Inflammation and tissue damage in mouse lung by single and repeated dosing of urban air coarse and fine particles collected from six European cities.

The authors have previously demonstrated heterogeneities in the inflammatory activities of urban air fine (PM(2.5-0.2)) and coarse (PM(10-2.5)) particulate samples collected from six European cities with contrasting air pollution situations. The same samples (10 mg/kg) were intratracheally instilled to healthy C57BL/6J mice either once or repeatedly on days 1, 3, and 6 of the study week. The lungs were lavaged 24 h after the single dose or after the last repeated dosing. In both size ranges, repeated dosing of particles increased the total cell number in bronchoalveolar lavage fluid (BALF) more than the respective single dose, whereas cytokine concentrations were lower after repeated dosing. The lactate dehydrogenase (LDH) responses increased up to 2-fold after repeated dosing of PM(2.5-0.2) samples and up to 6-fold after repeated dosing of PM(10-2.5) samples. PM(10-2.5) samples evoked a more extensive interstitial inflammation in the mouse lungs. The constituents with major contributions to the inflammatory responses were oxidized organic compounds and transition metals in PM(2.5-0.2) samples, Cu and soil minerals in PM(10-2.5) samples, and Zn in both size ranges. In contrast, poor biomass and coal combustion were associated with elevated levels of polycyclic aromatic hydrocarbons (PAHs) and a consistent inhibitory effect on the inflammatory activity of PM(2.5-0.2) samples. In conclusion, repeated intratracheal instillation of both fine and coarse particulate samples evoked enhanced pulmonary inflammation and cytotoxicity compared to single-dose administration. The sources and constituents of urban air particles responsible for these effects appear to be similar to those encountered in the authors' previous single-dose study.

Subchronic inhalation of mixtures of cigarette smoke constituents in Xpa-/-p53+/- knock-out mice: a comparison of intermittent with semi-continuous exposure to acetaldehyde, formaldehyde, and acrolein.

We investigated whether inhaling peak concentrations of aldehydes several times daily is more damaging than semi-continuously inhaling low-dose aldehydes. We exposed Xpa-/-p53+/- knock-out mice either intermittently or semi-continuously to mixed acetaldehyde, formaldehyde, and acrolein. The intermittent regimen entailed exposure to the aldehydes 7 min every 45 min, 12 times/day, 5 days/week, corresponding to concentrations inhaled by smokers. Semi-continuously exposed animals received half the dose of aldehydes in 8h/day, 5 days/week. Some mice in each group were sacrificed after 13 weeks of exposure; the rest breathed clean air until the end of 1 year. Mice injected intratracheally with benzo[a]pyrene formed a positive control group. The nasal cavity, lungs, and any macroscopically abnormal organs of all mice were analysed histopathologically. After 13 weeks of exposure, the subacute, overall, histopathological changes induced by the inhalation differed noticeably between the intermittently and semi-continuously treated Xpa-/-p53+/- knock-out mice. After 13 weeks of mixed aldehyde exposure, atrophy of the olfactory epithelium generally appeared, but disappeared after 1 year (adaptation and/or recovery). Respiratory epithelial metaplasia of the olfactory epithelium occurred at a higher incidence at 1 year. Except for a significantly greater number of tumours observed in knock-out mice compared to wild mice (semi-continuous aldehyde exposure and controls), no differences between the semi-continuous and intermittent exposure groups were observed.

Host genetics of Bordetella pertussis infection in mice: significance of Toll-like receptor 4 in genetic susceptibility and pathobiology.

The susceptibility to and the severity of Bordetella pertussis infections in infants and children varies widely, suggesting that genetic differences between individuals influence the course of infection. We have previously identified three novel loci that influence the severity of whooping cough by using recombinant congenic strains of mice: Bordetella pertussis susceptibility loci 1, 2, and 3 (Bps1, -2, and -3). Because these loci could not account for all genetic differences between mice, we extended our search for additional susceptibility loci. We therefore screened 11 inbred strains of mice for susceptibility to a pertussis infection after intranasal infection. Susceptibility was defined by the number of bacteria in the lungs, being indicative of the effect between the clearance and replication of bacteria. The most resistant (A/J) and the most susceptible (C3H/HeJ) strains were selected for further genetic and phenotypic characterization. The link between bacterial clearance and chromosomal location was investigated with 300 F2 mice, generated by crossing A/J and C3H/HeJ mice. We found a link between the delayed clearance of bacteria from the lung and a large part of chromosome 4 in F2 mice with a maximum log of the odds score of 33.6 at 65.4 Mb, which is the location of Tlr4. C3H/HeJ mice carry a functional mutation in the intracellular domain of Tlr4. This locus accounted for all detectable genetic differences between these strains. Compared to A/J mice, C3H/HeJ mice showed a delayed clearance of bacteria from the lung, a higher relative lung weight, and increased body weight loss. Splenocytes from infected C3H/HeJ mice produced almost no interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) upon ex vivo restimulation with B. pertussis compared to A/J mice and also showed a delayed gamma interferon (IFN-gamma) production. TNF-alpha expression in the lungs 3 days after infection was increased fivefold compared to uninfected controls in A/J mice and was not affected in C3H/HeJ mice. In conclusion, Tlr4 is a major host factor explaining the differences in the course of infection between these inbred strains of mice. Functional Tlr4 is essential for an efficient IL-1-beta, TNF-alpha, and IFN-gamma response; efficient clearance of bacteria from the lung; and reduced lung pathology.

