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Rationale: Enteral administration of probiotics may modify the gastrointestinal environment in a manner that preferentially favors the growth of minimally virulent species. It is unknown whether probiotic modification of the upper aerodigestive flora can reduce nosocomial infections.Objectives: To determine whether oropharyngeal and gastric administration of Lactobacillus rhamnosus GG can reduce the incidence of ventilator-associated pneumonia (VAP).Methods: We performed a prospective, randomized, double-blind, placebo-controlled trial of 146 mechanically ventilated patients at high risk of developing VAP. Patients were randomly assigned to receive enteral probiotics (n = 68) or an inert inulin-based placebo (n = 70) twice a day in addition to routine care.Measurements and Main Results: Patients treated with Lactobacillus were significantly less likely to develop microbiologically confirmed VAP compared with patients treated with placebo (40.0 vs. 19.1%; P = 0.007). Although patients treated with probiotics had significantly less Clostridium difficile-associated diarrhea than patients treated with placebo (18.6 vs. 5.8%; P = 0.02), the duration of diarrhea per episode was not different between groups (13.2 ± 7.4 vs. 9.8 ± 4.9 d; P = 0.39). Patients treated with probiotics had fewer days of antibiotics prescribed for VAP (8.6 ± 10.3 vs. 5.6 ± 7.8 d; P = 0.05) and for C. difficile-associated diarrhea (2.1 ± 4.8 SD d vs. 0.5 ± 2.3 d; P = 0.02). No adverse events related to probiotic administration were identified.Conclusions: These pilot data suggest that L. rhamnosus GG is safe and efficacious in preventing VAP in a select, high-risk ICU population. Clinical trial registered with www.clinicaltrials.gov (NCT00613795). [ABSTRACT FROM AUTHOR]

BACKGROUND: The assessment of asthma control is pivotal to the evaluation of treatment response in individuals and in clinical trials. Previously, asthma control, severity, and exacerbations were defined and assessed in many different ways. PURPOSE: The Task Force was established to provide recommendations about standardization of outcomes relating to asthma control, severity, and exacerbations in clinical trials and clinical practice, for adults and children aged 6 years or older. METHODS: A narrative literature review was conducted to evaluate the measurement properties and strengths/weaknesses of outcome measures relevant to asthma control and exacerbations. The review focused on diary variables, physiologic measurements, composite scores, biomarkers, quality of life questionnaires, and indirect measures. RESULTS: The Task Force developed new definitions for asthma control, severity, and exacerbations, based on current treatment principles and clinical and research relevance. In view of current knowledge about the multiple domains of asthma and asthma control, no single outcome measure can adequately assess asthma control. Its assessment in clinical trials and in clinical practice should include components relevant to both of the goals of asthma treatment, namely achievement of best possible clinical control and reduction of future risk of adverse outcomes. Recommendations are provided for the assessment of asthma control in clinical trials and clinical practice, both at baseline and in the assessment of treatment response. CONCLUSIONS: The Task Force recommendations provide a basis for a multicomponent assessment of asthma by clinicians, researchers, and other relevant groups in the design, conduct, and evaluation of clinical trials, and in clinical practice. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are important regulators of cell functions in asthma. We recently reported that regulator of G-protein signaling (RGS) 2, a selective modulator of Gq-coupled GPCRs, is a key regulator of airway hyper-responsiveness (AHR), the pathophysiologic hallmark of asthma. Because RGS2 protein levels in airway cells were significantly lower in patients with asthma compared with patients without asthma, we further investigated the potential pathological importance of RGS2 repression in asthma. The human RGS2 gene maps to chromosome 1q31. We first screened patients with asthma for RGS2 gene promoter single-nucleotide polymorphisms (SNPs) and found significant differences in the distribution of two RGS2 SNPs (A638G, rs2746071 and C395G, rs2746072) between patients with asthma and nonasthmatic subjects. These two SNPs are always associated with each other and have the same higher prevalence in patients with asthma (65%) as compared with nonasthmatic subjects (35%). Point mutations corresponding to these SNPs decrease RGS2 promoter activity by 44%. The importance of RGS2 down-regulation was then determined in an acute IL-13 mouse model of asthma. Intranasal administration of IL-13 in mice also decreased RGS2 expression in lungs by ∼50% and caused AHR. Although naive RGS2 knockout (KO) mice exhibit spontaneous AHR, acute IL-13 exposure further increased AHR in RGS2 KO mice. Loss of RGS2 also significantly enhanced IL-13-induced mouse airway remodeling, including peribronchial smooth muscle thickening and fibrosis, without effects on goblet cell hyperplasia or airway inflammation in mice. Thus, genetic variations and increased inflammatory cytokines can lead to RGS2 repression, which exacerbates AHR and airway remodeling in asthma.

