[Methodology of neuroepidemiological studies in tropical countries: a challenge?]. / Méthodologie des études neuroépidémiologiques dans les pays tropicaux : un challenge ?

The purpose of this paper is to highlight the difficulties of applying neuroepidemiological methods in low income countries or developing countries, which are mostly tropical countries, taking advantage of the experience of the Institute of Neuroepidemiology and Tropical Neurology, which was created in Limoges in 1982. These difficulties could be related to several aspects: methodological, logistical, political or economical, linked to ethical issues, even difficulties to publish the studies. However, concept and neuroepidemiological methods should stay the same worldwide, even if their translation into practice could sometimes raise some problems in developing countries. Study protocol should be more detailed. Some specific epidemiological methods could be useful. Collection of data should be standardized. True cooperation at every level is needed for these researches to be valid.

Multiple sclerosis and solar exposure before the age of 15 years: case-control study in Cuba, Martinique and Sicily.

Few studies report a protective role of childhood solar exposure to multiple sclerosis. Our objective was to confirm the protective role of childhood solar exposure in multiple sclerosis in Cuba, Martinique and Sicily. This was a matched case- control study, and cases met Poser criteria for clinically, laboratory (definite, probable) multiple sclerosis. Controls were resident population, without neurological disorder, living close to cases (within 100 km), matched for sex, age (+/-5 years), residence before age 15. We recruited 551 subjects during a 1-year period (193 cases, Cuba n = 95, Sicily n = 50, Martinique n = 48; 358 controls). Some (89%) met definite clinical multiple sclerosis criteria (relapsing remitting form (with and without sequel) (74%), secondary progressive (21%), primary progressive (5%)). Odds ratios in a uni-variate analysis were: family history of multiple sclerosis (5.1) and autoimmune disorder (4.0); wearing shirt (3.5), hat (2.7), pants (2.4); sun exposure causing sunburn (1.8); sun exposure duration (1 h more/day; weekends 0.91, weekdays 0.86); bare-chested (0.6); water sports (0.2). Independent factors in the multivariate analysis were family history of multiple sclerosis (4.8 (1.50-15.10)), wearing pants under sunlight (1.9 (1.10-3.20)), sun exposure duration (1 h more/ day, weekdays 0.90 (0.85-0.98), weekends 0.93 (0.87-0.99)), water sports (0.23 (0.13-0.40)). We conclude that outdoor leisure activities in addition to sun exposure reports are associated with a reduced multiple sclerosis risk, with evidence of dose response.

Respiratory tract responses to repeated inhalation of an oxidant and acid gas-particle air pollutant mixture.

The purpose of this study was to examine a broad range of toxicologic responses in rats exposed to a multi-component pollutant atmosphere. Cumulative and adaptive respiratory tract responses to 3 concentrations of an inhaled particle-oxidant mixture were examined in Fisher 344 N rats exposed 4 h/day, 3 days/week for 4 weeks. The mixtures contained O3, NO2, NH4HSO4, carbon particles, and HNO3 vapor. Irritant-induced, rapid-shallow breathing responses were present during the first 4-h exposure to medium and high concentrations. Successive exposures showed diminished responses in medium concentrations and exacerbated responses in high concentrations. At the end of 4 weeks, rats exposed to high concentrations exhibited lung lesions. Lavaged pulmonary macrophages showed dose-dependent depressions of Fc-receptor binding and phagocytosis. Lung tissue macrophages showed dose-dependent increases in acid phosphatase staining density and carbon particles. Respiratory tract clearance of tracer particles was not significantly affected by the exposures. Broncho-alveolar epithelial permeability was increased by the high concentration. Epithelial cell-proliferation labeling showed a dose-dependent increase at all levels of the respiratory tract. Progressively exacerbated breathing-pattern responses at high concentrations were associated with lung lesions and high cell-proliferation labeling in the nose transitional epithelium and terminal bronchioles. Attenuating or adaptive breathing-pattern responses occurred in the presence of smaller, but in many cases still significant, compromise of respiratory functions. Either attenuating or exacerbated breathing-pattern responses can occur in the presence of a significant dose-dependent compromise of other respiratory functions and lung tissue injury.