Dose dependency of adjuvant activity of particulate matter from five European sites in three seasons in an ovalbumin-mouse model.

Various particulate matter (PM) samples were tested for their adjuvant potency in an animal model of allergy (ovalbumin) in the European Union study entitled Respiratory Allergy and Inflammation Due to Ambient Particles. Coarse and fine ambient particles were collected during spring, summer, and winter in Rome, Oslo, Lodz, Amsterdam, and De Zilk. De Zilk, at the Dutch seaside, has mainly westerly winds and served as a negative pollution control. EHC-93 (Ottawa dust) was used as a positive control. We studied the adjuvant potency of the particle antibody responses to ovalbumin and histopathological changes in the lung. After a sensitization phase by coexposure to EHC-93 and ovalbumin, the antibody response to ovalbumin and inflammatory responses in the lung were huge. There was more adjuvant activity in reaction to 9-mg/ml samples than to 3-mg/ml samples. A best-fit analysis of these samples shows that the ambient coarse and fine particles at these sites, in combination with allergens, have severe to mild adjuvant activity in the order Lodz, Rome, Oslo, and Amsterdam. A high dose of the fine fraction was more potent than a high dose of the coarse fraction, except at De Zilk, where the reverse was true. Spring and winter PM was more potent than summer PM. Depending on the site, either a water-soluble or a water-insoluble fraction was responsible for the adjuvant activity. A concentration of 3 mg/ml is effective for screening high-activity samples, as is a concentration of 9 mg/ml for screening low-activity samples in the ovalbumin-mouse model.

Adjuvant activity of ambient particulate matter of different sites, sizes, and seasons in a respiratory allergy mouse model.

In the framework of an EU project entitled, "Respiratory Allergy and Inflammation due to Ambient Particles (RAIAP)", various ambient particulate matter samples were tested for their adjuvant potency in an animal allergy model to ovalbumin. Coarse (2.5-10 microm) and fine (0.15-2.5 microm) particles were collected during the spring, summer, and winter in Rome, Oslo, Lodz, and Amsterdam. Coarse and fine particles were also collected near a seaside location in the Netherlands, where prevailing winds are westerly. These latter particles served as a control, with a minimum contribution by traffic. Ottawa dust (EHC-93) was used as a standard reference sample. Immunoglobulins (IgE, IgG1, and IgG2a), histopathological changes in the lung, cytokines, and the number of cells and their differentiation in lung lavages were used as effect parameters to study the adjuvant potency of these particles. The particles (3 mg/ml) were mixed with ovalbumin (0.4 mg/ml) and intranasally administered during the sensitization or the challenge phase. Intranasal administration of ovalbumin only induced very little antibody response, but introduced a minor inflammatory response in the lung or BAL during the sensitization and challenge phase. On the contrary, after coexposure to EHC-93 and ovalbumin, a major increase was found in immunoglobulin levels specific for ovalbumin, and a major inflammatory response in lung and BAL was induced. Coexposure to ovalbumin with 4 out of 12 collected PM samples (3 mg/ml) resulted in an increase of mainly IgE and IgG1. The histopathological changes consisted of a small to severe peribronchial and perivascular inflammatory response, a hypertrophy of bronchiolar mucous cells and an increase in eosinophils and neutrophils in the BAL. Statistical evaluation of the above-mentioned parameters showed associations with PMx (coarse and fine), site, season, season x PMx, season x site and (PMx) x site. In addition, adjuvant activity of the PMx can be ranked as Lodz > Rome = Amsterdam > Oslo. When the different seasons were compared for IgE, PM from winter was found more active compared to PM from spring and summer. Only for the histopathological lesions, statistically significant difference in effects was found between coarse and fine (coarse > fine). No associations were found between the endotoxin content and the biological effects parameters, although endotoxin was much more confined to the coarse fraction. In conclusion, PM, both coarse and fine, and from various geographic sites, was found to differ in adjuvant activity; furthermore, winter was found more active than spring and summer.

Adjuvant activity of ambient particulate matter in macrophage activity-suppressed, N-acetylcysteine-treated, iNOS- and IL-4-deficient mice.

