banana by solvent fractionation and identified by chromatography, spectroscopy and high performance liquid chromatography as the flavonoid leucocyanidin. Dried unripe plantain banana powder, the extracted leucocyanidin and a purified synthetic leucocyanidin demonstrated a significant (P < 0.05) protective effect against aspirin-induced erosions.

Expression of stably transfected murine glutathione S-transferase A3-3 protects against nucleic acid alkylation and cytotoxicity by aflatoxin B1 in hamster V79 cells expressing rat cytochrome P450-2B1.

Aflatoxin B1 (AFB1) is activated to AFB1-8,9-oxide (AFBO), a potent mutagenic and carcinogenic metabolite of AFB1. In the mouse, AFBO has been shown to be most efficiently detoxified by a specific isozyme of alpha-class glutathione S-transferase (GST), mGSTA3-3 (mGST-Yc). A hamster V79 cell line (V79MZr2B1, originally designated V79/SD1) previously transfected with the rat cytochrome P450-2B1 was stably transfected with an mGSTA3-3 expression vector, to study the chemopreventive role of GST in protecting against cytotoxicity or genotoxicity of AFBO. Immunoblotting demonstrated strong expression of an alpha-class GST in the mGSTA3-3 transfected cell line, whereas no detectable alpha-class GST protein was observed in the control (empty vector-transfected) cells. Previous studies with the V79MZr2B1 cell line indicated that it can activate AFB1 to a mutagenic metabolite via a transfected rat P450-2B1 stably expressed in the cells. We examined the ability of the expressed mGSTA3-3 to protect against AFB1-induced cytotoxicity or [3H]-covalent adduct formation in cellular nucleic acids. Exposure of empty vector-transfected control cells and mGSTA3-3 expressing cells to up to 600 nM [3H]-AFB1 indicated that a 70-80% reduction in DNA and RNA adducts was afforded by the expression of mGSTA3-3 in the transfected cells. Clonogenic survival assays showed that the mGSTA3-3 cell line was 4.6-fold resistant to AFB1 cytotoxicity as compared with the empty vector-transfected control SD1 cells, with IC50 values of 69 and 15 microM, respectively. The results of these studies demonstrate that mGSTA3-3 confers substantial protection against nucleic acid covalent modification and cytotoxicity by AFB1 in this transgenic cell model system.

Critical care transport: outcome evaluation after interfacility transfer and hospitalization.

STUDY OBJECTIVE: To test the hypothesis that interfacility transfer is not associated with increased mortality, duration of stay, or readmission within 7 days. METHODS: We matched 3,298 patients who were hospitalized for chest pain or related complaints in Kaiser Permanente medical centers after transfer from the emergency department of a nonplan hospital (transported patients) with 3,298 patients of the same gender and age (+/-5 years) and with the same principal diagnosis who were hospitalized within 6 months without transfer in the same Kaiser Permanente medical center (directly admitted patients). Patients were compared in terms of outcome measures: in-hospital deaths, continued care in another facility, readmission within 7 days, in-patient length of stay (LOS), and LOS in special care units. RESULTS: The adjusted odds ratios for in-hospital mortality and readmission within 7 days were 1.0 (95% confidence interval,.8 to 1.4) and.9 (95% confidence interval,.7 to 1.2), respectively. The adjusted mean difference in LOS was -.1 days (95% confidence interval, -.2 to.1). Transported and directly admitted cardiac patients were also compared for all examined outcome measures at each of 10 medical centers. At a few medical centers, we observed significant difference in LOS, special care LOS, and continued care in another facility. However, all these differences were small, and most were probably random errors. CONCLUSION: Conservative patient selection criteria, pretransfer stabilization, and the use of appropriate equipment and medical personnel have resulted in the interfacility transfer program's achieving its goal of transferring high-risk patients without adverse impact on clinical outcomes or resource use.

Overexpression of stably transfected human glutathione S-transferase P1-1 protects against DNA damage by benzo[a]pyrene diol-epoxide in human T47D cells.

