Effect of inhaled inactivated Mycobacterium phlei in children with moderate asthma.

Bacillus Calmette-Guérin and other mycobacterial vaccines are important therapeutic methods in a series of chronic inflammatory disorders characterized by Th1/Th2 imbalance in which Th2 type cells and cytokines often increase. However, few studies have investigated whether it can reduce or prevent the symptoms and attacks in children with asthma. This study evaluated the effect of inactivated Mycobacterium phlei inhaled by an atomizing device placed on asthmatic children. In this randomized, single-center, Seretide-controlled study, children aged 4-12 years with newly diagnosed, moderate, persistent asthma were treated with either inhaled inactivated M. phlei or inhaled Seretide patch. The efficacy of inhaled inactivated M. phlei was related with the alleviation of asthma symptoms, improvement of lung function and reduction of bronchial hyper-responsiveness and total serum IgE, which was similar with Seretide. These findings may have important clinical value in confirming inhaled inactivated M. phlei as a new therapeutic method in moderately asthmatic children.

A comparison of olopatadine 0.2% ophthalmic solution versus fluticasone furoate nasal spray for the treatment of allergic conjunctivitis.

Allergic conjunctivitis is an inflammatory condition of the ocular surface characterized by ocular itching, redness, tearing, chemosis, and eyelid swelling. The purpose of this study was to assess the comparative efficacy of an ophthalmic antihistamine/mast cell stabilizer solution and an intranasal steroid at reducing the signs and symptoms of allergic conjunctivitis induced by the conjunctival allergen challenge (CAC) model. Sixty subjects were enrolled in a single center, randomized, placebo-controlled, parallel-treatment, four-visit CAC study. After titration and confirmation of the allergic reaction at visits 1 and 2, subjects were randomized at visit 3 into one of 4 treatment groups (olopatadine 0.2% ophthalmic solution, fluticasone furoate nasal spray, a tear substitute, or saline nasal spray), dosed with study medication, and challenged 15 minutes later, after which ocular allergic signs and symptoms were assessed. Subjects continued treatment of the assigned medication for 6 days. At visit 4, subjects underwent similar procedures to those performed at visit 3. Fifty-nine subjects completed the study. Olopatadine 0.2% ophthalmic solution showed statistical and clinical superiority over fluticasone furoate nasal spray at all post-CAC time points after a single dose (p < 0.001) and after a 1-week loading period (p < 0.01) for ocular itching, the primary end point. Similarly, olopatadine 0.2% showed statistical and clinical superiority over fluticasone furoate for the majority of time points for ocular redness, tearing, chemosis, and eyelid swelling. Olopatadine 0.2% ophthalmic solution was statistically and clinically superior to fluticasone furoate nasal spray for the relief of signs and symptoms of allergic conjunctivitis.

Emerging treatment of atopic dermatitis.

Atopic dermatitis is a chronically relapsing eczematous disease of the skin. A wide range of therapeutic regimens has been used for atopic dermatitis. A better understanding of its pathogenesis will also lead to the development of novel approaches to treating this disease. This article reviews the recent advances in allergen-specific sublingual immunotherapy and therapy with antileukotriene drugs, probiotics, mycophenolate mofetil, leflunomide, and intermittent fluticasone propionate ointment, which the authors expect will be clinically useful therapies in the near future.

Pregnancy outcomes in women using inhaled fluticasone during pregnancy: a case series.

BACKGROUND: The aim of the study was to report the maternal and fetal outcomes of women with respiratory illnesses who were treated with inhaled fluticasone during pregnancy. MATERIAL AND METHODS: We identified 12 cases treated with inhaled fluticasone during pregnancy out of women who received obstetric and teratogen-risk evaluation at the Korean Motherisk Program. A detailed medical and obstetric history was obtained and cases were followed-up until either spontaneous or voluntary pregnancy termination or delivery occurred. RESULTS: None of the participants had any obstetric complication. However, in addition to fluticasone, most of the 12 cases were simultaneously exposed to a variety of medications. There were 3 abortions (one spontaneous and 2 requested by the patients arguing personal reasons). Live born babies without any evidence of major congenital malformations included 8 singleton babies and 2 twins. Of them, 3 babies were born prematurely. CONCLUSIONS: Our results are in agreement with previous large studies where no increased rate of adverse outcomes was reported with the use of inhaled corticosteroids during pregnancy.

