Modulation of immune response by RAGE and TLR4 signalling in PBMCs of diabetic and non-diabetic patients.

Diabetes is associated with increased glucose levels and accumulation of glycated products. It is also associated with impairment in the immune response, such as increased susceptibility to infections. In this study, we assessed the possible interactions between TLR4 and RAGE signalling on apoptosis and on the expression of inflammatory cytokines in PBMC from individuals with and without diabetes. PBMCs were isolated from seven diabetic patients and six individuals without diabetes and stimulated in vitro with bacterial LPS (1 µg/ml) associated or not with BSA-AGE (200 µg/ml). This stimulation was performed for 6 h, both in the presence and in the absence of inhibitors of TLR4 (R. sphaeroides LPS, 20 µg/ml) and RAGE (blocking monoclonal antibody). Apoptosis at early and late stages was assessed by the annexin-V/PI staining using flow cytometry. Regulation of TNF-α and IL-10 gene expression was determined by RT-qPCR. PBMCs from diabetes patients tended to be more resistant apoptosis. There were no synergistic or antagonistic effects with the simultaneous activation of TLR4 and RAGE in PBMCs from either diabetes or non-diabetes group. Activation of TLR4 is more potent for the induction of TNF-α and IL-10; RAGE signalling had a negative regulatory effect on TNF-α expression induced by LPS. TLR and RAGE do not have relevant roles in apoptosis of PBMCs. The activation of TLR has greater role than RAGE in regulating the gene expression of IL-10 and TNF-α.

Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates.

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500,000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients' urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

Longitudinal Association between Late-Life Depression (LLD) and Frailty: Findings from a Prospective Cohort Study (MiMiCS-FRAIL).

OBJECTIVES: The aim of the present study was to investigate whether late-life depression (LLD) is associated with incident frailty over time. DESIGN: Prospective cohort study, one-year follow-up. SETTING: Geriatric outpatient clinic, Southwestern of Brazil. PARTICIPANTS: 181 follow-up participants aged 60 years or over. MEASUREMENTS: Depressive disorders were classified as Major Depressive disorder (MDD) or Subthreshold Depression (STD) according to DSM-5 criteria. Depressive symptoms were assessed with validated versions of 15-item Geriatric Depression Scale (GDS-15) and 9-item Patient Health Questionnaire (PHQ-9). We performed binary logistic regressions to estimate the odds ratio (OR) for frailty in LLD adjusting for multiple confounders. Participants who were frail at baseline were excluded from the analyses according to measures of frailty (FRAIL questionnaire and 36-item Frailty Index, FI-36). We also estimated the risk ratio or relative risk (RR) and the risk difference (RD) for incident frailty. RESULTS: We observed a 2 to 4-fold increased risk for incident frailty among participants with LLD. The presence of a depressive disorder was significantly associated with the onset of frailty (adjusted OR for FRAIL and FI-36: 3.07 [95% CI = 1.03 - 9.17] and 3.76 [95% CI = 1.09 - 12.97], respectively. Notably, the risk for frailty due to LLD was significantly higher with the FI-36 compared to the FRAIL (RR: 3.03 versus 2.23). RD was of 17.3% and 12.7% with the FRAIL and the FI-36, respectively. CONCLUSION: Our data support the association between LLD and incident frailty over one year among geriatric outpatients, reinforcing longitudinal evidence from population-based studies.

Workshop summary: phosgene-induced pulmonary toxicity revisited: appraisal of early and late markers of pulmonary injury from animal models with emphasis on human significance.