Evidence suggests that estrogen signaling is involved in sex differences in the prevalence rates and control of asthma, but the expression patterns of estrogen receptor variants and estrogen function in the lung are not well established. We investigated the expression of major estrogen receptor variants occurring naturally and after the development of allergen-induced airway hyperreactivity in a murine model of allergic asthma, along with the role of estrogen signaling in small-airway ciliary motion and smooth muscle contraction. Female BALB/c mice were sensitized with ovalbumin, and estrogen receptor expression patterns were examined by immunofluorescence and Western blot analysis. Time-lapse video and photodiode-based displacement measurement systems were used to assess the effects of estrogen signaling on airway ciliary beat frequency and smooth muscle contraction. We found that a novel variant of estrogen receptor (ER)-α, ER-α36, is expressed in airway epithelial and smooth muscle cells. ER-α36 was predominately localized on the plasma membranes of airway cells. After sensitization to allergen, the expression levels of ER-α36 increased significantly (P < 0.01), whereas the expression of ER-β and ER-α66 did not significantly change. Estrogen treatment in vitro resulted in a rapid increase in airway cilia motion in a dose-dependent fashion, but did not exert any effect on airway smooth muscle contraction. We speculate that the up-regulation of estrogen receptor expression associated with allergen-induced airway hyperresponsiveness may constitute a protective mechanism to facilitate the clearance of mucus. The identification and localization of specific estrogen receptor subtypes in the lung could lead to newer therapeutic avenues aimed at addressing sex differences of asthma susceptibility.

Asthmatic airway remodeling is characterized by goblet cell hyperplasia, angiogenesis, smooth muscle hypertrophy, and subepithelial fibrosis. This study evaluated whether acquired changes in fibroblast phenotype could contribute to this remodeling. Airway and parenchymal fibroblasts from control or chronically ovalbumin (OVA)-sensitized and challenged "asthmatic" mice were assessed for several functions related to repair and remodeling +/- exogenous transforming growth factor (TGF)-beta. All OVA-challenged mouse fibroblasts demonstrated augmented gel contraction (P < 0.05) and chemotaxis (P < 0.05); increased TGF-beta(1) (P < 0.05), fibronectin (P < 0.05), and vascular endothelial growth factor (P < 0.05) release; and expressed more alpha-smooth muscle actin (P < 0.05). TGF-beta(1) stimulated both control and asthmatic fibroblasts, which retained all differences from control fibroblasts for all features(P < 0.05, all comparisons). Parenchymal fibroblasts proliferated more rapidly (P < 0.05), while airway fibroblasts proliferated similarly compared with control fibroblasts (P = 0.25). Thus, in this animal model, OVA-challenged mouse fibroblasts acquire a distinct phenotype that differs from control fibroblasts. The augmented profibrotic activity and mediator release of asthmatic fibroblasts could contribute to airway remodeling in asthma.

Anti-immunoglobulin E (anti-IgE) (omalizumab), a humanized monoclonal anti-IgE antibody that binds to circulating IgE, has been studied in several large double-blind, randomized, placebo-controlled clinical trials to determine its pharmacokinetic characteristics, efficacy, and safety in ragweed- or birch pollen-induced seasonal allergic rhinitis (SAR). The consequences of readministering omalizulab after a lapse of time have also been studied. These studies have confirmed that serum-free IgE declines in a dose-related manner with such treatment and that omalizumab-induced declines in IgE correlate with symptom improvement. Whether omalizumab is administered intravenously or subcutaneously, its pharmacokinetics do not differ. A Phase II dose-ranging study demonstrated that the optimum efficacious dose of omalizumab for the treatment of seasonal allergic rhinitis is 300 mg administered subcutaneously. The dosing frequency, in terms of whether the antibody is administered every 3 or 4 wk, is based on the patient's baseline IgE level. With adequate dosing, nasal and ocular symptoms are significantly reduced, and quality of life is significantly improved. Omalizumab is safe and well tolerated and can be safely readministered in subsequent pollen seasons.