In previous studies, we have shown strong adjuvant activity for Ottawa dust (EHC-93) after coexposure of the BALB/c mouse to EHC-93 and ovalbumin. Mice were intranasally sensitized at days 0 and 14 with 200 microg ovalbumin and 150 microg EHC-93, and challenged with ovalbumin at days 35, 38, and 41 with 200 microg ovalbumin. Mice were autopsied at day 42. This adjuvant activity was shown for the antibody response to ovalbumin (immunoglobulins E, G1, and G2a), histopathological lesions in the lung, cytokines, and the numbers of eosinophils in lung lavages. To study the mechanisms of this adjuvant activity, mice (BALB/cC.D2-Vil6) with natural-resistance-associated macrophage protein (Nramp1s), BALB/c mice pretreated with the antioxidant N-acetylcysteine (NAC), mice (B6.129P2-Nos2tmLau) deficient in inducible nitric oxide synthase (iNOS), and mice with interleukin-4 (IL-4) deficiency (BALB/cIl4< tm2Nnt) were coexposed to ovalbumin and EHC-93. Our studies have shown that the adjuvant activity induced after such coexposure does not change if the macrophage activation of the mice is disturbed or if the mice have been pretreated with N-acetylcysteine. In addition, the adjuvant activity does not develop through the pathway in which inducible nitric oxide synthase is involved. Because the histopathological lesions are statistically significant less in the IL-4 knockout strain in comparison with the wild type, we conclude that interleukin-4 might play an important role in the adjuvant activity caused by EHC-93.

Intratracheal instillation of cytoplasmic granules from Phleum pratense pollen induces IgE- and cell-mediated responses in the Brown Norway rat.

BACKGROUND: Release of cytoplasmic granules from grass pollen upon contact with water is thought to be an important source of airborne allergens. OBJECTIVES: To investigate the humoral and cellular responses to intratracheal instillation of Phleum pratense (timothy grass) pollen cytoplasmic granules (PCG) in the Brown Norway rat. METHODS: PCG were purified from timothy grass pollen by filtration through 5-microm-mesh filters. Rats were sensitized (day 0) and challenged (day 21) intratracheally with purified PCG suspended in saline (6 x 10(6) PCG/rat). Rats were then challenged 4 weeks later (1.5 x 10(6) PCG/rat). Blood samples, bronchial lymph nodes and lungs were collected from the rats 4 days after the second challenge. PCG-specific IgE and IgG1 levels and specificity were determined by ELISA and Western blotting. Pollen, pollen extract and PCG-induced proliferation of lymph node cells were monitored by [(3)H]-thymidine incorporation in a lymph node assay. Histopathological examination was carried out on the lungs. RESULTS: Specific IgE and IgG1 were present in the sera. Cultured lymph node cells proliferated in the presence of pollen, pollen extract and PCG. Western blots showed that all major pollen allergens are recognized by IgE and IgG1 from PCG-treated rats. Histopathological examination revealed features of a mild allergic reaction. CONCLUSIONS: In our rat model of allergy, purified timothy grass PCG instillation induced specific antibodies and lymph node cell responses, comparable to those obtained with intact pollen.

Timing of infection and prior immunization with respiratory syncytial virus (RSV) in RSV-enhanced allergic inflammation.

Respiratory syncytial virus (RSV) infection has been shown to be a risk factor for the development of allergy in humans and mice. The allergy-enhancing properties of RSV may be dependent on atopic background and an individual's history of RSV infection. We examined the influence of the timing of infection and prior inoculation with RSV in a mouse model of allergic asthma. Mice were sensitized to and challenged with ovalbumin (OVA) and were inoculated with RSV either before or during the sensitization or challenge period. One group of mice was inoculated with RSV both before sensitization to OVA and during challenge with OVA. Increased pulmonary expression of interleukin (IL)-4, IL-5, and IL-13 mRNA and aggravated alveolitis and hypertrophy of mucus-producing cells were observed only when OVA-sensitized mice were inoculated with RSV shortly before or during challenge with OVA. Despite protection against viral replication, prior inoculation with RSV did not abrogate RSV-enhanced, OVA-induced expression of T helper 2 (Th2) cytokines in the lung. In conclusion, inoculation with RSV enhances allergic disease only when the immune system has already been Th2-primed by the allergen (i.e., OVA). This RSV-enhanced allergy is not completely abrogated by prior inoculation with RSV.

Phleum pratense pollen starch granules induce humoral and cell-mediated immune responses in a rat model of allergy.