A workshop was held February 14, 2007, in Arlington, VA, under the auspices of the Phosgene Panel of the American Chemistry Council. The objective of this workshop was to convene inhalation toxicologists and medical experts from academia, industry and regulatory authorities to critically discuss past and recent inhalation studies of phosgene in controlled animal models. This included presentations addressing the benefits and limitations of rodent (mice, rats) and nonrodent (dogs) species to study concentration x time (C x t) relationships of acute and chronic types of pulmonary changes. Toxicological endpoints focused on the primary pulmonary effects associated with the acute inhalation exposure to phosgene gas and responses secondary to injury. A consensus was reached that the phosgene-induced increased pulmonary extravasation of fluid and protein can suitably be probed by bronchoalveolar lavage (BAL) techniques. BAL fluid analyses rank among the most sensitive methods to detect phosgene-induced noncardiogenic, pulmonary high-permeability edema following acute inhalation exposure. Maximum protein concentrations in BAL fluid occurred within 1 day after exposure, typically followed by a latency period up to about 15 h, which is reciprocal to the C x t exposure relationship. The C x t relationship was constant over a wide range of concentrations and single exposure durations. Following intermittent, repeated exposures of fixed duration, increased tolerance to recurrent exposures occurred. For such exposure regimens, chronic effects appear to be clearly dependent on the concentration rather than the cumulative concentration x time relationship. The threshold C x t product based on an increased BAL fluid protein following single exposure was essentially identical to the respective C x t product following subchronic exposure of rats based on increased pulmonary collagen and influx of inflammatory cells. Thus, the chronic outcome appears to be contingent upon the acute pulmonary threshold dose. Exposure concentrations high enough to elicit an increased acute extravasation of plasma constituents into the alveolus may also be associated with surfactant dysfunction, intra-alveolar accumulation of fibrin and collagen, and increased recruitment and activation of inflammatory cells. Although the exact mechanisms of toxicity have not yet been completely elucidated, consensus was reached that the acute pulmonary toxicity of phosgene gas is consistent with a simple, irritant mode of action at the site of its initial deposition/retention. The acute concentration x time mortality relationship of phosgene gas in rats is extremely steep, which is typical for a local, directly acting pulmonary irritant gas. Due to the high lipophilicity of phosgene gas, it efficiently penetrates the lower respiratory tract. Indeed, more recent published evidence from animals or humans has not revealed appreciable irritant responses in central and upper airways, unless exposure was to almost lethal concentrations. The comparison of acute inhalation studies in rats and dogs with focus on changes in BAL fluid constituents demonstrates that dogs are approximately three to four times less susceptible to phosgene than rats under methodologically similar conditions. There are data to suggest that the dog may be useful particularly for the study of mechanisms associated with the acute extravasation of plasma constituents because of its size and general morphology and physiology of the lung as well as its oronasal breathing patterns. However, the study of the long-term sequelae of acute effects is experimentally markedly more demanding in dogs as compared to rats, precluding the dog model to be applied on a routine base. The striking similarity of threshold concentrations from single exposure (increased protein in BAL fluid) and repeated-exposure 3-mo inhalation studies (increased pulmonary collagen deposition) in rats supports the notion that chronic changes depend on acute threshold mechanisms.

Evaluation of an rK39-based immunochromatographic test for the diagnosis of visceral leishmaniasis in human saliva.

Visceral leishmaniasis (VL) is a tropical neglected disease endemic in 98 countries and affects more than 58 000 individuals per year. Several serological tests are available for VL diagnosis, including an immunochromatographic (IC) test with the rK39 antigen and finger prick-collected blood, a rapid and low-invasive test. Here, we investigate the possibility to use saliva as a non-invasive source of biological material for the rK39 IC test. Blood samples from 84 patients with suspected VL were screened by the rK39 IC test, and 29 were confirmed as being infected by a positive rK39 IC test and the presence of amastigotes on smears slides or parasite DNA (detected using PCR-RFLP) from bone marrow aspirate. The rK39 IC test using saliva samples was positive for 17 of the 29 confirmed VL cases (58.6%). The amount of Leishmania-specific IgG or total IgG, as evaluated by an immunoenzymatic assay, was higher in the saliva of patients who had rK39 IC test positivity using saliva, whereas the amount of Leishmania-specific IgA or total IgA was similar to the healthy donors. These results suggest that saliva is not an appropriated material for diagnosing VL with this test.

(1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol: a potent new neuroprotectant which blocks N-methyl-D-aspartate responses.