Lung injury, inflammation, and inflammatory stimuli in rats exposed to ozone.

The effects of ozone (O3) on airway epithelia, inflammation, and expression of inflammatory stimuli were investigated to delineate the mechanisms of inflammatory reactions relevant to lung injury. Because the airway responses to O3 develop gradually, this investigation included a time-sequence analysis. Rats exposed for 3 h to 1 ppm O3 were studied at 4-h intervals up to 20 h postexposure. Bronchoalveolar lavage fluid (BAL) was analyzed for albumin as an indicator of increased permeability, polymorphonuclear leukocytes (PMNs) to assess the inflammatory status, macrophage inflammatory protein-2 (MIP-2, an inflammatory chemokine), and cell adhesion molecules for their role in inflammation and PMN functions. The time-related increase in albumin was matched by a similar significant increase for PMNs, MIP-2, and intercellular adhesion molecule-1 (ICAM-1). However, no marked change occurred for beta-2 integrin (CD-18) and leukotriene B4 (LTB4). The results establish a temporal correlation of epithelial permeability with changes in inflammatory activity and stimuli responsible for PMN recruitment in the lung. The observations of elevated MIP-2 and ICAM-1 levels are consistent with their role in injury and inflammation. An early expression of MIP-2 mRNA in BAL cells, that is, immediately post O3 exposure, and the peak increase in BAL MIP-2 levels 4 h later support the chemotactic role of MIP-2 in PMN recruitment at 4- and 12-h time points. The rapid drop in MIP-2 and ICAM-1 levels appears to signal the termination of inflammatory cell recruitment, which is accompanied by an onset of recovery.

Toxicity of chemical components of ambient fine particulate matter (PM 2.5) inhaled by aged rats.

The toxicity of two important chemical components of fine ambient particulate matter (PM 2.5)-ammonium bisulfate (ABS) and elemental carbon (C)-was studied using aged (senescent) rats. The study tested the hypotheses that fine particle exposure can damage lungs and impair host defenses in aged rats and that ozone would potentiate the toxicity of these particles. Ammonium bisulfate aerosols were generated by nebulization of dilute aqueous solutions. Elemental carbon was generated from an aqueous suspension of carbon black. Carbon and ABS mixtures were generated by nebulization of a suspension of carbon black in a dilute aqueous solution of ABS. Rats were exposed, nose-only, for 4 h a day, three consecutive days a week, for 4 weeks. The rats were exposed to one of six atmospheres: (1) purified air; (2) C, 50 microg m(-3), 0.3 microm mass median aerodynamic diameter (MMAD); (3) ABS, 70 microg m(-3), 0.3 microm MMAD; (4) O3, 0.2 ppm; (5) ABS + C, 0.46 microm MMAD; and (6) ABS + C + O3, 0.45 microm MMAD. Data were analyzed using ANOVA and Tukey multiple comparison tests; a two-tailed significance level of 0.05 was used. The nuclei of lung epithelial and interstitial cells were examined to determine the labeling of the DNA of dividing cells by 5-bromo-2-deoxyuridine and to identify the location of injury-repair-related cell replication. Increased labeling of both epithelial and interstitial lung cells occurred following all pollutant exposures. Although epithelial cells are most likely impacted by inhaled particles first, the adjacent interstitial cells were the cells that showed the greatest degree of response. Exposure to the ABS + C + O3 mixture resulted in losses of lung collagen and increases in macrophage respiratory burst and phagocytic activities that were statistically significant. Our results demonstrate that ozone can increase the toxicity of inhaled particles (or vice versa), and suggest that detailed study of mixtures could provide a more comprehensive understanding of the mechanisms by which inhaled pollutants adversely affect human health.

Attenuation of ozone-induced lung injury by interleukin-10.