The mechanisms by which mediators and cytokines stimulate neutrophils to migrate across the lung epithelium are still unclear. We hypothesized that neutrophil transepithelial migration depends upon polarity of the epithelium. We therefore compared neutrophil migration through human lung Type II-like alveolar epithelial cell line (A549) monolayers grown on the upper versus lower surface of permeable filters to simulate apical-to-basal and basal-to-apical movement of neutrophils, respectively. The classic chemoattractants formyl-methionylleucylphenylalanine (FMLP), leukotriene B4 (LTB4), and interleukin-8 (IL-8) induced equivalent neutrophil transepithelial migration in the apical-to-basal and basal-to-apical directions. However, the degree of neutrophil transepithelial migration was significantly greater in the basal-to-apical direction in response to either IL-1beta or tumor necrosis factor-alpha (TNF-alpha). Enhanced TNF-alpha-induced neutrophil migration through A549 monolayers in the basal-to-apical direction occurred regardless of whether the TNF-alpha was above or below the filter/monolayer complex. Actinomycin D pretreatment of A549 monolayers had no effect on FMLP-induced neutrophil transepithelial migration, but markedly (about 75%) inhibited both TNF-alpha- and IL-1beta-induced neutrophil transepithelial migration, regardless of monolayer orientation. Thus, in contrast to classic chemoattractants, IL-1beta and TNF-alpha induced greater neutrophil transepithelial migration in a basal-to-apical direction, and this occurred independently of the cytokine location, but depended upon intact metabolic capacity of the A549 cells. These data suggest that the mechanisms important for neutrophil transepithelial migration in response to classic chemoattractants differ from those important for migration in response to inflammatory cytokines.

To better understand the mechanisms by which neutrophils migrate to the airway lumen during an inflammatory response, we constructed an in vitro model system to examine the interactions of human neutrophils, human lung epithelial cells, mediators, and proinflammatory cytokines. We directly compared neutrophil movement through three lung epithelial cell lines, A549, H441, and 16-HBE-14o, in response to three chemoattractants, FMLP, LTB4, and IL-8, and the proinflammatory cytokines IL-1 alpha and beta and TNF alpha. While there was variation in the responses to the chemotaxins, there was no correlation between the transmonolayer electrical resistance and the ability of the neutrophils to migrate across the epithelia in response to the agents used. FMLP, IL-8, and LTB4 induced dose- and time-dependent neutrophil migration across all three epithelia. However, TNF alpha- and IL-1-induced neutrophil migration occurred only through monolayers that produced soluble chemoattractants in response to these cytokines. Although all three epithelia produced low amounts of IL-8 constitutively, the capacity of IL-1 and TNF alpha to induce transepithelial migration was directly associated with the ability of the epithelia to produce large amounts of IL-8 in response to IL-1 and TNF alpha. We conclude that the phenotype of the epithelial cell (e.g., capacity to produce IL-8) affects stimulated neutrophil transepithelial migration.