(1S,2S)-1-(4-Hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (20, CP-101,606) has been identified as a potent and selective N-methyl-D-aspartate (NMDA) antagonist through a structure activity relation (SAR) program based on ifenprodil, a known antihypertensive agent with NMDA antagonist activity. Sites on the threo-ifenprodil skeleton explored in this report include the pendent methyl group (H, methyl, and ethyl nearly equipotent; propyl much weaker), the spacer group connecting the C-4 phenyl group to the piperidine ring (an alternating potency pattern with 0 and 2 carbon atoms yielding the greatest potency), and simple phenyl substitution (little effect). While potent NMDA antagonists were obtained with a two atom spacer, this arrangement also increased alpha 1 adrenergic affinity. Introduction of a hydroxyl group into the C-4 position on these piperidine ring resulted in substantial reduction in alpha 1 adrenergic affinity. The combination of these observations was instrumental in the discovery of 20. This compound potently protects cultured hippocampal neurons from glutamate toxicity (IC50 = 10 nM) while possessing little of the undesired alpha 1 adrenergic affinity (IC50 approximately 20 microM) of ifenprodil. Furthermore, 20 appears to lack the psychomotor stimulant effects of nonselective competitive and channel-blocking NMDA antagonists. Thus, 20 shows great promise as a neuroprotective agent and may lack the side effects of compounds currently in clinical trials.

Ozone-induced tissue injury and changes in antioxidant homeostasis in normal and ascorbate-deficient guinea pigs.

It has been reported previously that ozone (O3) toxicity from acute (4 hr) exposure is enhanced by ascorbate (AH2) deficiency in guinea pigs. We hypothesized that lung injury from continuous 1-week O3 exposure would also be increased under conditions of AH2 deficiency because of (1) a diminished antioxidant pool to counteract the oxidant challenge, (2) impaired reparation of tissue injury, and/or (3) altered antioxidant redox homeostasis. Female Hartley guinea pigs (260-330 g) were made AH2 deficient by providing a diet similar to guinea pig chow, but having no AH2. The dietary regimen was started 1 week prior to exposure and was continued during exposure to O3 (0, 0.2, 0.4, or 0.8 ppm, 23 hr/day, 7 days) as well as 1 week post-exposure. Bronchoalveolar lavage (BAL) and tissue AH2 were measured in subgroups at the beginning of exposure (1 week on the AH2-deficient diet), at its termination and 1 week post-exposure. AH2 measured in ear tissue punches proved to be an easy and effective monitor for AH2 deficiency. One week on the AH2-deficient diet caused a 70-80% drop in ear, lung and liver AH2, while AH2 in BAL was decreased by 90%. Immediately after the exposure, total BAL protein and albumin (markers of lung permeability) were increased (approximately 50%) at 0.8 ppm with no difference between the dietary groups. O3 caused an increase in total BAL cells and neutrophils in a concentration-dependent manner with only a slight augmentation due to diet. Exposure to O3 caused an increase in lung and BAL AH2 in normal guinea pigs. Glutathione and uric acid were also increased in the lung and BAL after O3 exposure (40-570%) in both dietary groups, and the levels remained elevated during the recovery period. Lung alpha-tocopherol was not changed due to O3. A significant overall diet-related decrease was seen in AH2-deficient guinea pigs, immediately after the exposure and recovery. In summary, lung injury/inflammation following 1 week O3 exposure and recovery were minimally affected by AH2 deficiency. Antioxidants also appeared to increase in response to O3 exposure despite the deficiency in AH2.

Bioavailable transition metals in particulate matter mediate cardiopulmonary injury in healthy and compromised animal models.