Lipoteichoic acid induces nuclear factor-kappaB activation and nitric oxide synthase expression via phosphatidylinositol 3-kinase, Akt, and p38 MAPK in RAW 264.7 macrophages.

We previously demonstrated that lipoteichoic acid (LTA) might activate phosphatidylcholine-phospholipase C (PC-PLC) and phosphatidylinositol-phospholipase C (PI-PLC) to induce protein kinase C activation, which in turn initiates nuclear factor-kappaB (NF-kappaB) activation and finally induces inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release in RAW 264.7 macrophages. In this study, we further investigated the roles of tyrosine kinase, phosphatidylinositiol 3-kinase (PI3K)/Akt, and p38 mitogen-activated protein kinase (MAPK) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Tyrosine kinase inhibitors (genistein and tyrphostin AG126), PI3K inhibitors (wortmannin and LY 294002), and a p38 MAPK inhibitor (SB 203580) attenuated LTA-induced iNOS expression and NO release in concentration-dependent manners. Treatment of RAW 264.7 macrophages with LTA caused time-dependent activations of Akt and p38 MAPK. The LTA-induced Akt activation was inhibited by wortmannin, LY 294002, genistein, and tyrphostin AG126. The LTA-induced p38 MAPK activation was inhibited by genistein, tyrphostin AG126, wortmannin, LY 294002, and SB 203580. The LTA-induced formation of an NF-kappaB-specific DNA-protein complex in the nucleus was inhibited by wortmannin, LY 294002, genistein, tyrphostin AG126, and SB 203580. Treatment of macrophages with LTA caused an increase in kappaB-luciferase activity, and this effect was inhibited by tyrphostin AG126, wortmannin, LY 294002, the Akt dominant negative mutant (AktDN), and SB 203580. Based on those findings, we suggest that LTA might activate the PI3K/Akt pathway through tyrosine kinase to induce p38 MAPK activation, which in turn initiates NF-kappaB activation, and ultimately induces iNOS expression and NO release in RAW 264.7 macrophages.

Subset-dependent modulation of dendritic cell activity by circovirus type 2.

Viral interactions with dendritic cells (DCs) have important consequences for immune defence function. Certain single-stranded DNA viruses that associate with a number of species, including humans and pigs, exhibit interesting characteristics in this context. Porcine circovirus type 2 (PCV2) can persist within myeloid DCs in the absence of virus replication. Internalization was observed with both conventional blood DCs and plasmacytoid DCs [natural interferon-producing cells (NIPCs)], as well as DC precursors. This PCV2-DC interaction neither induced nor inhibited DC differentiation. The maturation of myeloid DCs induced by a cocktail of interferon-alpha/tumour necrosis factor-alpha (IFN-alpha/TNF-alpha), and the ability to process and present antigen to T lymphocytes, remained intact in the presence of PCV2. The virus was clearly internalized by the DCs, a process noted with both mature and immature cells. This suggested a non-macropinocytic uptake, confirmed by an insensitivity to wortmannin but sensitivity to cytochalasin D, chlorpromazine and bafilomycin. Nevertheless, PCV2 was immunomodulatory, being effected through the reaction of NIPC to danger signals. When NIPCs responded to the CpG-oligonucleotide (CpG-ODN), their costimulatory function which induces myeloid DC maturation was clearly impaired by the presence of PCV2. This was caused by a PCV2-induced inhibition of the IFN-alpha and TNF-alpha normally produced following interaction with CpG-ODN. Thus, the immunomodulatory activity of PCV2 is mediated through the disruption of NIPC function. This would impair the maturation of associated myeloid DC and have major implications for the efficient recognition of viral and bacterial danger signals, favouring the establishment of infections additional to that of PCV2.