Ozone (O3), an oxidant air pollutant, is capable of producing pulmonary inflammation and injury. Exposure to O3 results in the release of inflammatory cytokines including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) by alveolar macrophages. In addition, O3 exposure results in an increased expression of the inducible isoform of nitric oxide synthetase (iNOS). Interleukin-10 (IL-10) is an anti-inflammatory cytokine which inhibits the synthesis of TNF-alpha and IL-1 by macrophages and decreases the expression of iNOS. To test the protective properties of IL-10 in vivo, on the pulmonary injury induced by O3 exposure, we intratracheally instilled rat recombinant IL-10 1 h prior to O3 exposure (0.8 ppm x 3 h). Approximately 10-12 h following exposure, the animals were sacrificed and the bronchoalveolar lavage fluid (BALF) collected. The quantification of albumin, protein and fibronectin in the BALF provided a means of assessing pulmonary injury while the analysis of the BALF cells reflected the inflammatory response. Ozone exposure resulted in a significant (P<0.05) increase in BALF albumin, protein and fibronectin content as compared to air-exposed controls. In addition, significant increases in the percentage of BALF polymorphonuclear leukocytes (PMNs) and tissue expression of fibronectin mRNA were observed. The intratracheal instillation of IL-10 prior to O3 exposure resulted in a significant reduction in BALF albumin, protein and fibronectin content, and lung fibronectin mRNA as compared to O3 exposure alone. The data shows that IL-10, when given intratracheally, significantly reduces the pulmonary injury following O3 exposure in the rat. However, since the PMNs and the levels of albumin, protein and fibronectin in the IL-10 treated group did not reach baseline values, we conclude that other mediators of inflammation and injury not regulated by IL-10 also contribute to the pathophysiology of O3-induced lung injury.

Alteration of epithelial integrity, alkaline phosphatase activity, and fibronectin expression in lungs of rats exposed to ozone.

The deleterious effects of ozone (O3), an oxidant air pollutant, in the lung are dependent on dose and exposure duration and generally evolve with time postexposure. This study characterized the time sequence of epithelial injury and fibronectin expression in the lungs of rats exposed to O3. Bronchoalveolar lavage (BAL) fluid was analyzed for alkaline phosphatase and total protein as markers of epithelial injury and increased permeability, and fibronectin for its role in inflammation and lung injury. The results revealed a time-related increase in total protein in the BAL fluid following a 3-h exposure of rats to 1 ppm O3. The increased protein concentrations peaked at 12 h and then declined, but remained significantly higher than control at 24 h postexposure. A similar time-related significant increase also occurred for BAL fibronectin and alkaline phosphatase activity. However, the return of alkaline phosphatase levels to baseline prior to a comparable reduction in protein levels suggests repair of injured cells, but a delay in the formation of epithelial junctions that limit the transfer of serum proteins to air spaces. By cytochemistry, alkaline phosphatase activity was detected in association with lung type II epithelial cells and in BAL polymorphonuclear leukocytes (PMNs), but not in macrophages. While a significant increase in cytochemically detectable alkaline phosphatase resulted from the increase in PMN number following O3 exposure, mononuclear cells constituted the primary cell type responsible for fibronectin mRNA upregulation. While the cytochemical observations support the role of inflammatory cells in the injury process, the comparability of temporal changes in BAL protein, fibronectin, and alkaline phosphatase suggests a mechanistic role for fibronectin in lung injury.

Acute peritoneal dialysis in neonates: comparison of two types of peritoneal access.

A total of 23 sessions of peritoneal dialysis (PD) were given to 20 neonates with acute renal failure. Intravenous cannula (Biovalve 14G Vygon) was used for PD access in 13 procedures and guide wire-inserted femoral vein catheter (Medcomp-pediatric) in 10 procedures. Intraperitoneal bleed was seen in 1 procedure each in both groups. Dialysate leak and catheter blockade were more common with intravenous cannula [3 (23.1%), 8 (61.5%)] than guide wire-inserted femoral vein catheter [1 (10%), 4 (40%), P-NS]. Due to repeated catheter blockade, 5 (38.4%) PD sessions could not be completed with intravenous cannula and had to be prematurely closed; this complication was not seen with guide wire-inserted femoral vein catheter (P<0.05). Percentage reduction of serum creatinine per PD session was significantly higher in neonates being dialyzed with guide wire-inserted femoral vein catheter (51.7%+/-8.5%) than those dialyzed with intravenous cannula (38.3%+/-5.2%). Incidence of peritonitis was not significantly different [2 (15.4%) vs. 1 (10%)]. To conclude, for an effective PD in neonates, guide wire-inserted femoral vein catheter is safe and is associated with fewer access-related problems.