Many epidemiologic reports associate ambient levels of particulate matter (PM) with human mortality and morbidity, particularly in people with preexisting cardiopulmonary disease (e.g., chronic obstructive pulmonary disease, infection, asthma). Because much ambient PM is derived from combustion sources, we tested the hypothesis that the health effects of PM arise from anthropogenic PM that contains bioavailable transition metals. The PM samples studied derived from three emission sources (two oil and one coal fly ash) and four ambient airsheds (St. Louis, MO; Washington; Dusseldorf, Germany; and Ottawa, Canada). PM was administered to rats by intratracheal instillation in equimass or equimetal doses to address directly the influence of PM mass versus metal content on acute lung injury and inflammation. Our results indicated that the lung dose of bioavailable transition metal, not instilled PM mass, was the primary determinant of the acute inflammatory response for both the combustion source and ambient PM samples. Residual oil fly ash, a combustion PM rich in bioavailable metal, was evaluated in a rat model of cardiopulmonary disease (pulmonary vasculitis/hypertension) to ascertain whether the disease state augmented sensitivity to that PM. Significant mortality and enhanced airway responsiveness were observed. Analysis of the lavaged lung fluids suggested that the milieu of the inflamed lung amplified metal-mediated oxidant chemistry to jeopardize the compromised cardiopulmonary system. We propose that soluble metals from PM mediate the array of PM-associated injuries to the cardiopulmonary system of the healthy and at-risk compromised host.

Rodent models of cardiopulmonary disease: their potential applicability in studies of air pollutant susceptibility.

The mechanisms by which increased mortality and morbidity occur in individuals with preexistent cardiopulmonary disease following acute episodes of air pollution are unknown. Studies involving air pollution effects on animal models of human cardiopulmonary diseases are both infrequent and difficult to interpret. Such models are, however, extensively used in studies of disease pathogenesis. Primarily they comprise those developed by genetic, pharmacologic, or surgical manipulations of the cardiopulmonary system. This review attempts a comprehensive description of rodent cardiopulmonary disease models in the context of their potential application to susceptibility studies of air pollutants regardless of whether the models have been previously used for such studies. The pulmonary disease models include bronchitis, emphysema, asthma/allergy, chronic obstructive pulmonary disease, interstitial fibrosis, and infection. The models of systemic hypertension and congestive heart failure include: those derived by genetics (spontaneously hypertensive, Dahl S. renin transgenic, and other rodent models); congestive heart failure models derived by surgical manipulations; viral myocarditis; and cardiomyopathy induced by adriamycin. The characteristic pathogenic features critical to understanding the susceptibility to inhaled toxicants are described. It is anticipated that this review will provide a ready reference for the selection of appropriate rodent models of cardiopulmonary diseases and identify not only their pathobiologic similarities and/or differences to humans but also their potential usefulness in susceptibility studies.

Functional evidence of persistent airway obstruction in rats following a two-hour inhalation exposure to methyl isocyanate.

Pulmonary function was assessed in male, F344 rats 1,2,4,7, and 13 weeks after a single 2-hr exposure to 0, 3, 10, or 30 ppm methyl isocyanate. No significant changes were observed in the rats exposed to 3 ppm through 13 weeks. Diffusing capacity (DLco), quasistatic lung compliance, and homogeneity of ventilation, as determined by multibreath nitrogen washout, were depressed in the rats exposed to 10 and 30 ppm by 1 week after exposure. None of the rats exposed to 30 ppm survived beyond 1 week. By 13 weeks, dramatic increases in lung volumes were observed in the rats exposed to 10 ppm, while DLco and lung compliance were only mildly affected. However, volume-specific DLco and compliance were depressed in the rats exposed to 10 ppm, suggesting that lung hyperinflation or other compensatory means of increasing lung size occurred in response to the methyl isocyanate-induced lung lesion. This group also exhibited increased expiratory times during tidal breathing and severely impaired distribution of ventilated air. Collectively, these results suggest the development and likely progression of a severe, obstructive airway lesion with associated gas trapping, and the existence of a pronounced concentration-response relationship between 3 and 10 ppm methyl isocyanate exposures.

Cardiovascular and systemic responses to inhaled pollutants in rodents: effects of ozone and particulate matter.