Identification of CD13, CD107a, and CD164 as novel basophil-activation markers and dissection of two response patterns in time kinetics of IgE-dependent upregulation.

Using two-colour flow cytometry >200 antibodies submitted to the 8th International Workshop of Human Leukocyte Differentiation Antigens (HLDA8) have been analyzed for their reactivity with resting and activated CD203c+ basophils. Four antibodies either non-reactive or weakly reactive with resting basophils exhibited an increased reactivity with basophils activated by anti-IgE-mediated cross-linking of the high affinity IgE receptor (FceRI). These include antibodies against CD164 (WS-80160, clone N6B6 and WS-80162, clone 67D2), as well as two reagents with previously unknown specificities that were identified as CD13 (WS-80274, clone A8) and CD107a (WS-80280, clone E63-880). The activation patterns followed either the "CD203c-like" or "CD63-like" activation profile. The CD203c profile is characterized by a rapid and significant upregulation (of CD13, CD164, and CD203c), reaching maximum levels after 5-15 min of stimulation. The Phosphoinositide-3-kinase (PI3K)-specific inhibitor Wortmannin inhibited the upregulation of these markers whereas 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced a rapid and FceRI-independent upregulation within 1-2 min. In the CD63 profile, maximum upregulation (of CD63 and CD107a) was detected only after 20-40 min, and upregulation by TPA reached maximum levels after 60 min. In summary, our data identify CD13, CD107a, and CD164 as novel basophil-activation antigens. Based on time kinetics of upregulation, we hypothesize that molecules of the "CD203c group" and the "CD63 group" are linked to two different mechanisms of basophil activation.

Pneumolysin potentiates oxidative inactivation of alpha-1-proteinase inhibitor by activated human neutrophils.

This study was designed to investigate the effects of the Streptococcus pneumoniae-derived, pro-inflammatory toxin, pneumolysin (8.37 and 41.75 ng/ml), on the oxidative inactivation of alpha-1-protease inhibitor (API) by chemoattractant-activated human neutrophils in vitro. The elastase inhibitory capacity (EIC) of API in supernatants from unstimulated neutrophils, neutrophils treated with pneumolysin only, or with the chemoattractant FMLP (1 microM) only, or the combination of the toxin with FMLP was measured by a colorimetric procedure based on the activity of added porcine elastase. The EIC of API was unaffected by exposure to pneumolysin only, unstimulated neutrophils, or neutrophils treated with pneumolysin only. However, exposure to FMLP-activated neutrophils resulted in a reduction of the EIC of API, which was significantly (P<0.05) augmented by pneumolysin (mean reductions of 16%, 43% and 83% for FMLP only and in combination with 8.37 and 41.75 ng/ml pneumolysin, respectively), and was attenuated by wortmannin (1 microM), an inhibitor of NADPH oxidase, the oxidant-scavenger methionine (100 microM), and depletion of Ca2+ from the cell-suspending medium. These pro-proteolytic interactions of pneumolysin with chemoattractant-activated neutrophils may contribute to the invasiveness of the pneumococcus.

Effects of fluticasone on systemic markers of inflammation in chronic obstructive pulmonary disease.

Systemic inflammation is present in chronic obstructive pulmonary disease (COPD), which has been linked to cardiovascular morbidity and mortality. We determined the effects of oral and inhaled corticosteroids on serum markers of inflammation in patients with stable COPD. We recruited 41 patients with mild to moderate COPD. After 4 weeks during which inhaled corticosteroids were discontinued, patients were assigned to fluticasone (500 mcg twice a day), oral prednisone (30 mg/day), or placebo over 2 weeks, followed by 8 weeks of fluticasone at 500 mcg twice a day and another 8 weeks at 1,000 mcg twice a day. Withdrawal of inhaled corticosteroids increased baseline C-reactive protein (CRP) levels by 71% (95% confidence interval [CI], 16-152%). Two weeks with inhaled fluticasone reduced CRP levels by 50% (95% CI, 9-73%); prednisone reduced it by 63% (95% CI, 29-81%). No significant changes were observed with the placebo. An additional 8 weeks of fluticasone were associated with CRP levels that were lower than those at baseline (a 29% reduction; 95% CI, 7-46%). Inhaled and oral corticosteroids are effective in reducing serum CRP levels in patients with COPD and suggest their potential use for improving cardiovascular outcomes in COPD.