Tumor necrosis factor alpha (TNF alpha) is a potentially important cytokine in allergic respiratory reactions since it is released by mast cells and eosinophils, and it can promote mediator and cytokine release, adhesion molecule expression, and granulocyte migration. Therefore, we induced an IgE-mediated response in human lung samples and studied: (1) whether TNF alpha was produced in sufficient quantities to promote granulocyte migration; and (2) which cells expressed mRNA for TNF alpha using in situ hybridization. Lung fragments (from thoracotomy) were treated for 30 min with either anti-IgE, 1:100 dilution, or buffer (control). Anti-IgE treatment of 16 lungs resulted in greater than 4-fold increase in histamine release and the significant production of chemotactic activity. The chemotactic activity generated induced dose-responsive neutrophil and eosinophil migration through naked filters and endothelial and pulmonary epithelial monolayers. Fourteen of 16 samples had a significant increase in TNF alpha subsequent to anti-IgE treatment (P < 0.05). Anti-TNF alpha antibody (4 micrograms/ml) inhibited about 25% of the neutrophil chemotactic activity in supernatants from anti-IgE treated lungs. TNF alpha at a concentration measured after anti-IgE treatment of lung samples (50 pg/ml) induced neutrophil transendothelial migration. Finally, we found that anti-IgE treatment led to an increase in TNF alpha mRNA-positive cells by in situ hybridization (1.6/ mm2 experimental versus 0.5/mm2 control), some of which were eosinophils. Thus, human lung IgE-mediated responses in vitro results in: (1) release of TNF alpha in amounts sufficient to effect a biologic response, granulocyte chemotaxis: and (2) upregulation of mRNA for TNF alpha in eosinophils and other cells. These findings suggest that TNF alpha is an important effector molecule in the pathogenesis of allergic respiratory reactions.

Interleukin-1 alpha (IL-1 alpha) is a cytokine with a myriad of potent proinflammatory effects. Neutrophils are important immune effector cells in allergic and inflammatory lung diseases. We examined the effects of IL-1 alpha on human neutrophil migration across naked filters and human umbilical vein endothelial (HUVE) cell and type II-like pulmonary epithelial cell (A549) monolayers cultured on these filters. IL-1 alpha from 10(-13) to 10(-9) M induced dose-dependent neutrophil migration through both HUVE and A549 cellular monolayers but not through naked filters. Neutrophil migration was consistently greater through A549 monolayers compared with HUVE monolayers. IL-1 alpha-induced neutrophil migration was also time dependent, and the kinetics of neutrophil migration through HUVE and A549 monolayers were similar. Significant migration through either monolayer was not observed until 2 h, and maximal migration occurred at 3 h through A549 and 5 h though HUVE cellular monolayers. Supernatants of IL-1 alpha (10(-11) M)-stimulated HUVE and A549 monolayers induced significantly more migration of neutrophils across naked filters than 10(-11) M IL-1 alpha itself, suggesting the release of soluble secondary chemotactic factor(s). Pretreatment of HUVE and A549 monolayers with actinomycin D inhibited both IL-1 alpha-induced production of soluble chemotactic factor(s) and transcellular migration by > 90%. Supernatants from IL-1 alpha-treated HUVE and A549 cells contained significant concentrations of interleukin 8 (IL-8), and coincubation of these supernatants with anti-IL-8 inhibited approximately 50% of supernatant-induced chemotaxis.(ABSTRACT TRUNCATED AT 250 WORDS)

The efficacy and safety of single oral doses of the leukotriene D4-receptor antagonist, ICI 204,219, were tested in subjects with acute seasonal allergic rhinitis. Subjects who were enrolled in the double-blind, placebo-controlled trial spent 8 h/d for two consecutive days in a park at the peak of ragweed season (counts > 1,000 grains/m3). Subjects (n = 164) who had sufficient symptoms during a 3-h baseline period on Day 1 were randomized to treatment with 10 (n = 33), 20 (n = 33), 40 (n = 33), or 100 mg (n = 32) of ICI 204,219 or placebo (n = 33). Rhinitis symptoms (nasal congestion, sneezing, rhinorrhea, itchy nose, throat and palate, and eye symptoms) were recorded hourly in the park and three times each evening at home. Blood samples were collected twice daily to determine plasma levels of ICI 204,219. Nasal congestion improved (p < 0.01) most consistently from the evening of Day 1 through Day 2 after treatment with 20- and 40-mg doses of ICI 204,219 versus placebo. Sneezing and rhinorrhea (p < or = 0.05) also improved on Day 2 for subjects who received 20- and 40-mg doses of ICI 204,219 compared with placebo. Mean symptoms scores for the entire day showed that 20 mg of ICI 204,219 was the minimally effective dose in this trial. The onset of action for all treatment groups, including placebo, was within the first 2 h of dosing. No serious adverse events were reported during the trial. ICI 204,219 was well tolerated and relieved symptoms of acute seasonal allergic rhinitis.