The (+)-anti enantiomer of benzo[a]pyrene-7,8-dihydrodiol-9, 10-epoxide (BPDE) is a potent mutagenic and carcinogenic metabolite of benzo[a]pyrene (BP), and a major fraction is conjugated with glutathione in vivo. The chemopreventive role of glutathione S-transferases (GSTs) in protecting against covalent modification of DNA and other cellular macromolecules by BPDE was modeled in human T47D and MCF-7 cell lines previously stably transfected with human GSTpi1 (hGSTP1). Cells were exposed to [3H]BPDE (30-600 nM). Dose-response experiments indicated that the high level of expression of hGSTP1-1 in the T47Dpi cell line (4411 +/- 183 milliunits/mg of cytosolic protein, using 1-Cl-2,4-dinitrobenzene as substrate), resulted in 70-90% reduction in the covalent 3H-adduct formation in DNA or RNA isolated from the GSTP1-transfected T47Dpi cell line. The lower level of hGSTP1-1 expression in the transfected MCF-7 cell line (91 milliunits/mg) provided only marginal protection against [3H]BPDE adduct formation and did not affect sensitivity to BPDE-induced cytotoxicity. Protection against BPDE-induced cytotoxicity was observed only in the T47Dpi cell line, which had an IC50 value 5.8-fold greater than that of the T47Dneo control cell line. Measurement of glutathione conjugates of BPDE indicated that the total conjugation was 5-fold higher in the GSTpi-transfected T47D line, most of which was exported into the culture medium over the 20-min exposure period. These results indicate that hGSTP1-1 protects effectively against DNA and RNA modification by BPDE, but moderate to high level expression may be required for strong protection against BPDE-induced genotoxicity and cytotoxicity.

Chemoprotective functions of glutathione S-transferases in cell lines induced to express specific isozymes by stable transfection.

The authors have shown that expression of mGSTM1-1 or hGSTP1-1 in MCF-7 cells protects against DNA alkylation by 4-nitroquinoline-1-oxide (NQO) in an isozyme-specific manner and is commensurate with relative specific activity. Expression of GSTs also conferred protection against both DNA strand breaks and sister-chromatid exchange induced by NQO. Interestingly, GST expression did not protect against NQO cytotoxicity in transfected MCF-7 cell lines, although resistance to NQO cytotoxicity was observed in a T47D pi transfectant line, expressing much higher specific activity of the transfected hGSTP1-1. However, high level expression of hGSTP1-1 or mGSTM1-1 in V79 transfectants did not confer resistance to cytotoxicity, indicating that expression of GST alone is not sufficient. The authors have also shown protection against AFB1 in cell lines expressing transfected rat CYP2B1 (V79MZr2B1) and transfected mGST-Yc (mGSTA3-3). Protection was observed against both alkylation of DNA (3-fold) by [3H]AFB1 and against AFB1 cytotoxicity (7-fold). Similarly, V79MZr1A1 cells that express CYP1A1 and either transfected human or murine GSTP1-1 (< 5000 mIU/mg, CDNB) exhibited > 70% decrease in covalent labeling of total nucleic acids by [3H]BPDE. However, no protection against the cytotoxicity of BPDE was conferred by expression of hGSTP1-1. Overall, these results indicate that in some (NQO or BPDE), but not all (AFB1) cases, protection by GST expression against DNA damage is more effective than protection against cytotoxicity. In addition, there is evidence to indicate that additional factor(s) other than high GST isozyme expression level and good substrate efficacy affect the degree of protection against cytotoxicity of reactive electrophiles. This includes the differential protection against NQO cytotoxicity in T47D pi, but not V79 Xh pi-33 cells and also the recent studies which showed that expression of the MRP GS-X conjugate efflux transporter confers synergistic protection against NQO cytotoxicity when co-expressed with transfected human GSTP1-1 in MCF-7 cells. Thus, protective efficacy conferred by GST expression can vary with different cellular targets and/or experimental end-points, as well as with variations in relative specific activity or in different cellular phenotypic contexts.