Ozone-induced lung inflammation and mucosal barrier disruption: toxicology, mechanisms, and implications.

The airway epithelial lining serves as an efficient barrier against penetration of exogenous particles and macromolecules. Disruption of this barrier following O3 exposure represents a state of compromised epithelial defenses leading to increased transmucosal permeability. Although the barrier disruption following an acute exposure is transient in nature, the brief period of disruption caused by O3, an oxidant air pollutant, provides an opportunity for facilitated entry of a potentially toxic particulate copollutant(s) across the airway epithelia. The subsequent deposition and retention of the copollutant(s) in the subepithelial compartment for prolonged periods adds the risk of injury due to chronic exposure following an acute episode. Toxicological studies from several laboratories have demonstrated alterations in epithelial permeability, suggestive of barrier disruption, in animals and humans exposed to O3. Inflammatory cells represent another important component of pulmonary defenses, but upon activation these cells can both induce and sustain injury. The recruitment of these cells into the lung following O3 exposure presents a risk of tissue damage through the release of toxic mediators by activated inflammatory cells. Several studies have reported concomitant changes in permeability and recruitment of the inflammatory cells in the lung following O3 exposure. In these studies, an inflammatory response, as detected by an increase in the number of polymorphonuclear leukocytes in the bronchoalveolar lavage (BAL) or in lung parenchyma, was accompanied by either an increased tracer transport across the airway mucosa or an elevation in the levels of total protein and/or albumin in the BAL. The magnitude of response and the time at which the permeability changes and inflammatory response peaked varied with O3 concentration, exposure duration, and the mode of analysis. The responsiveness to O3 also appeared to vary with the animal species, and increased under certain conditions such as physical activity and pregnancy. Some of the effects seen after an acute exposure to O3 were modified upon repeated exposures. The responses following repeated exposures included attenuation, persistence, or elevation of permeability and inflammation. Mechanistic studies implicate chemotactic factors, cellular mediators, and cell-surface-associated molecules in the induction of inflammation and lung injury. In discussing these studies, this review serves to introduce the mucosal barrier functions in the lung, evaluates inflammatory and permeability consequences of O3, addresses mechanisms of inflammatory reactions, and offers alternate viewpoints.

The influence of polymorphonuclear leukocytes on altered pulmonary epithelial permeability during ozone exposure.

Ozone (O3), a pulmonary irritant, and a major toxic component of photochemical smog, is capable of inducing pulmonary inflammation characterized by recruitment of polymorphonuclear leukocytes (PMNs) into the lung. The recruited PMNs, in turn, can release toxic mediators and produce lung injury. The mechanism of ozone-induced changes in lung permeability remains unknown. It is our hypothesis that PMNs migrating into the lung play a significant role in the pathophysiology following O3 exposure and that increasing the number of PMNs coming into the lung will exaggerate the changes in lung permeability. To test this hypothesis, we induced an influx of PMNs into the lungs of Sprague-Dawley rats by intratracheal instillation of 1% rabbit serum and then exposed the animals to either 0.8 ppm O3 or filtered air for 3 h. Control animals were intratracheally instilled with phosphate-buffered saline (PBS) and simultaneously exposed to O3 or filtered air in the same manner as the serum-treated animals. The animals were sacrificed and the lungs lavaged 10-12 h after exposure. The bronchoalveolar lavage fluid (BALF) was analyzed for albumin and protein, as indicators of permeability. In addition, BALF from the various groups was tested for its ability to alter epithelial resistance of pulmonary type II cells in culture. O3 exposure resulted in a significant increase in albumin and protein levels in the BALF as compared to air-exposed controls. The instillation of serum resulted in a significant increase in airway PMNs, but no significant elevations in albumin levels in both the O3 and air-exposed groups, as compared to PBS instillation. In vitro studies did not reveal a differential BALF effect on epithelial resistance. The data demonstrate that an excessive neutrophilia in the lung is not matched by a comparable amplification of epithelial injury. It is therefore suggested that a simple elevation in PMN number in the air spaces, as that induced by serum instillation, does not necessarily augment the lung pathophysiology, but that a more complex interaction with O3 may be required for cellular activation and release of toxic products.

