Cell surface adhesive glycoproteins are principal regulators of nearly all aspects of immune/inflammatory responses. Using monoclonal antibodies to individual adhesion molecules, the expression and contribution of specific molecules in the pathogenesis of allergen-induced airway hyperresponsiveness in monkeys has been deciphered. Results confirm the importance of cell adhesion and demonstrate that antagonism of a single adhesion molecule may provide a novel therapeutic approach.

Cell surface adhesive glycoproteins are principal regulators of nearly all aspects of immune/inflammatory responses. Using monoclonal antibodies to individual adhesion molecules, the expression and contribution of specific molecules in the pathogenesis of allergen-induced airway hyperresponsiveness in monkeys has been studied. Results confirm the importance of cell adhesion and demonstrate that antagonism of a single adhesion molecule may provide a novel therapeutic approach.

Using various animal models of toxic or antigenic-induced airway inflammation, we have demonstrated that adhesion molecules play an important role in the recruitment, retention, and site-specific activation of inflammatory cells within the airways. Furthermore, we have shown that cytokines may contribute to inflammatory responses in the airways by enhancing the expression of adhesion molecules on respiratory epithelial cells.

BACKGROUND:The action of 5-lipoxygenase on arachidonic acid generates potent inflammatory mediators that may contribute to the pathophysiology of asthma.METHODS:Using the potent and selective 5-lipoxygenase inhibitor BI-L-239, we have examined the role of 5-lipoxygenase products in three animal models of asthma.RESULTS:In vitro BI-L-239 inhibited 5-lipoxygenase product generation from human lung mast cells, alveolar macrophages, and peripheral blood leukocytes with a concentration that would provide 50% inhibition values of 28 to 340 nmol/L. A 36-fold selectivity for immunoreactive leukotriene C4 versus immunoreactive prostaglandin D2 inhibition was demonstrated in mast cells. In anesthetized cynomolgus monkeys, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced immunoreactive leukotriene C4 release (maximum, 73%; bronchoalveolar lavage [BAL], 20 minutes), late-phase bronchoconstriction (maximum, 41%; +6 to 8 hours), and neutrophil infiltration (maximum, 63%; BAL, +8 hours). In conscious sheep, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced late-phase bronchoconstriction (maximum, 66%; +6 to 8 hours) and increase in airway responsiveness (maximum, 82%; carbachol, +24 hours). The acute bronchoconstriction was shortened, and neutrophil infiltration diminished (maximum, 61%; BAL, +8 hours) in this model. Finally in conscious actively sensitized guinea pigs pretreated with pyrilamine and indomethacin, inhaled BI-L-239 attenuated acute bronchoconstriction (maximum, 80%; +5 to 15 minutes), leukocyte infiltration (58%; BAL, +3 days) and increase in airway responsiveness (100%; methacholine, +3 days) induced by three alternate-day ovalbumin inhalations.CONCLUSIONS:In conclusion, results in these three animal models indicate that 5-lipoxygenase products may be major contributors to the bronchoconstriction (especially late phase), leukocyte infiltration, and airway hyperresponsiveness that characterize asthma.

In the quest to understand the events involved with an inflammatory response, research has focused on understanding the role of single mediators such as leukotrienes, thromboxane, platelet activating factor, histamine, etc. Whereas each of these single mediators undoubtedly contributes to the complexity of disease, illustrating individual pathophysiologic or therapeutic modalities by selectively antagonizing or inhibiting the activity of each has proven difficult. In light of these findings attention has focused on understanding the types of cells and their pattern of migration into inflamed tissue. It is thought that by inhibiting either the presence or function of pro inflammatory cells in the lungs it is possible to eliminate a whole range of potentially adverse mediators and promote homeostasis.