Striking similarities have been observed in a number of extrapulmonary responses of rodents to seemingly disparate ambient pollutants. These responses are often characterized by primary decreases in important indices of cardiac and thermoregulatory function, along with secondary decreases in associated parameters. For example, when rats are exposed to typical experimental concentrations of ozone (O(3), they demonstrate robust and consistent decreases in heart rate (HR) ranging from 50 to 100 beats per minute, whereas core temperature (T(co) often falls 1.5-2.5 degrees C. Other related indices, such as metabolism, minute ventilation, blood pressure, and cardiac output, appear to exhibit similar deficits. The magnitudes of the observed decreases may be modulated by changes in experimental conditions and appear to vary inversely with both ambient temperature and body mass. More recent studies in which both healthy and compromised rats were exposed to either particulate matter or its specific components yielded similar results. The agents studied included representative examples of ambient, combustion, and natural source particles, along with individual or combined exposures to their primary metallic constituents. In addition to the substantial decreases in HR and T(co), similar to those seen with the O(3)-exposed rats, these animals also displayed numerous adverse changes in electrocardiographic waveforms and cardiac rhythm, frequently resulting in fatal outcomes. Although there is only limited experimental evidence that addresses the underlying mechanisms of these responses, there is some indication that they may be related to stimulation of pulmonary irritant receptors and that they may be at least partially mediated via the parasympathetic nervous system.

Cardiopulmonary effects in awake rats four and six months after exposure to methyl isocyanate.

Cardiopulmonary function was assessed four and six months after Fischer 344 rats were exposed to 2 hr to 0, 3, or 10 ppm methyl isocyanate (MIC). During assessment, the rats were challenged with 4 and 8% carbon dioxide (CO2) to stimulate ventilatory drive. Minute ventilation (VE) during CO2 challenge was increased in MIC-treated rats compared to controls when examined 4 months after exposure to 10 ppm MIC, suggesting a ventilation/perfusion inequality. An increase in maximum expiratory flow and a decrease in expiratory time indicated increased lung recoil in these rats. Evidence of pulmonary hypertension was observed in electrocardiograms (ECGs) and supported by postmortem analysis that showed a positive association between increased ECG abnormalities and increased right ventricular weights in the rats treated with 10 ppm MIC. At 6 months, forced expiratory flow-volume curves indicated persistent airway obstruction; however, no changes in inspiratory or expiratory resistance were evident. Decreased dynamic compliance and changes in two new measures of lung function (volume and time at zero expiratory intrapleural pressure) suggest that MIC-induced lung dysfunction also exhibited elements of a restrictive disease.

Acute pulmonary toxicity of particulate matter filter extracts in rats: coherence with epidemiologic studies in Utah Valley residents.

Epidemiologic reports by C.A. Pope III et. al. demonstrated that in the Utah Valley, closure of an open-hearth steel mill over the winter of 1987 was associated with reductions in respiratory disease and related hospital admissions in valley residents. To better examine the relationship between plant-associated changes in ambient particulate matter (PM) and respiratory health effects, we obtained total suspended particulate filters originally collected near the steel mill during the winter of 1986 (before closure), 1987 (during closure), and 1988 (after plant reopening). PM subcomponents were water-extracted from these filters and Sprague-Dawley rats were intratracheally instilled with equivalent masses of extract. Data indicated that 24 hr later, rats exposed to 1986 or 1988 extracts developed significant pulmonary injury and neutrophilic inflammation. Additionally, 50% of rats exposed to 1986 or 1988 extracts had increased airway responsiveness to acetylcholine, compared to 17 and 25% of rats exposed to saline or the 1987 extract, respectively. By 96 hr, these effects were largely resolved except for increases in lung lavage fluid neutrophils and lymphocytes in 1986 extract-exposed rats. Analogous effects were observed with lung histologic assessment. Extract analysis using inductively coupled plasma-mass spectroscopy demonstrated in all three extracts nearly 70% of the mass appeared to be sodium-based salts derived from the glass filter matrix. Interestingly, relative to the 1987 extract, the 1986/1988 extracts contained more sulfate, cationic salts (i.e., calcium, potassium, magnesium), and certain metals (i.e., copper, zinc, iron, lead, strontium, arsenic, manganese, nickel). Although total metal content was (3/4) 1% of the extracts by mass, the greater quantity detected in the 1986 and 1988 extracts suggests metals may be important determinants of the pulmonary toxicity observed. In conclusion, the pulmonary effects induced by exposure of rats to water-based extracts of local ambient PM filters were in good accord with the cross-sectional epidemiologic reports of adverse respiratory health effects in Utah Valley residents.

