Adolph J. Januszkiewicz

Pro-inflammatory alterations and status of blood plasma iron in a model of blast-induced lung trauma.

Impact of blast shock waves (SW) with the body wall produces blast lung injuries characterized by bilateral traumatic hemorrhages. Such injuries often have no external signs, are difficult to diagnose, and therefore, are frequently underestimated. Predictive assessment of acute respiratory distress syndrome outcome in SW-related accidents should be based on experimental data from appropriate animal models. Blood plasma transferrin is a major carrier of blood iron essential for proliferative “emergency” response of hematopoietic and immune systems as well as injured tissue in major trauma. Iron-transferrin complexes ([Fe3+]TRF) can be quantitatively analyzed in blood and tissue samples with low-temperature EPR techniques. We hypothesized that use of EPR techniques in combination with assays for pro-inflammatory cytokines and granulocytes in the peripheral blood and BAL would reveal a pattern of systemic sequestration of [Fe3+]TRF that could be useful for development of biomarkers of the systemic inflammatory response to lung injury. With this goal we (i) analyzed time-dependent dynamics of [Fe3+]TRF in the peripheral blood of rats after impacts of SW generated in a laboratory shock-tube and (if) assayed the fluctuation of granulocyte (PMN) counts and expression of CD11b adhesion molecules on the surface of PMNs during the first 24 h after SW-induced injury. Sham-treated animals were used as control. Exposure to sw led to a significant decrease in the amount of blood [Fe3+]TRF that correlated with the extent of lung injury and developed gradually during the first 24 h. Thus, sequestration of [Fe3+]TRF occurred as early as 3 h post-exposure. At that time, the steady state concentration of [Fe3+]TRF in blood samples decreased from 19.7±0.6 μM in controls to 7.5±1.3 μM in exposed animals. The levels of [Fe3+]TRF remained decreased thoughout the entire study period. PMN counts increased 5-fold and 3.5-fold over controls respectively, at 3 and 6 h postexposure. These effects were accompanied by an increase in expression of CD11b on the surface membrane of PMNs. Extensive release of cytokines IL-1, IL-6, MCP-1, and MIP-2 was observed in BAL fluid and blood plasma during 24 h postexposure. We conclude that EPR monitoring of blood [Fe3+]TRF can be a useful approach for assessment of systemic pro-inflammatory alterations due to SW-induced lung injury.

Effects of Nitrogen Dioxide on the Expression of Intercellular Adhesion Molecule-1, Neutrophil Adhesion, and Cytotoxicity: Studies in Human Bronchial Epithelial Cells

Nitrogen Dioxide (NO2) is a product of high-temperature combustion and an environmental oxidant of concern. We have recently reported that early changes in NO2-exposed human bronchial epithelial cells are causally linked to increased generation of proinflammatory mediators, such as nitric oxide/nitrite and cytokines like interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-8. The objective of the present in vitro study was to further delineate the cellular mechanisms of NO2-mediated toxicity, and to define the nature of cell death that ensues upon exposure of normal human bronchial epithelial (NHBE) cells to a brief high dose of NO2. Our results demonstrate that the NHBE cells undergo apoptotic cell death during the early post-NO2 period, but this is independent of any significant increase in caspase-3 activity. However, necrotic cell death was more prevalent at later time intervals. Interestingly, an increased expression of HO-1, a redox-sensitive stress protein, was observed in NO2-exposed NHBE cells at 24 h. Since neutrophils (PMNs) play an active role in acute lung inflammation and resultant oxidative injury, we also investigated changes in human PMN–NHBE cell interactions. As compared to normal cells, increased adhesion of PMNs to NO2-exposed cells was observed, which resulted in an increased NHBE cell death. The latter was also increased in the presence of IL-8 and TNF-α + interferon (IFN)-γ, which correlated with upregulation of intercellular adhesion molecule-1 (ICAM-1). Our results confirmed an involvement of nitric oxide (NO) in NO2-induced cytotoxicity. By using NO synthase inhibitors such as L-NAME and 3-aminoguanidine (AG), a significant decrease in cell death, PMN adhesion, and ICAM-1 expression was observed. These findings indicate a role for the L-arginine/NO synthase pathway in the observed NO2-mediated toxicity in NHBE cells. Therapeutic strategies aimed at controlling excess generation of NO and/or inflammatory cytokines may be useful in alleviating NO2-mediated adverse effects on the bronchial epithelium.