The 5(R) and 5(S) hydroxyeicosatetraenoic acids (5[R]-HETE, 5[S]-HETE) are important inflammatory mediators in lung diseases: they increase mucus, induce airway contraction, and potentiate neutrophil chemotaxis. Neutrophils are important cells in allergic and inflammatory lung diseases. Therefore, we examined the effects of both 5(R)-HETE and 5(S)-HETE on human neutrophil migration across naked filters and human umbilical vein endothelial (HUVE) cell and human type II-like pulmonary epithelial cell (A549) monolayers cultured on these filters. Time courses for both 5(R)-HETE and 5(S)-HETE show significant neutrophil migration at 40 min and maximal migration at 60 to 90 min through all three barriers. Checkerboard analysis showed that migration was chemotactic. Dose-response curves for both isomers through cellular monolayers had the same shapes, but 5(R)-HETE was more potent than 5(S)-HETE. There was greater migration through cellular barriers than through naked filters. Actinomycin D pretreatment of the cellular monolayers slightly inhibited the neutrophil transcellular chemotactic response to both 5-HETEs equally. Enhanced transcellular migration was not due to the production of a soluble chemotactic factor. Thus, although both isomers of 5-HETE were potent chemotactic agents, 5(R)-HETE was slightly more potent. Moreover, relevant endothelial and epithelial monolayers enhance both dose- and time-dependent neutrophil migration stimulated by 5(R)-HETE and 5(S)-HETE. These data indicate that (1) both 5(R)-HETE and 5(S)-HETE are important in mediating lung inflammatory processes, and (2) 5(R)-HETE may play a more important role in neutrophil-rich lung inflammatory responses than 5(S)-HETE because it is a more potent inducer of neutrophil migration through endothelial and epithelial barriers.

Eosinophils are important immune effector cells in a variety of allergic responses and inflammatory lung diseases. Bacterial products and inflammatory mediators have been implicated in inducing an influx of eosinophils into the respiratory tract subsequent to an acute inflammatory response. Therefore, to better understand the role of eosinophils in lung inflammation, we compared the ability of three known chemoattractants, formylmethionylleucylphenylalanine (FMLP), leukotriene B4 (LTB4), and platelet-activating factor (PAF), to induce human eosinophils to migrate across 3.0-microns-pore naked filters and human umbilical vein endothelial cells (HUVEC) and A549 human pulmonary type II-like epithelial (A549) cells cultured in monolayers on these filters. Kinetic experiments indicated that eosinophil migration through all three barriers occurred by 60 min and plateaued by 2 h. Each of these chemoattractants induced eosinophil migration in dose-responsive fashion across all three barriers. Although similar maximal eosinophil migration was observed, the doses at which this occurred varied, indicating that the rank order of potency through naked filters is FMLP > PAF > or = LTB4. However, their relative chemotactic potency through cellular barriers was different, with FMLP > LTB4 > PAF. In contrast to previous studies with neutrophils, the rank order of potency of the three chemoattractants was not influenced by the barrier through which the eosinophil migrated. Thus, these and previous data show that FMLP, LTB4, and PAF are eosinophil and neutrophil chemoattractants. Therefore, it is likely that these three agents are important mediators of granulocytic inflammatory responses in the lung, albeit with different potency profiles.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokines are proinflammatory protein mediators produced by many cells, including mast cells, T lymphocytes, eosinophils, airway epithelial cells, and macrophages. There are numerous in vitro and in vivo animal and human studies showing that cytokines are released as a result of allergic reactions. Cytokines mediate allergic inflammation by activating eosinophils, promoting mast cell development, regulating immunoglobulin isotype switching to immunoglobulin E, modulating adhesion molecule regulation, and promoting both neutrophil and eosinophil chemotaxis. Furthermore, there are data that show the pro-inflammatory effects of cytokines may be blocked by cytokine antagonists. This report reviews the in vitro and in vivo animal and human studies of allergic models of cytokine production and regulation. It also discusses the specific roles of cytokines in the allergic inflammatory response and asthma.