BACKGROUND: Timothy grass (Phleum pratense) pollen allergens are an important cause of allergic symptoms. However, pollen grains are too large to penetrate the deeper airways. Grass pollen is known to release allergen-bearing starch granules (SG) upon contact with water. These granules can create an inhalable allergenic aerosol capable of triggering an early asthmatic response and are implicated in thunderstorm-associated asthma. OBJECTIVE: We studied the humoral (IgE) and bronchial lymph node cells reactivities to SG from timothy grass pollen in pollen-sensitized rats. METHODS: Brown-Norway rats were sensitized (day 0) and challenged (day 21) intratracheally with intact pollen and kept immunized by pollen intranasal instillation by 4 weeks intervals during 3 months. Blood and bronchial lymph nodes were collected 7 days after the last intranasal challenge. SG were purified from fresh timothy grass pollen using 5 microm mesh filters. To determine the humoral response (IgE) to SG, we developed an original ELISA inhibition test, based on competition between pollen allergens and purified SG. The cell-mediated response to SG in the bronchial lymph node cells was determined by measuring the uptake of [3H]thymidine in a proliferation assay. RESULTS: An antibody response to SG was induced, and purified SG were able to inhibit the IgE ELISA absorbance by 45%. Pollen extract and intact pollen gave inhibitions of 55% and 52%, respectively. A cell-mediated response was also found, as pollen extract, intact pollen and SG triggered proliferation of bronchial lymph node cells. CONCLUSIONS: It was confirmed that timothy grass pollen contains allergen-loaded SG, which are released upon contact with water. These granules were shown to be recognized by pollen-sensitized rats sera and to trigger lymph node cell proliferation in these rats. These data provide new arguments supporting the implication of grass pollen SG in allergic asthma.

Optimization of route of administration for coexposure to ovalbumin and particle matter to induce adjuvant activity in respiratory allergy in the mouse.

Epidemiological and experimental studies have not only shown that air pollution induces increased pulmonary morbidity, and mortality, but also that air pollution components may potentiate allergic responses. The respiratory allergy model to ovalbumin in the mouse has been shown a useful tool to characterize the adjuvant potency of air pollution components. However, the choice for the most effective route of administration for testing small amounts of air pollution component is hampered by the diversity of routes of administration used. To test the adjuvant activity of airborne particles (Ottawa dust EHC-93), we studied the optimal route of respiratory administration: intranasally (in) and aerosol (aero) in comparison with responses observed by intraperitoneal (ip) with diesel exhaust particles (DEP) as a positive control. Our results show that the combination of in/aero with ovalbumin caused almost similar immunoglobulin (Ig)E and inflammatory responses compared to the ip/aero. In/in application induced less responses for IgE, less inflammation in the lung, and less increased numbers of eosinophils in the bronchoalveolar lavage (BAL). This response increased dramatically when ovalbumin was coadministered with DEP. Subsequently, EHC-93, which is made up of airborne particles, was tested via the in/in route of administration. EHC-93 induced similar IgE responses, inflammation, and eosinophilic response in BAL compared to DEP. In addition, EHC-93 increased the airway responsiveness of the ovalbumin-sensitized mice measured in unrestrained condition and not in nonsensitized control mice. It is concluded that intranasal sensitization with intranasal challenge with airborne particles (EHC-93) is an effective route of administration to show potency of adjuvant activity of airborne particles.

Adjuvant activity of various diesel exhaust and ambient particles in two allergic models.

In the framework of an EU study entitled "Respiratory Allergy and Inflammation Due to Ambient Particles" (RAIAP), various collected particulate matter samples were to be tested for their adjuvant potency in two animal models of allergy. A pollen allergy model in the Brown Norway (BN) rat and an ovalbumin model in the BALB/c mouse were used in this study to compare the discriminatory value of these two models and to evaluate them for later studies of collected RAIAP-samples. Two different sources of diesel exhaust particles (DEP I and DEP II ), a residual oil fly ash source (ROFA), and two sources of ambient particles (Ottawa dust, EHC-93, and road tunnel dust, RTD) were tested. Rats were sensitized intratracheally with Timothy grass pollen (Phleum pratense, 200 microl, 10 mg/ml) on d 0, challenged on d 21, and examined on d 25. Mice were sensitized intranasally at d 0 and 14, challenged intranasally at d 35, 38, and 41 (50 microl, 0.4 mg ovalbumin/ml), and examined at d 42. Particulate matter (PM) was administered either during the sensitization phase only or during the sensitization and challenge phases (for mice only) or during the challenge phase only. In the pollen model, only DEP I, but not DEP II, ROFA, EHC-93, and RTD, stimulated the immunoglobulin (Ig) E and IgG1 response in serum to pollen allergens. In addition to this adjuvant effect noted, no other biomarkers in lung or bronchoalveolar lavage (BAL) revealed adjuvant activity in the pollen model. In the BAL of BN rats exposed to a combination of pollen and PM, the percentages of eosinophilic granulocytes were decreased compared to the BAL of BN rats immunized with pollen only. In the ovalbumin model, the IgE levels in serum were increased in mice after coexposure to ovalbumin and PM (including DEPI, DEPII, ROFA, EHC-93, and RTD) in the sensitization phase but not after coexposure during the challenge phase only. The inflammatory response was greater in the lung, predominantly the influx of eosinophilic granulocytes, as was observed by both histopathological examination and BAL analysis. In addition, BAL levels of inflammatory interleukin (IL)-4 were increased. Based on the IgE antibody response to ovalbumin, the ovalbumin model ranked the adjuvant capacity of the particles in the following order: RTD > ROFA > EHC-93 > DEPI > DEPII. In conclusion, the ovalbumin model is a sensitive system to detect adjuvant activity of airborne particles, whereas the pollen-induced allergy model in rat was less sensitive.