Nitrogen dioxide-induced acute lung injury in sheep

Lung mechanics, hemodynamics and blood chemistries were assessed in sheep (Ovis aries) before, and up to 24 h following, a 15-20 min exposure to either air (control) or approximately 500 ppm nitrogen dioxide (NO2). Histopathologic examinations of lung tissues were performed 24 h after exposure. Nose-only and lung-only routes of exposure were compared for effects on NO2 pathogenesis. Bronchoalveolar lavage fluids from air- and NO2-exposed sheep were analyzed for biochemical and cellular signs of NO2 insult. The influence of breathing pattern on NO2 dose was also assessed. Five hundred ppm NO2 exposure of intubated sheep (lung-only exposure) was marked by a statistically significant, albeit small, blood methemoglobin increase. The exposure induced an immediate tidal volume decrease, and an increase in both breathing rate and inspired minute ventilation. Pulmonary function, indexed by lung resistance and dynamic lung compliance, progressively deteriorated after exposure. Maximal lung resistance and dynamic lung compliance changes occurred at 24 h post exposure, concomitant with arterial hypoxemia. Bronchoalveolar lavage fluid epithelial cell number and total protein were significantly increased while macrophage number was significantly decreased within the 24 h post-exposure period. Histopathologic examination of lung tissue 24 h after NO2 revealed patchy edema, mild hemorrhage and polymorphonuclear and mononuclear leukocyte infiltration. The NO2 toxicologic profile was significantly attenuated when sheep were exposed to the gas through a face mask (nose-only exposure). Respiratory pattern was not significantly altered, lung mechanics changes were minimal, hypoxemia did not occur, and pathologic evidence of exudation was not apparent in nose-only, NO2-exposed sheep. The qualitative responses of this large animal species to high-level NO2 supports the concept of size dependent species sensitivity to NO2. In addition, when inspired minute ventilation was used as a dose-determinant, a linear relationship between NO2 dose and lung resistance was found. The importance of these findings, NO2 dose-determinants, and the utility of sheep as a large animal inhalation model are discussed.

Consequences of Brief Exposure to High Concentrations of Carbon Monoxide in Conscious Rats

Exposure to high-concentration carbon monoxide (CO) is of concern in military operations. Experimentally, the physiologic manifestations of a brief exposure to elevated levels of CO have not been fully described. This study investigated the development of acute CO poisoning in conscious male Sprague-Dawley rats (220–380 g). Animals were randomly grouped (n = 6) and exposed to either air or 1 of 6 CO concentrations (1000, 3000, 6000, 10,000, 12,000, or 24,000 ppm) in a continuous air/CO dynamic exposure chamber for 5 min. Respiration was recorded prior to and during exposures. Mixed blood carboxyhemoglobin (COHb) and pH were measured before and immediately after exposure. Before exposure the mean baselines of respiratory minute volumes (RMVs) were 312.6 ± 43.9, 275.2 ± 40.8, and 302.3 ± 39.1 ml/min for the 10,000, 12,000 and 24,000 ppm groups, respectively. In the last minute of exposure RMVs were 118.9 ± 23.7, 62.1 ± 10.4, and 22.0 ± 15.1% (p < .05) of their mean baselines in these 3 groups, respectively. Immediately after exposure, blood COHb saturations were elevated to 60.16, 63.42, and 69.37%, and blood pH levels were reduced to 7.43 ± 0.09, 7.25 ± 0.05, and 7.13 ± 0.04 in the 3 groups, respectively. Mortality during exposure was 1/12 in the 12,000 ppm group and 4/12 in the 24,000 ppm group. Deaths occurred close to the end of 5 min exposure. In each animal that died by exposure, pH was <6.87 and COHb saturation was >82%. Blood pH was unaltered and no death occurred in rats exposed to CO at concentrations <6000 ppm, although COHb saturations were elevated to 14.52, 29.94, and 57.24% in the 1000, 3000, and 6000 ppm groups, respectively. These results suggest that brief exposure to CO at concentrations <10,000 ppm may produce some significant physiological changes. However, exposure to CO at concentrations >10,000 ppm for brief periods as short as 5 min may change RMV, resulting in acute respiratory failure, acidemia, and even death.