BACKGROUND:Diarrheal disease is a leading cause of morbidity and mortality globally, especially in low- and middle-income countries. High-throughput and low-cost approaches to identify etiologic agents are needed to guide public health mitigation. Nanoliter-qPCR (nl-qPCR) is an attractive alternative to more expensive methods yet is nascent in application and without a proof-of-concept among hospitalized patients.METHODS:A census-based study was conducted among diarrheal patients admitted at two government hospitals in rural Bangladesh during a diarrheal outbreak period. DNA was extracted from stool samples and assayed by nl-qPCR for common bacterial, protozoan, and helminth enteropathogens as the primary outcome.RESULTS:A total of 961 patients were enrolled; stool samples were collected from 827 patients. Enteropathogens were detected in 69% of patient samples; More than one enteropathogen was detected in 32%. Enteropathogens most commonly detected were enteroaggregative Escherichia coli (26.0%), Shiga toxin-producing E.coli (18.3%), enterotoxigenic E. coli (15.5% heat stable toxin positive, 2.2% heat labile toxin positive), Shigella spp. (14.8%), and Vibrio cholerae (9.0%). Geospatial analysis revealed that the median number of pathogens per patient and the proportion of cases presenting with severe dehydration were greatest amongst patients residing closest to the study hospitals."CONCLUSIONS:This study demonstrates a proof-of-concept for nl-qPCR as a high-throughput low-cost method for enteropathogen detection among hospitalized patients.

Bronchoalveolar lavage (BAL) and blood lymphocytes were gated and analyzed by flow cytometry during the onset (day 0 to 10; antigen [Ag] on day 3, 5, 7) and sustaining (day 10 to 24; Ag on day 10, 12, 14, 17, 19, 21) of Ag-induced airway hyperresponsiveness as well as recovery (day 24 to 32) post-Ag in 11 adult male cynomolgus monkeys. Airway responsiveness was assessed by determining the concentration of nebulized and inhaled methacholine that induced a 100% increase in respiratory system resistance (PC100). The BAL CD4 + lymphocytes doubled by day 10 and tended to decline slightly thereafter (Table 1) . Interestingly, CD8+ lymphocytes also tended to increase by day 10, reaching significance at day 24 and remaining elevated through day 32. The IL2 receptor positive (IL2R+) lymphocytes were undetectable at day 0, rose to 12% at day 10, and significantly decreased during the recovery period. Blood CD4+ cells did not change, while CD8 + cells were decreased at day 24 (51.11±2.03 to 35.53 ±2.37; p<0.01).

We have examined the effects of a PAF receptor antagonist, WEB 2170, on several indices of acute and chronic airway inflammation and associated changes in lung function in a primate model of allergic asthma. A single oral administration WEB 2170 provided dose related inhibition of the release of leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) recovered and quantified in bronchoalveolar lavage (BAL) fluid obtained during the acute phase response to inhaled antigen. In addition, oral WEB 2170 treatment in dual responder primates blocked the acute influx of neutrophils into the airways as well as the associated late-phase airway obstruction occurring 6 h after antigen inhalation. In contrast, a multiple dosing regime with WEB 2170 (once a day for 7 consecutive days) failed to reduce the chronic airway inflammation (eosinophilic) and associated airway hyperresponsiveness to inhaled methacholine that is characteristic of dual responder monkeys. Thus, we conclude that the generation of PAF following antigen inhalation contributes to the development of lipid mediators, acute airway inflammation and associated late-phase airway obstruction in dual responder primates; however, PAF does not play a significant role in the maintenance of chronic airway inflammation and associated airway hyperresponsiveness in this primate model.

Platelet-activating factor (PAF) is a potent pro-inflammatory mediator that may play a role in the pathogenesis of airway hyper-responsiveness and asthma. In man, a single inhalation of PAF induces a small but prolonged increase in airway responsiveness in some individuals. The purpose of this study was to determine the effects of single and multiple inhalations of PAF on airway cell composition and responsiveness in monkeys. Anaesthetized and intubated adult male cynomolgus monkeys were studied. Airway cell composition was measured by bronchoalveolar lavage (BAL). Airway responsiveness was measured by determining the concentration (PC100) of inhaled methacholine that caused a 100% increase in respiratory system resistance (Rrs). Airway cell composition (BAL) and responsiveness (PC100) were determined 1 day before and 20 hr after a single inhalation of PAF (approximately 200 micrograms) or 3 days before (Day 0) and 3 days after (Day 10) 3-alternate-day (Days 3, 5 and 7) inhalations of PAF (each approximately 600 micrograms). The single inhalation of PAF (n = 8) caused an acute increase in Rrs (147 +/- 69%), an increase in BAL granulocytes, and a decrease in PC100 in four of eight animals that was moderate (greater than eight fold) in only one animal. The mean +/- s.e. change in log PC100 was -0.29 +/- 0.18. The multiple inhalations of PAF (n = 8) caused acute increases in Rrs (143 +/- 38%, 175 +/- 44% and 156 +/- 39%, respectively), an increase in BAL granulocytes, and a decrease in PC100 in four of eight animals that was moderate in two animals. The mean +/- s.e. change in log PC100 was -0.43 +/- 0.22.