Impact of the hypothermic response in inhalation toxicology studies.

Previous studies from this laboratory showed that the decreases in Tco and associated functional parameters often observed in rodents following exposure to xenobiotic agents are capable of modulating the subsequent toxic response and that the magnitude of this induced hypothermic response may itself be modified by a number of experimental conditions. A moderate hypothermic response, characterized by a temperature drop of approximately 2 degrees C, appears to afford the optimal protection. Studies in which exposures occur through inhalation of harmful gases or particles present a special set of problems. In such studies, the dose of the toxic agent to which the animal is exposed is a function of the concentration of the agent in the atmosphere and the minute ventilation of the animal. Although ambient concentrations is generally held constant in laboratory studies, minute ventilation varies directly with metabolism, and both of these parameters may change significantly across experimental conditions. Thus, at low Tas, metabolism and minute ventilation are relatively high and uptake of inhalable toxic agents is increased. However, the development of the hypothermic response during the exposure entails a directly correlated reduction in these parameters and, presumably, in dose. For the most part, inhalation toxicological studies are conducted using resting animals or exercising humans. Animals are sometimes concurrently exposed to CO2 to simulate the increased ventilation of exercise and more closely mimic human studies. The experimental protocols employed in the above inhalation studies permitted examination of (1) the impact of species, size, handling stress, and changes in Ta on both the induced hypothermic response and the concomitant pulmonary toxicity; (2) the additive impact of exercise stress on O3 toxicity; and (3) the toxicity of ambient-derived particulate matter in normal rats and in rats with preexisting pulmonary inflammation. The results of these studies demonstrate that the magnitude of the induced hypothermic response is directly proportional to the uptake of the toxic agent by the lung and inversely proportional to the mass of the animal and the ambient temperature at which the exposure is conducted. The hypothermic response is sensitive to a number of experimental stresses including handling and changes in cage conditions. Exercise attenuates the hypothermic response, whereas CO2-stimulated increases in ventilation employed as an exercise surrogate may potentiate the response. Toxic exposures conducted in animals with lung disease or compromised pulmonary function may induce a severe hypothermic response while comparable exposures in normal animals produce only mild or moderate responses. In general, the development of the hypothermic response in the presence of ambient pollutants serves to decrease the minute ventilation of the animal and therefore limits the uptake and dose of the airborne toxicant. The results of these inhalation studies support our previous conclusions concerning the impact of the hypothermic response on toxicity and emphasize the need to monitor and incorporate these changes in functional parameters into analyses of toxicological data. Furthermore, because humans do not demonstrate a robust hypothermic response following exposure to toxic agents, extrapolation of the results obtained from animal studies and comparisons with data from human studies are considerably more complicated.

Ozone exposure alters tracheobronchial mucociliary function in humans.

Mucociliary function is a primary defense mechanism of the tracheobronchial airways, and yet the response of this system to an inhalational hazard, such as ozone, is undefined in humans. Utilizing noninvasive techniques to measure deposition and retention of insoluble radiolabeled particles on airway mucous membranes, we studied the effect on mucus transport of 0.2 and 0.4 ppm ozone compared with filtered air (FA) in seven healthy males. During 2-h chamber exposures, subjects alternated between periods of rest and light exercise with hourly spirometric measurement of lung function. Mechanical and mucociliary function responses to ozone by lung airways appeared concentration dependent. Reduction in particle retention was significant (P less than 0.005) (i.e., transport of lung mucus was increased during exposure to 0.4 ppm ozone and was coincident with impaired lung function; e.g., forced vital capacity and midmaximal flow rate fell by 12 and 16%, respectively, and forced expiratory volume at 1 s by 5%, of preexposure values). Regional analysis indicated that mucus flow from distal airways into central bronchi was significantly increased (P less than 0.025) by 0.2 ppm ozone. This peripheral effect, however, was buffered by only a marginal influence of 0.2 ppm ozone on larger bronchi, such that the resultant mucus transport for all airways of the lung in aggregate differed only slightly from FA exposures. These data may reflect differences in regional diffusion of ozone along the respiratory tract, rather than tissue sensitivity. In conclusion, mucociliary function of humans is acutely stimulated by ozone and may result from fluid additions to the mucus layer from mucosal and submucosal secretory cells and/or alteration of epithelial permeability.