Enhancement of fibronectin expression in rat lung by ozone and an inflammatory stimulus.

This study investigated the relationship of fibronectin expression and induction of pulmonary inflammation by ozone (O3). Rats were exposed to 0.8 parts/million O3 to induce lung inflammation. A second inflammatory stimulus, rabbit serum, was applied intratracheally to augment O3-induced inflammation. Bronchoalveolar lavage fluid (BALF) and lung tissues were analyzed for fibronectin protein and mRNA expression. Blood plasma was analyzed to investigate the potential of a minimally invasive procedure in predicting lung inflammation and fibronectin levels. Significant increases in the levels of fibronectin protein in the BALF and lung tissue after O3 exposure were further enhanced by pretreatment with normal serum. An increase in fibronectin mRNA following O3 exposure was also enhanced by serum pretreatment, which by itself had no effect on lung fibronectin mRNA expression. Plasma fibronectin levels were comparable in air-PBS and O3-PBS groups but increased in the O3-serum group. The results suggest leakage of fibronectin from blood plasma into the lung following intratracheal application of rabbit serum and upregulation of local synthesis following O3 exposure.

Effects of ozone on macrophage adhesion in vitro and epithelial and inflammatory responses in vivo: the role of cytokines.

Inhalation exposure to ozone (O3) is known to induce epithelial and inflammatory changes in the lungs, characterized by neutrophilia and changes in epithelial permeability. Several cell types and their soluble mediators, including interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha), are involved in the evolution of these responses. In this study, we have compared the effects of the combination of anti-IL-1 alpha and anti-TNF-alpha on in vitro and in vivo responses to inhaled O3. Male, Sprague-Dawley rats were exposed, nose-only, to 0.8 ppm O3 for 3 h and the in vitro and in vivo parameters were measured 8-12 h following exposure. The in vitro studies revealed that the adherence of inflammatory cells, primarily macrophages, harvested from the lungs of O3-exposed rats to cultured lung epithelial cells (ARL-14) was significantly greater than the adherence of macrophages from air-exposed controls. Furthermore, this adherence was significantly reduced in antibody-treated cells as compared to cells treated with preimmune rabbit serum. In vivo, elevations were found in the percentage of neutrophils in bronchoalveolar lavage fluid (BALF), transport of 99mTc-diethylenetriaminepentaacetate (DTPA) across the tracheal epithelium, and concentrations of total protein and albumin in BALF following O3 exposure. However, these effects were not significantly altered by treatment with the anti-IL-1 alpha/anti-TNF-alpha combination. Therefore, it was concluded that O3 affects the early stages of the inflammatory response, particularly with respect to macrophage activation and adherence to epithelial cells, and that this early response may be mediated by IL-1 alpha and/or TNF-alpha. The results also suggest that the in vivo effects of O3 are controlled by complex mechanisms involving factors other than IL-1 alpha and TNF-alpha, even though these cytokines are capable of modifying macrophage function as revealed by the in vitro adherence studies.

Alteration of alveolar macrophage chemotaxis, cell adhesion, and cell adhesion molecules following ozone exposure of rats.

Ozone (O3) exposure of humans and animals induces an inflammatory response in the lung, which is associated with macrophage stimulation, release of chemotactic agents, and recruitment of polymorphonuclear leukocytes (PMNs). This study was designed to investigate the functional aspects of the macrophages that impact inflammatory processes in the lung. Macrophages recovered by bronchoalveolar lavage (BAL) from rats exposed to purified air or 0.8 ppm O3 were studied for their chemotactic activity, adhesive interactions with alveolar epithelial cells in culture, surface morphology, and surface expression of cell adhesion molecules. The macrophages isolated from O3-exposed rats exhibited a greater motility in response to a chemotactic stimulus than the macrophages isolated from rats exposed to purified air. The macrophages from O3-exposed animals also displayed greater adhesion when placed in culture with epithelial cells isolated from adult rat lung (ARL-14) than the macrophages from control rats. Both chemotactic motility and cell adhesion stimulated by O3 exposure were attenuated when the macrophages were incubated in the presence of monoclonal antibodies to leukocyte adhesion molecules, CD11b, or epithelial cell adhesion molecules, ICAM-1. Flow cytometry revealed a modest increase in the surface expression of CD11b but no change in ICAM-1 expression in macrophages from O3-exposed rats when compared to those from the air-exposed controls. The results demonstrate an alteration of macrophage functions following O3 exposure and suggest the dependence of these functions on the biologic characteristics, rather than the absolute expression, of the cell adhesion molecules.