Acute effect of air pollution on respiratory complaints, exhaled NO and biomarkers in nasal lavages of allergic children during the pollen season.

During 2 months of the pollen season, the acute and putative adjuvant effect of traffic-related air pollution on respiratory health was investigated in children sensitised to grass pollen or house dust mite (HDM). Respiratory complaints were objectified via measurement of exhaled NO and inflammatory mediators in nasal lavage (NAL). During the study children, skin prick negative (n = 31) or positive to grass pollen (n = 22), HDM (n = 34) or grass pollen + HDM (n = 32), kept a daily diary on respiratory symptoms, and NAL and exhaled air was sampled twice a week. The level of air pollutants and pollen was monitored continuously. Like children sensitised to HDM, those sensitised to pollen reported respiratory complaints (shortness of breath, itchy eyes or blocked nose) more frequently than non-sensitised children during (but not before) the pollen season; the respiratory complaints of sensitised children were independent of the pollen level. In addition, exposure to increased levels of PM(10) induces 'shortness of breath' in pollen- and HDM-sensitised children, whereas ozone induces a blocked nose in HDM-sensitised children. Combined exposure to PM(10) + pollen and O(3) + pollen induces a blocked nose in both HDM-sensitised children and children sensitised to pollen + HDM. Significant positive associations were found between eNO and the levels of NO(2), CO, PM(2.5) and pollen in both sensitised and non-sensitised children. At the start of the pollen season, the NAL concentration of eosinophils and ECP in pollen-sensitised children was increased compared to winter, but their levels were not further affected by increased exposure to pollen or air pollution. In conclusion, during the pollen season, sensitised children continuously report a high prevalence of respiratory complaints which coincides with increased levels of upper and lower airway inflammatory markers. No additional pro-inflammatory effect of air pollution was observed, which indicates that air pollution does not facilitate allergen-induced inflammatory responses.

Exhaled NO level and number of eosinophils in nasal lavage as markers of pollen-induced upper and lower airway inflammation in children sensitive to grass pollen.

OBJECTIVES: This study investigates the upper and lower inflammatory response induced by natural exposure to grass pollen in atopic and non-atopic children. METHODS: After children's atopic profile had been assessed, their nasal lavage fluid (NAL) and exhaled air was sampled once before and once during the pollen season. Level of nitric oxide (NO) was determined in exhaled air, and the following mediators were measured in NAL: ECP, IL-6, IL-8, albumin, uric acid, and urea. The number of eosinophils in NAL was determined after Giemsa staining. During the experiment ozone and pollen levels were measured continuously. RESULTS: During the pollen season the level of grass pollen was 95 pollen grains per cubic metre. At baseline, 8.0% and 5.4% of total cells in NAL of children sensitive to, respectively, house dust mite (HDM) and pollen + HDM were eosinophils, whereas virtually no eosinophils were observed in NAL of non-atopic children. In contrast to the non-atopic and HDM groups, in children sensitive only to grass pollen, grass pollen induced a threefold increase in the percentage of NAL eosinophils and a 2.5-fold increase in the NAL level of ECP ( P<0.05). In all groups, the NAL levels of albumin, uric acid, urea, IL-6 and IL-8 were not significantly increased by pollen exposure. At baseline, children sensitive to HDM showed significantly higher exhaled nitric oxide (eNO) values than non-atopic subjects and children sensitive only to pollen (79 to 141% increase). During pollen exposure eNO of children sensitive only to pollen increased from 35.8 to 64.5 ppb ( P<0.05), whereas no increase in eNO was observed in the other children. CONCLUSION: Pollen-sensitive children show a season-dependent upper and lower airway inflammatory response, resembling the continuous inflammation in HDM-sensitive children.

Pulmonary effects of ultrafine and fine ammonium salts aerosols in healthy and monocrotaline-treated rats following short-term exposure.