Identification of mediator specific Cardiovascular waveforms using a back propagation neural networks

Abstract A diverse milieu of inhaled substances induce the release of endogenous mediators. These mediators are responsible for the symptoms manifested by a wide spectrum of allergic and toxic scenarios. Platelet activating factor (PAF) and prostaglandin H 2 analog (PGH) are two such mediators. A sheep was exposed to varied doses of PAF and PGH. Five parameters were monitored to characterize the cardiovascular response. A back propagation network was used to analyze the response data. The network was trained using three different groups of 21 of the 28 total data sets. In each case, seven data sets were withheld from training and used to assess the performance of the networks. In each case, the back propagation network (BPN) qualitatively and quantitatively (error

The use of back propagation neural networks to identify mediator-specific cardiovascular waveforms

Exposure to xenobiotics characteristically initiates the release of cascade of endogenous mediators which trigger diverse cardiopulmonary responses. Platelet activating factor (PAF) and prostaglandin H2 analog (PGH) are two mediators which have demonstrated distinct effects on the cardiovascular and pulmonary systems. The biological responses to these mediators have been implicated in the etiology of various disease states. A heteroassociative back propagation neural network (BPN) was used to identify injected mediators by analyses of complex cardiopulmonary waveforms. In all cases, the BPN qualitatively and quantitatively identified the mediator substance which induced the response. The results demonstrate the potential for deciphering complex waveforms into mediator-specific events. The BPN can isolate complex physiological patterns into component parts. Therefore, BPN has the potential to be a powerful investigative tool for deciphering etiologic mechanisms of xenobiotic insults

Morbidity and mortality resulting from acute inhalation exposures to hydrogen fluoride and carbonyl fluoride in rats

Abstract Objective: Experiments were undertaken to compare morbidity and mortality from brief inhalation exposures to high levels of hydrogen fluoride (HF) and carbonyl fluoride (COF2). Methods: Rats from both sexes were exposed for durations of 5 and 10 min to nominal concentrations of 10,000 to 57,000 ppm HF or 500 to 10,000 ppm COF2. Respiration was monitored before, during, and after exposure. Animals were observed up to 6 days post-exposure. Terminal blood samples were collected for routine clinical chemistry and hematology. Post-mortem lung fluoride concentrations and lung weights were measured, and gross pathology noted. Results: Both gases produced respiratory depression independent of concentration or exposure duration with minute ventilation decreasing to approximately 50% of baseline. Estimated mixed-gender HF and COF2 10-min LC50’s were 48,661 ppm and 1083 ppm, respectively. HF mortalities were generally delayed 3 to 4 days post-exposure, while COF2 mortalities occurred during or briefly after exposure. Lung fluoride levels increased with COF2 dose, though elevated lung weights occurred only at the mid-level exposures. Lung weights were unaffected in the HF-exposed animals, and their lung fluoride concentrations were variable. Clinical chemistry and hematology had few consistent trends with the exception of hemoconcentration primarily in HF-exposed males. These short-term exposure experiments conclude that COF2 is nearly 45 times more lethal than HF in rats. Conclusions: These experiments suggest that hydrolysis to HF cannot solely explain COF2 toxicity. Although HF and COF2 may have common injury mechanisms, they are expressed to markedly different degrees and temporal occurrence.