Role of tachykinins in ozone-induced acute lung injury in guinea pigs.

To examine the hypothesis that the acute reversible changes caused by ozone (O3) exposure are mediated by tachykinin release, guinea pigs were depleted of tachykinins by use of repeated capsaicin (CAP) injections before O3 exposure in an attempt to prevent O3-induced functional changes. Unexpectedly, CAP pretreatment caused divergent results in the functional responses to O3. Ventilatory measurements obtained from CAP-pretreated O3-exposed (CAP-O3) animals were exacerbated rather than diminished compared with the effects of O3 alone. Similarly, lavage fluid protein accumulation was enhanced in the CAP-O3 group compared with the O3-exposed group. In better agreement with our initial hypothesis, the CAP-O3 group was less responsive than the O3-exposed animals to histamine aerosol challenge. Additionally, Evans blue dye accumulation, a hallmark of tachykinin release, was increased in O3-exposed animals and was partially blocked in the CAP-O3 group. These data suggest that tachykinin-containing sensory fibers are unlikely to mediate the acute effects of O3 exposure on tidal breathing and lavage fluid protein accumulation but may play a role in causing post-O3 airway hyperreactivity and protein extravasation into the trachea.

Ozone toxicity in the rat. III. Effect of changes in ambient temperature on pulmonary parameters.

Pulmonary toxicity of ozone (O3) was examined in adult male Fischer 344 rats exposed to 0.5 parts/million O3 for either 6 or 23 h/day over 5 days while maintained at an ambient temperature (Ta) of either 10, 22, or 34 degrees C. Toxicity was evaluated by using changes in lung volumes and the concentrations of constituents of bronchoalveolar lavage fluid that signal lung injury and/or inflammation. Results indicated that toxicity increased as Ta decreased. Exposures conducted at 10 degrees C were associated with the greatest decreases in body weight and total lung capacity and the greatest increases in lavageable protein, lysozyme, alkaline phosphatase activity, and percent neutrophils. O3 effects not modified by Ta included increases in residual volume and lavageable potassium, glucose, urea, and ascorbic acid with exposure at 34 degrees C. Most effects were attenuated during the 5 exposure days and/or returned to normal levels after 7 air recovery days, regardless of prior O3 exposure or Ta. It is possible that Ta-induced changes in metabolic rate may have altered ventilation and, therefore, the O3 doses among rats exposed at the three different Ta levels.

Cardiac arrhythmia induction after exposure to residual oil fly ash particles in a rodent model of pulmonary hypertension.

Recent epidemiological studies have reported a positive association between exposure to ambient concentrations of particulate matter (PM) and the incidence of cardiopulmonary-related morbidity and mortality. The present study examined the effects of fugitive residual oil fly ash (ROFA) PM on cardiac arrhythmia induction in healthy and cardiopulmonary-compromised rodents. Male Sprague-Dawley rats were implanted with radiotelemetry transmitters capable of monitoring the electrocardiogram and were subjected to one of two treatment regimens. Rats in the first treatment regimen (n = 16) served as normal control animals whereas rats in the second treatment regimen (n = 16) were injected with monocrotaline (MCT, 60 mg/kg, ip) to induce pulmonary vascular inflammation and hypertension and served as a model of cardiopulmonary disease. Rats within each treatment regimen were equally divided into four dose groups (0.0, 0.25, 1.0, 2.5 mg ROFA), instilled intratracheally, and monitored for 96 h. In the animals in the first treatment regimen, ROFA instillation caused dose-related increases in the incidence and duration of serious arrhythmic events that appeared to be associated with impaired atrioventricular conduction and myocardial hypoxia. There were no lethalities in the normal animals following ROFA instillation. The frequency and severity of arrhythmias were greatly exacerbated in the MCT-treated animals in the second treatment regimen and were accompanied by one, three, and two deaths in the low-, medium-, and high-dose groups, respectively. The results of the present study demonstrate substantial cardiac effects in normal and compromised rats after exposure to ROFA PM and implicate both conductive and hypoxemic arrhythmogenic mechanisms in the observed cardiac-related lethalities. These results support previous epidemiological studies that suggest a link between preexisting cardiopulmonary disease and potentiation of adverse health effects following exposure to anthropogenic particulates.