In the present study the effects of a 3-day inhalation exposure to model compounds for ambient particulate matter were investigated: ammonium bisulfate, ammonium ferrosulfate, and ammonium nitrate, all components of the secondary aerosol fraction of ambient particulate matter (PM), and carbon black (CB, model aerosol for primary PM). The objective of this study was to test the hypothesis that secondary model aerosols exert acute pulmonary adverse effects in rats, and that rats with pulmonary hypertension (PH), induced by monocrotaline (MCT), are more sensitive to these components than normal healthy animals. An additional aim was to test the hypothesis that fine particles exert more effects than ultrafines. Healthy and PH rats were exposed to ultrafine (mass median diameter [MMD] approximate, equals 0.07-0.10 microm; 4 x 10(5) particles/cm(3)) and fine (MMD approximate, equals 0.57-0.64 micro;m; 9 x 10(3) particles/cm(3)) ammonium aerosols during 4 h/day for 3 consecutive days. The mean mass concentrations ranged from 70 to 420 microg/m(3), respectively, for ultrafine ammonium bisulfate, nitrate, and ferrosulfate and from 275 to 410 microg/m(3) for fine-mode aerosols. In an additional experiment, simultaneous exposure to a fine CB aerosol (0.6 microm; 2-9 mg/m(3)) and ammonium nitrate (0.4-18 mg/m(3)) was performed. Bronchoalveolar lavage fluid (BALF) analysis and histopathological examination were performed on animals sacrificed 1 day after the last exposure. Histopathology of the lungs did not reveal test atmosphere-related abnormalities in either healthy or PH rats exposed to the ammonium salts, or to a combination of CB + nitrate. Alveolar macrophages in rats exposed to CB only revealed the presence of black material in their cytoplasm. There were no signs of cytotoxicity due to the aerosol exposures (as measured with lactate dehydrogenase [LDH], protein, and albumin contents in BALF). Macrophages were not activated after MCT treatment or the test atmospheres, since no changes were observed in N-acetyl glucosaminidase (NAG). Cell differentiation profiles were inconsistent, partly caused by an already present infection with Haemophilus sp. However, we believe that the test atmospheres did not affect cell differentiation or total cell counts. The results show that at exposure levels of ammonium salts at least one order of magnitude higher than ambient levels, marked adverse health effects were absent in both healthy and PH rats.

Dietary fatty acid composition during pregnancy and lactation in the rat programs growth and glucose metabolism in the offspring.

AIMS/HYPOTHESIS: We investigated of the effects of fatty acid composition of the maternal diet on fetal and postnatal growth, morphology of the pancreas and glucose metabolism and muscle hexosamine concentrations in the adult offspring of rats. METHODS: High-fat diets enriched with either saturated or unsaturated fatty acids were fed to female adult rats 2 weeks before mating until the end of the weaning period. After weaning, the offspring was maintained on a diet with a balanced fatty acid content. At 3 months of age, pancreatic Langerhans islet size and number were assessed by morphometric analysis and oral glucose tolerance tests (OGTT) were carried out. RESULTS: The unsaturated fatty acid diet showed lower birth weight and reduced postnatal weight gain. Furthermore, this group showed increased pancreatic islet numbers without affected glucose tolerance at the age of 12 weeks. The offspring of the saturated fatty acid diet group showed a reduced number of large pancreatic islets. Moreover, a faster and higher insulin response was observed after an oral glucose load in these animals. Muscle hexosamine concentrations were not different between groups. CONCLUSION/INTERPRETATION: Maternal diets enriched with either saturated fatty acids or unsaturated fatty acids had opposite effects on pancreatic islet development in rat offspring, with consequences for the insulin response at 12 weeks of age. Therefore, maternal dietary fatty acid composition plays a role in programming growth, pancreatic development and glucose metabolism in the offspring.

Attenuation and recovery of pulmonary injury in rats following short-term, repeated daily exposure to ozone.

Controlled human and epidemiology studies have demonstrated that during repeated exposure to ozone (O(3)) attenuation of lung function responses may occur. It is yet unknown whether inflammatory and biochemical effects in lower airways of humans, as observed upon single O(3) exposure, also show a diminutive response following repeated exposure to O(3). The aim of this study was to investigate inflammatory, permeability, and histopathological responses in lungs of rats following repeated daily O(3) exposure and to study the time course of attenuation and recovery of these effects using single O(3) challenges at various postexposure times. To aid in animal-to-human extrapolation, this study and a previously reported human study (Devlin et al., 1997) were designed with similar protocols. Wistar rats were exposed for 5 consecutive days to 0.4 ppm O(3) for 12 h/night. Subsequently, the time course of postexposure recovery was determined by a single challenge of 12 h to 0.4 ppm O(3) after a 5-, 10-, 15-, or 20-day recovery period. Bronchoalveolar lavage (BAL) examination and histopathology were performed 12 h after this O(3) challenge. To quantify the magnitude of the O(3) response, results were compared with a group exposed only once for 12 h to 0.4 ppm O(3) and sacrificed simultaneously. The results demonstrate that a single exposure of 0.4 ppm O(3) causes marked permeability and inflammatory responses in lower airways of rats, as evidenced by enhanced BAL fluid levels of proteins, fibronectin, interleukin (IL)-6, and inflammatory cells. However, 5 days of exposure to 0.4 ppm O(3) for 12 h/night resulted in a complete disappearance of these responses, resulting in BAL fluid values that were not different from those observed in unexposed controls. Postexposure analyses of pulmonary response to O(3) challenges demonstrated that these attenuated responses show a gradual recovery. The data indicate that with respect to BAL fluid levels of albumin, IL-6, and number of macrophages and neutrophils, the period for lung tissue to regain its full susceptibility and responsiveness to O(3) following a 5-day preexposure period is approximately 15-20 days. Remarkably, the total protein and fibronectin responses in BAL fluid still exhibited an attenuated response to an O(3) challenge at 20 days postexposure. Morphometry (number of BrdU-labeled cells in terminal bronchiolar epithelium, and number of alveolar macrophages) showed that after a recovery of 5-10 days following a 5-day preexposure the response to a challenge was identical to that after a single exposure. These results suggest that complete repair from lower airway inflammation caused by short-term, repeated exposure to O(3) may take longer than previously assumed.