DEC-205lo Langerinlo neonatal Langerhans' cells preferentially utilize a wortmannin-sensitive, fluid-phase pathway to internalize exogenous antigen.

Antigen treatment of neonatal epidermis results in antigen-specific immune suppression. Compared with adult counterparts, neonatal Langerhans' cells (LC) demonstrate an impaired ability to transport antigen to the lymph node (LN). As it is possible that neonatal LC have a reduced ability to endocytose antigen, we evaluated the acquisition of endocytic function, the expression of uptake receptors and the internalization of soluble and small particulate antigens in neonatal, juvenile and adult mice. Although LC from 4-day-old mice were weakly positive for the mannose-type receptor, Langerin, they were capable of internalizing fluorescein isothiocyanate (FITC)-dextran, but to a lesser extent than LC from 6-week-old mice. However, when ratio data were calculated to account for variations in fluorescence intensity at 4 degrees, it was demonstrated that neonatal LC continued to internalize antigen over a longer period of time than adult mice and, as the ratios were much higher, that neonatal cells were also relatively more efficient in antigen uptake. When receptors for mannan and mannose were competitively blocked, LC from neonatal mice, but not adult mice, could still efficiently internalize FITC-dextran. Consequently, the uptake of FITC-dextran, in part, occurred via alternative receptors or a receptor-independent fluid-phase pathway. A feasible pathway is macropinocytosis, as LC from 4-day-old mice demonstrated a reduction in FITC-dextran internalization by the macropinocytosis inhibitor, wortmannin. Evidence of a functional macropinocytosis pathway in neonatal LC was further supported by internalization of the soluble tracer Lucifer Yellow (LY). We conclude that neonatal LC preferentially utilize a wortmannin-sensitive, fluid-phase pathway, rather than receptor-mediated endocytosis, to internalize antigen. As neonatal LC are capable of sampling their environment without inducing immunity, this may serve to avoid inappropriate immune responses during the neonatal period.

Effects of montelukast on surrogate inflammatory markers in corticosteroid-treated patients with asthma.

We evaluated whether montelukast conferred additive effects in patients with asthma receiving fluticasone/salmeterol (FP/SM) combination and FP alone. Twenty-two patients with mild to moderate asthma completed a double-blind, placebo-controlled study. After a 2-week run-in using FP 250 microg/SM 50 microg 1 puff twice daily, patients entered a randomized crossover period to receive additional montelukast 10 mg daily or placebo for 3 weeks each. For the first 2 weeks, they received FP/SM 1 puff BID, and then they received FP 250 microg 1 puff BID for the 3rd week. The primary outcome was adenosine monophosphate challenge threshold and recovery time; secondary outcomes included surrogate inflammatory markers and lung function. Compared with FP/SM run-in, adding montelukast to FP/SM was better (p < 0.05) than placebo for inflammatory markers but not for lung function. For adenosine monophosphate threshold, recovery, exhaled nitric oxide, and blood eosinophils, there were 1.4 (95% confidence interval, 1.1-1.8) geometric mean fold, 10 minutes (3-17 minutes), 2.1 parts per billion (0.2-3.9 parts per billion), and 88 (34-172) x 10(6)/L differences, respectively. The combination of FP plus montelukast was superior to FP/SM for inflammatory markers but was inferior for lung function. Thus, in patients taking FP/SM or FP, montelukast conferred complimentary effects on surrogate inflammatory markers, which were dissociated from lung function. Further studies are required to evaluate whether these effects of montelukast translate into clinical benefits.

Cytokine induction in pulmonary airways of horses with heaves and effect of therapy with inhaled fluticasone propionate.