Modification of macrophage adhesion by ozone: role of cytokines and cell adhesion molecules.

The inflammatory response in the lungs following an inhalation exposure of animals and humans to ozone (O3) is associated with macrophage stimulation, release of chemotactic agents, and neutrophilia. This study investigated the adhesive behavior of the alveolar macrophages and its relevance to the inflammatory processes in the lung. Macrophages recovered by BAL from rats exposed to purified air or 0.8 ppm O3 were studied in vitro for their adhesion to epithelial cells derived from ARL-14. The macrophages from O3-exposed animals displayed greater adhesion to the epithelial cells than the macrophages from control rats exposed to purified air. The O3-induced adhesion was attenuated in the macrophages treated with a combination of interleukin-1 alpha and tumor necrosis factor-alpha antibodies (anti-IL-1+anti-TNF). The cell adhesion stimulated by O3 exposure was also attenuated when the macrophages were incubated in the presence of antibodies to leukocyte adhesion molecules, CD11b, or epithelial cell adhesion molecules, ICAM-1. A marginal increase in the surface expression of CD11b was noticed in macrophages from the rats exposed to O3. A similar change in the ICAM-1 expression was, however, not observed. The results suggest that the O3-induced modifications of macrophages are mediated by IL-1 and TNF, and that these modifications are accompanied by a minimal change in the expression of the cell-adhesion molecules.

Surfactant dysfunction after inhalation of nitric oxide.

To study whether nitric oxide (NO) affects surfactant function, 36 young rats inhaled one of the following humidified environments for 24 h: 1) air; 2) 95% O2; 3) air and 100 parts/million (ppm) NO; and 4) 95% O2 and 100 ppm NO. The treatments did not change the recovery of phospholipid from bronchoalveolar lavage (BAL). Exposure to NO of animals that breathed either air or 95% O2 increased the minimum surface tension of surfactant from BAL at low (1.5 mumol/ml), but not at high (4 mumol/ml), phosphatidylcholine concentration. After inhaled NO, the nonsedimentable protein of BAL decreased the surface activity of surfactant (1 mumol phosphatidylcholine/ml) more than the protein from the controls. NO treatment of animals that breathed either air or 95% O2 affected neither the quantity nor the molecular weight distribution of nonsedimentable protein. Hyperoxia increased the amount of the nonsedimentable protein, whereas NO increased the iron saturation of transferrin. The surfactant fraction and the nonsedimentable protein from BAL were separately exposed to 80 ppm NO in vitro. NO exposure had no effect on the surface activity of surfactant fraction. NO exposure of nonsedimentable protein from the control animals (no NO) increased the inhibition of the surface activity and changed the adsorption spectrum of the protein, suggesting conversion of hemoglobin to methemoglobin. Nonsedimentable protein from NO-exposed animals contained methemoglobin. We propose that surfactant dysfunction caused by inhaled NO is in part due to alteration of protein(s) in epithelial lining fluid that in turn inactivates surfactant.

Effects of ozone on the epithelial and inflammatory responses in the airways: role of tumor necrosis factor.