Effects of diesel exhaust enriched concentrated PM2.5 in ozone preexposed or monocrotaline-treated rats.

Epidemiological studies have observed statistical associations between short-term exposure to increased ambient particulate air pollution and increased hospital admissions, medication use, pulmonary morbidity, and mortality. To examine the effects of particle air pollution in animals, rats with a preexisting pulmonary inflammation (induced by 1600 microg/m(3) ozone) or hypertension (induced by monocrotaline, MCT) were nose-only exposed to concentrated freshly generated diesel exhaust particles (DEP) mixed with ambient air (CDP). It was hypothesized that a single 6-h exposure to PM exacerbates respiratory inflammatory processes, which affects health parameters in the blood. Histopathology of lung and nose, bronchiolar lavage (BAL), and blood analyses were performed at 1, 2, and 4 days after of the CDP exposure. Morphometry of BrdU-labeled cells in lung and nose was performed at 4 days postexposure. One day after ozone exposure, a mild inflammatory reaction in the centriacinar area was present, consisting of an increase in cellularity of septa and in the number of alveolar macrophages, decreasing in time. Additional CDP exposure did not influence this pattern, except for alveolar macrophages that were loaded with CDP. The only effect seen in the nose after ozone exposure was a slight hypertrophy of the septal mucous cells. Additional exposure to CDP did not change this appearance. MCT-treated rats showed hypertrophy of the media of the pulmonary muscular arteries that was not effected by CDP. BrdU labeling of predominantly Clara cells in the terminal bronchioles was significantly increased after ozone exposure as well as after MCT treatment, whereas this labeling index was markedly enhanced after an additional exposure to CDP. However, no increases in Clara cell protein (CC16) levels were measured of Clara cell protein (CC16) in either BAL or blood. BrdU labeling in the nasal epithelium was not influenced by exposure to ozone or ozone + CDP. CDP exposures did not induce significant toxic effects in the lungs. CDP exposures clearly induced an oxidative stress that was indicated by increasing glutathione levels in BAL with time. In addition, blood fibrinogen levels were enhanced in pulmonary hypertensive rats exposed to CDP. The present study demonstrates that very high CDP concentrations are needed to result in pulmonary changes in animal models with a preexisting pulmonary inflammation or hypertension that continue for days after a single exposure. In addition, CDP has the potential to induce changes in blood. It has not yet been determined how the effects seen with CDP would compare to similar levels of ambient particles.

Time study on development and repair of lung injury following ozone exposure in rats.

The aim of this study was to investigate the time course of lung injury in rats during acute and subchronic ozone exposure and during postexposure recovery. Rats were continuously exposed to 0.4 ppm ozone ( approximately 0.8 mg O(3)/m(3)) for 1, 3, 7, 28, or 56 days. Recovery from 3 days of exposure was studied at day 7, 14, and 28; recovery from 7 days of exposure was studied at day 14, 28, and 56, recovery from 28 days of exposure was studied at day 35 and 56, and recovery from 56 days of exposure was studied at day 136. The study included a correlated biochemical and morphological analysis of inflammatory responses, structural changes, and collagen content. The acute inflammatory response, as measured by an increase of polymorphonuclear cells and plasma protein in bronchoalveolar lavage (BAL) fluid, reached a maximum at day 1 and resolved largely within 6 days during ongoing exposure. Numbers of macrophages in BAL fluid increased progressively up to day 56, and slowly returned to near control levels when exposure was followed by postexposure recovery. Histological examination and morphometry of the lungs revealed centriacinar inflammatory responses throughout ozone exposure. Centriacinar thickening of septa was observed at day 7. Ductular septa, thickened progressively at days 7, 28, and 56 of exposure, showed increased collagen upon exposure at day 28, which was further enhanced at exposure at day 56. Increased collagen content in lungs, as measured biochemically by hydroxyproline concentration, was observed at exposure day 56. Collagen content was not different from control at day 56 when 7 or 28 days of exposure was followed by postexposure recovery. After continuous ozone exposure, respiratory bronchioles were present in an increasing degree, and remained present after a recovery period. The results of this study clearly show that after continuous exposure to O(3) some acute effects, such as protein and albumin content, and neutrophil influx in BAL fluid, returned to control levels within a few days. However, other parameters, such as the alveolar macrophage response and structural changes such as the presence of terminal bronchioles, thickening of ductular septa by enhanced cellularity, and collagen formation, persisted or progressively increased during continued exposure. Postexposure recovery seems to partly resolve these subchronic responses (macrophages response, septal cellularity), whereas other effects (collagen increase and respiratory bronchioles formation) do not disappear.