The development of selective PAF receptor antagonists may provide a novel approach to the treatment of human bronchial asthma. In preclinical animal models of human asthma, PAF receptor antagonists have been found to be efficacious in blocking antigen-induced changes in lung function. However, the majority of these models involve acute inflammatory events and transient changes in lung function and, therefore, their relevance to human asthma is questionable. In a recent study with a primate model of chronic airway inflammation and hyperresponsiveness, we have shown that treatment with a PAF receptor antagonist had no effect on reducing chronic inflammation and hyperresponsiveness. Similarly, recent studies in human asthmatics with PAF receptor antagonists have failed to show efficacy in blocking allergen-induced airway responses or to have any steroid sparing effects in patients with ongoing asthma. Thus, it seems that PAF may not be a key mediator which can be blocked and thereby provide therapy for bronchial asthma.

We have examined the role of intercellular adhesion molecule-1 (ICAM-1) in chronic airway inflammation and airway hyperresponsiveness in a primate model of asthma. Airway cellular composition was assessed by bronchoalveolar lavage (BAL) and airway responsiveness was measured as the bronchoconstrictor response to inhaled methacholine. In animals with chronic airway inflammation (increased BAL eosinophils) and sustained airway hyperresponsiveness, a 7 day dosing scheme with a murine anti-human ICAM-1 monoclonal antibody (R6.5, 2 mg/kg/day; i.v.) did not reduce the existing airway inflammation or airway hyperresponsiveness. In contrast, a similar dosing scheme with dexamethasone (0.2 mg/kg/day, i.m.) was found to significantly reduce both the airway eosinophilia and hyperresponsiveness. However, one week after cessation of dexamethasone treatment, the airway inflammation and hyperresponsiveness returned to pre-treatment levels. In further experiments where animals were first treated with dexamethasone (7 days) followed by a 7 day treatment with R6.5, the reoccurrence of airway inflammation and subsequent increase in airway responsiveness was prevented. We conclude that the efficacy of ICAM-1 is primarily associated with inhibition of the influx of inflammatory cells into the airways and subsequent reduction in airway responsiveness. These data suggest that in lungs with pre-existing inflammation the modulation of ICAM-1 following treatment with glucocorticoids may be a novel and more selective long-term treatment for control of the chronic airway inflammation and hyperresponsiveness associated with bronchial asthma.

We have examined the proinflammatory cell influx as well as the levels of eosinophil and neutrophil-derived granule proteins in BAL fluid obtained from monkeys undergoing acute and late-phase (dual) or single acute bronchoconstriction following antigen inhalation. Prior to antigen inhalation, there was a significantly higher number (and percentage) of eosinophils in BAL fluid from dual responder monkeys as compared with single responders. The late-phase response (LPR) (6 to 8 h postantigen) was associated with a decrease in the number of BAL eosinophils and an increase in the levels of BAL fluid EPO that returned to baseline levels by 24 h postantigen inhalation. In contrast, the number of BAL neutrophils prior to antigen inhalation were low. Concurrent with the LPR, the number of BAL neutrophils and the concentration of EPO in BAL fluid were significantly increased above that occurring in single responders. Chronic treatment (7 days) with dexamethasone significantly reduced the number of BAL eosinophils and the BAL levels of EPO prior to antigen inhalation in dual responder (LPR) monkeys and significantly blocked the dual response and both the associated neutrophil influx into the airways and an increase in BAL fluid EPO during the LPR. We conclude that, in this primate model, eosinophil activation and a large influx of neutrophils into the airways is associated with the occurrence of the antigen-induced late-phase airway obstructive response.