Interleukin-8 (IL-8) is an 8,000 D protein produced by many cells and has potent neutrophil chemoattractant and activating properties. Indeed, there is substantial data supporting a role for IL-8 in neutrophilic lung inflammatory responses. In vivo, neutrophils must adhere to and then migrate across endothelial and epithelial cell barriers in order to reach inflammatory foci. Therefore, we examined IL-8-induced neutrophil migration through naked filters and through human umbilical vein endothelial (HUVE) cells and human pulmonary type II-like epithelial cells (A549) cultured on these filters. IL-8 induced both dose- and time-dependent migration of neutrophils across all three barriers. At IL-8 concentrations greater than 10(-8) M, neutrophil migration across both endothelial and epithelial cell barriers was significantly greater than that observed across naked filters. In addition, time-course experiments indicated that neutrophil migration continued to occur for up to 3 h across both cellular barriers while neutrophil migration across naked filters plateaued by approximately 60 to 90 min. Migration of neutrophils through all barriers was completely inhibited by anti-IL-8 neutralizing antibody. The increased migration observed through both cellular barriers was not due to either changes in chemotactic gradients or the production of other soluble chemotactic factors by IL-8-stimulated HUVE and A549 cells versus naked filters. Furthermore, pretreatment of monolayers with actinomycin-D had no effect on the degree of transcellular migration. Thus, the facilitation of neutrophil migration through HUVE and A549 monolayers is not dependent upon new protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulated migration of eosinophils out of the bloodstream and into the lung is key in the development of tissue eosinophilia and inflammation in asthma. Platelet-activating factor (PAF) has been implicated as an important inflammatory mediator in asthma pathogenesis in part because of its chemotactic capacity. We therefore studied the ability of PAF to induce human peripheral blood eosinophil migration through naked filters and human umbilical vein endothelial cells (HUVECs) cultured on these filters. PAF induced eosinophil migration through both barriers in a time-dependent fashion, with maximal eosinophil migration occurring at 180 min. Significant eosinophil migration was observed at PAF concentration > or = 0.1 microM and was dose dependent up to 10.0 microM. No significant differences in eosinophil chemotactic responses were noted between naked filter and HUVEC barriers. The PAF receptor antagonist, WEB 2086, inhibited (> 85%) eosinophil transendothelial migration when co-incubated with PAF or when used as a pretreatment of either the eosinophils or HUVECs. However, WEB 2086 pretreatment of HUVECs did not inhibit PAF-induced neutrophil transendothelial migration, nor did it affect leukotriene B4-induced neutrophil or eosinophil transendothelial migration. Thus, the data indicate that the endothelial cell plays an important role in PAF-induced eosinophil inflammatory processes. Moreover, these data suggest that PAF's pathogenic role in asthma may in part be due to its ability to stimulate eosinophil migration across endothelial barriers and into the airways.

Stimulated neutrophil migration across lung endothelial and epithelial barriers is important in lung inflammatory processes. To better understand the interaction between chemoattractants, neutrophils, and endothelium and epithelium, we compared the ability of leukotriene B4 (LTB4), formylmethionylleucylphenylalanine (FMLP), and platelet-activating factor (PAF) to induce human neutrophil migration across 3-microns-pore filters alone and human umbilical vein endothelial (HUVE) cells and two different epithelial cell types, Madin-Darby canine kidney (MDCK) cells and human lung A549 cells, cultured in monolayers on these filters. LTB4, FMLP, and PAF induced neutrophil migration through naked filters, endothelial cells, and epithelial cells in a dose-related fashion. At optimal chemoattractant doses, LTB4, FMLP, and PAF induced relatively equivalent neutrophil migration through filters and endothelial and epithelial monolayers. However, the doses at which optimal neutrophil migration was observed to occur as well as the degree of neutrophil migration through the three barriers varied depending upon the chemoattractant. Based on dose-response experiments, the relative rank order of potency for the three chemoattractants was: LTB4 = FMLP greater than PAF for filter alone barrier; LTB4 greater than FMLP greater than PAF for HUVE cell barrier; and FMLP greater than LTB4 greater than PAF for MDCK and A549 epithelial cell barriers. Our data suggest that neutrophil chemotactic and subsequent lung inflammatory responses are interrelatedly influenced by both the quantity and type of chemoattractant present and the barrier through which the neutrophil must migrate.