A pollen model in the rat for testing adjuvant activity of air pollution components.

During the last decades, the prevalence of allergy has increased worldwide. Allergic rhinitis ("hay fever") and asthma are two of the most common allergic diseases. A possible cause for increased allergy to pollen is air pollution. The increase of industrialization and the number of diesel engines associated with diesel exhaust particles (DEP) in the air parallel the increase in allergic airway diseases. To investigate the adjuvant effect of DEP in pollen allergy, Brown Norway (BN) rats were sensitized intranasally or intratracheally with timothy grass pollen (Phleum pratense) with or without DEP (3 mg/ml). Intranasal sensitization (200 microl, 10 mg/ml) was performed daily for 5 consecutive days and intratracheal sensitization (200 microl, 10 mg/ml) was performed once. Challenge with pollen was performed at day 21 similarly to the sensitization protocol. Blood samples were taken at day 28 after the first sensitization. The binding of DEP to pollen grains was studied by scanning electron microscopy and the inflammatory response in the lung was studied by light microscopy. Immunoglobulin E (IgE) and IgG(1) responses against pollen grains were measured by digoxigenin (DIG) enzyme-linked immunosorbent assay (ELISA). Scanning electron microscopy revealed a mixture of free DEP and DEP associated with pollen grains. Both intranasal and intratracheal routes of administration of pollen grains induced inflammatory reactions in the lung with an influx of macrophages, eosinophilic granulocytes, and granuloma formation. Pollen grains were localized in the alveoli after both intranasal and intratracheal administration and were surrounded by macrophages. The number and localization of pollen grains were similar for both routes of administration. After coexposure with DEP, DEP-loaded macrophages were found around the pollen. Localization, inflammatory reaction, and integrity of pollen were similar to those seen without DEP. At day 28, specific IgE and IgG(1) antibodies were found in serum of rats immunized intranasally or intratracheally. IgE antibody response was higher in rats immunized with pollen grains and DEP than in rats immunized with pollen only (dilution mean +/- SEM: 59.4 +/- 4.6 vs. 27 +/- 5.1). The IgG(1) antibody response was much higher compared to the IgE response (factor of 10(4)), but the level of IgG(1) antibodies was only slightly increased by DEP (dilution mean +/- SEM: 24.2 +/- 2.0 x 10(4) vs. 16.1 +/- 2.1 x 10(4)). In conclusion, the intranasal application of pollen in the BN rat is a suitable and elegant method to evoke inflammatory reactions in the lung and pollen-specific IgE responses measured by DIG ELISA. Finally, this model gives similar results on adjuvant activity of DEP found in the ovalbumin models presented previously.

Pathological and immunological effects of respirable coal fly ash in male Wistar rats.

In this study the effects of inhalatory exposure to coal fly ash on lung pathology and the immune system in rats were examined. Rats were exposed to 0, 10, 30, or 100 mg/m(3) coal fly ash (6 h/day, 5 days/wk) for 4 wk, or to 0 and 100 mg/m(3) for 1 wk, and for 1 wk followed by a recovery in clean air of 3 wk. A concentration-related increase in lung weight was found starting from 30 mg/m(3) coal fly ash. After exposure to 100 mg/m(3), a time-related deposition of free particles in the lungs was observed as well as a time-related number of coal fly ash particles phagocytized in alveolar macrophages. Histological examination revealed increased cellularity in alveolar septa, consisting mainly of mononuclear cell infiltrate, proliferated type II cells, and a slight fibrotic reaction. After a recovery period of 3 wk the histological picture was identical to that after 1 wk of exposure, indicating no significant recovery. No toxicological significant changes were found in the hematological, clinical chemistry, or urine parameters. Effects both on nonspecific defense mechanisms and on specific immune responses were noted. With regard to the immune function in the draining lymph nodes of the lung, a significantly increased number of both T and B lymphocytes was observed. The ratio of both cell types was not changed in either of the groups. In serum of exposed rats a significant increase of up to 150% of the immunoglobulin A (IgA) content was found. The number and phagocytic capacity of macrophages were significantly increased, while the killing of Listeria bacteria per cell ex vivo/in vitro remained unchanged. Natural killer (NK) activity in pulmonary cell suspensions was slightly stimulated in rats exposed for 4 wk to 10 and 30 mg/m(3), whereas an exposure to 100 mg/m(3) resulted in a slight decrease; however, both changes were not significant. In conclusion, the alterations in lung histopathology and immunity, observed in a dose and exposure time relation at concentrations up to and including 100 mg/m(3) coal fly ash, may be considered an adverse response of the host to inhalation of particulate matter. Whether these observed alterations may effect the host resistance must be learned from infection studies.