Oil fly ash-induced elevation of plasma fibrinogen levels in rats.

Particulate matter air pollution (PM) has been associated with morbidity and mortality from ischemic heart disease and stroke in humans. It has been hypothesized that alveolar inflammation, resulting from exposure to PM, may induce a state of blood hypercoagulability, triggering cardiovascular events in susceptible individuals. Previous studies in our laboratory have demonstrated acute lung injury with alveolar inflammation in rats following exposure to residual oil fly ash (ROFA), an emission source particulate. In addition, increased mortality has been documented following exposure to ROFA in rats with preexistent cardiopulmonary disease. ROFA's toxicity derives from its soluble metal content, which appears also to drive the toxicity of ambient PM. The present study was conducted to test the hypothesis that exposure of rats to a toxic PM, like ROFA, would adversely alter hemostatic parameters and cardiovascular risk factors thought to be involved in human epidemiologic findings. Sixty-day-old male Sprague-Dawley rats were exposed by intratracheal instillation (IT) to varying doses (0.3, 1. 7, or 8.3 mg/kg) of ROFA, 8.3 mg/kg Mt. Saint Helen's volcanic ash (MSH, control particle), or 0.3 ml saline (SAL, control). At 24 h post-IT, activated partial thromboplastin time (APTT), prothrombin time (PT), plasma fibrinogen (PF), plasma viscosity (PV), and complete blood count (CBC) were performed on venous blood samples. No differences from control were detected in APTT and PT in ROFA-exposed rats; however, ROFA exposure did result in elevated PF, at 8.3 mg/kg only. In addition, PV values were elevated in both ROFA and MSH-exposed rats relative to SAL-control rats, but not significantly. Although no changes were detected in APTT and PT, alteration of important hematologic parameters (notably fibrinogen) through PM induction of an inflammatory response may serve as biomarkers of cardiovascular risk in susceptible individuals.

Cardiovascular and thermoregulatory effects of inhaled PM-associated transition metals: a potential interaction between nickel and vanadium sulfate.

Recent epidemiological studies have shown an association between daily morbidity and mortality and ambient particulate matter (PM) air pollution. It has been proposed that bioavailable metal constituents of PM are responsible for many of the reported adverse health effects. Studies of instilled residual oil fly ash (ROFA) demonstrated immediate and delayed responses, consisting of bradycardia, hypothermia, and arrhythmogenesis in conscious, unrestrained rats. Further investigation of instilled ROFA-associated transition metals showed that vanadium (V) induced the immediate responses, while nickel (Ni) was responsible for the delayed effects. Furthermore, Ni potentiated the immediate effects caused by V when administered concomitantly. The present study examined the responses to these metals in a whole-body inhalation exposure. To ensure valid dosimetric comparisons with instillation studies, 4 target exposure concentrations ranging from 0.3-2.4 mg/m(3) were used to incorporate estimates of total inhalation dose derived using different ventilatory parameters. Rats were implanted with radiotelemetry transmitters to continuously acquire heart rate (HR), core temperature (T(CO)), and electrocardiographic data throughout the exposure. Animals were exposed to aerosolized Ni, V, or Ni + V for 6 h per day x 4 days, after which serum and bronchoalveolar lavage samples were taken. Even at the highest concentration, V failed to induce any significant change in HR or T(CO). Ni caused delayed bradycardia, hypothermia, and arrhythmogenesis at concentrations > 1.2 mg/m(3). When combined, Ni and V produced observable delayed effects at 0.5 mg/m(3) and potentiated responses at 1.3 mg/m(3), greater than were produced by the highest concentration of Ni (2.1 mg/m(3)) alone. These results indicate a possible synergistic relationship between inhaled Ni and V, and provide insight into potential interactions regarding the toxicity of PM-associated metals.