We have investigated the possibility that tumor necrosis factor alpha (TNF) plays a role in the increased airway permeability and an inflammatory response following an acute ozone (O3) exposure. Male Sprague-Dawley rats were injected, intraperitoneally, with either rabbit anti-mouse antibody to TNF (anti-TNF) or preimmune rabbit serum, 2 h before a 3-h exposure to O3 or purified air. Permeability, as determined by [99mTc] diethylenetriamine pentaacetate (DTPA) transport, total protein and albumin concentrations in the bronchoalveolar lavage (BAL), and the inflammatory cell response in the BAL were assessed 10 h after the exposure was completed. The O3-exposed group that was injected with anti-TNF showed a significant decrease in permeability to DTPA in comparison to the O3- exposed group injected with preimmune rabbit serum. There was no difference between the anti-TNF group and the purified air group. In contrast, the total protein and albumin levels in the BAL were significantly greater in both of the O3-exposed groups than in the purified air group. The concentrations of protein and albumin in the anti-TNF group did, however, show an attenuating trend when compared to the preimmune O3-exposed group. The polymorphonuclear leukocytes (PMNs) in BAL of the anti-TNF group also showed an attenuating trend when compared to the preimmune O3-exposed group, but both of these O3-exposed groups were significantly greater than the purified air group. Lung sections stained with naphthol AS-D chloroacetate esterase showed an increase in the number of stained PMNs in the anti-TNF group in comparison to the preimmune O3- and air-exposed groups. These data suggest that TNF plays a role in the increase in tracheal permeability as determined by DTPA transport, while the contributing role that TNF plays in bronchoalveolar permeability and the inflammatory response seen following an acute exposure to 0.8 ppm O3 is less evident.

Cytoskeletal regulation of Caco-2 intestinal monolayer paracellular permeability.

An abnormal increase in intestinal paracellular permeability may be an important pathogenic factor in various intestinal diseases. The intracellular factors and processes that regulate and cause alteration of intestinal paracellular permeability are not well understood. The purpose of this study was to examine some of the intracellular processes involved in cytoskeletal regulation of intestinal epithelial paracellular permeability using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin-b and colchicine were used to disrupt the cytoskeletal elements, actin microfilaments, and microtubules. Cytochalasin-b (5 micrograms/ml) and colchicine (2 x 10(-5) M) at the doses used caused marked depolymerization and disruption of actin microfilaments and microtubules, respectively. Cytochalasin-b-induced disruption of actin microfilaments resulted in perturbation of tight junctions and desmosomes and an increase in Caco-2 monolayer paracellular permeability. The cytochalasin-b-induced disruption of actin microfilaments and subsequent changes in intercellular junctional complexes and paracellular permeability were not affected by inhibitors of protein synthesis (actinomycin-D or cycloheximide) or microtubule function (colchicine), but were inhibited by metabolic energy inhibitors (2,4-dinitrophenol or sodium azide). The cytochalasin-b-induced disturbance in Caco-2 actin microfilaments and intercellular junctional complexes and increase in paracellular permeability were rapidly reversed. The paracellular pathway "re-tightening" following cytochalasin-b removal was not affected by actinomycin-D, cycloheximide, or colchicine, but was inhibited by 2,4-dinitrophenol and sodium azide. The colchicine-induced disruption of microtubules did not have significant effect on actin microfilaments, intercellular junctions, or paracellular permeability. These findings suggest that cytochalasin-b-induced increase in Caco-2 monolayer paracellular permeability was due to actin microfilament mediated perturbation of intercellular junctional complexes. The re-tightening of paracellular pathways (following removal of cytochalasin-b) resulted from energy-mediated re-assembly of pre-existing actin microfilaments and intercellular junctional complexes. This re-closure process did not require protein synthesis or microtubule-mediated shuttling process.

Alteration of ozone-induced airway permeability by oxygen metabolites and antioxidants.

This study determined the interactive effects of O3 and enzymatically-generated oxidants and antioxidants in the lung. Rats treated with dimethylthiourea (DMTU) or H2O2, generated by glucose/glucose oxidase, were exposed for 2 h to 0.6 or 0.8 ppm O3. A significant increase in the flux of total albumin in the bronchoalveolar lavage (BAL) and a concomitant elevation in the transport of 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA) from trachea to blood occurred after O3 exposure. Pretreatment of rats with DMTU prevented the albumin flux in the BAL. Intratracheal instillation of glucose/glucose oxidase produced a localized response in trachea, but it did not affect the broncho-alveolar permeability. The results demonstrate an additive effect of O3 and an enzymatically-generated oxidant, and an antagonistic effect of an antioxidant in rats exposed to O3. The observations support the suggestion that a balance of oxidant-antioxidant system may be critical in maintaining respiratory integrity following O3 exposure.