Work in humans and laboratory animals has identified a central role for cytokines and chemokines in development and persistence of lower airway inflammation. The objectives of this study were to determine interleukin (IL)-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha induction in bronchoalveolar lavage (BAL) of control horses and horses with heaves both during remission and exacerbation of the disease, and to determine the effect of therapy with inhaled fluticasone propionate on the cytokine profile of horses with heaves. IL-1 beta and TNF-alpha mRNA expression was significantly higher in horses with heaves after exposure to moldy hay compared to either values obtained during clinical remission or to healthy controls. IL-8 mRNA expression and protein concentrations were significantly higher in horses with heaves than in controls. Both IL-4 and IFN-gamma mRNA expression was increased at various times in heaves-susceptible horses compared to controls. IL-2, IL-5 and IL-10 mRNA expression was not detected in BAL cells of either group. Therapy with inhaled fluticasone propionate after induction of a severe heaves exacerbation resulted in complete resolution of clinical signs, normalization of pulmonary function tests, and significant decrease in BAL neutrophilia. This was associated with a significant decrease in IL-4 mRNA expression and increase in IFN-gamma/IL-4 ratio in horses with heaves. These results demonstrate the clinical efficacy of inhaled fluticasone propionate for the treatment of heaves and suggest a role for cytokines in the development of lower airway inflammation in heaves-susceptible horses.

Effect of an inhaled glucocorticoid on reactive oxygen species production by bronchoalveolar lavage cells from smoking COPD patients.

Oxidative stress in the lung is important in the pathogenesis of COPD. Published data indicate that glucocorticoids inhibit blood cells in their capacity to produce reactive oxygen species (ROS). We investigated the effect of Fluticasone propionate (FP) on the ROS production capabilities of pulmonary cells. Bronchoalveolar lavage (BAL) was performed in smoking COPD patients, before and after a six month, placebo-controlled treatment with FP. BAL cells were stimulated with phorbol myristrate acetate (PMA) alone, and together with superoxide dismutase (SOD). From kinetic plots of ferricytochrome-c conversion we calculated the maximal rate of superoxide production: V(max). We also examined BAL cell subsets and performed correlation analyses on ROS production and relevant clinical determinants. Paired results were obtained from 6 FP- and 9 placebo-treated patients. No significant change of V(max) was found in both patient groups. Also BAL cellularity was unchanged. Correlation analyses showed a significant (inverse) association of V(max) with the number of cigarettes smoked per day. We concluded that a potent inhaled glucocorticoid had no effect on the ROS production capability of BAL cells from smoking COPD patients. Apparently, heavy smoking impaired the ability of alveolar macrophages to produce ROS, which was not further decreased by FP.

A sensitive radioimmunoassay, incorporating solid-phase extraction, for fluticasone 17-propionate in plasma.

Antibodies were produced in rabbits immunized with fluticasone 17-propionate (FP) conjugated to bovine thyroglobulin via its 3-carboxymethoxime with isobutylchloroformate. The antibodies were used to develop a sensitive and specific radioimmunoassay (RIA) for FP in human plasma. The limit of quantitation of the RIA is 50 pg per assay tube. This translates to 50 pg ml-1 plasma when a solid-phase extraction preceded the RIA. The interassay and intraassay relative standard deviations were < 15% in the centre of the assay concentration range rising to < 25% at the lower and upper limits (50 and 250 pg ml-1). No appreciable binding was seen between the antibodies and the metabolite of FP that has been identified in man. The RIA was used to study the time course of plasma concentrations in man following inhalation of FP.

Decreased immunological responses by wortmannin-containing rice culture of Fusarium oxysporum and by purified wortmannin in avian species.

Immunological assays were performed in young chicken and duck after they had been fed wortmannin-containing culture of Fusarium oxysporum or purified wortmannin for 2 weeks. The culture significantly decreased humoral response to sheep red blood cell, cell-mediated cutaneous hypersensitivity to phytohemagglutinin and phagocytic activity in isolated peritoneal exudate adherent cells, but only when the concentration was high enough to cause concurrent reduction in body weight gain and hematocrit. Increased dietary metabolizable energy and protein did not affect the toxicity of the culture. On the other hand, purified wortmannin (1 mg/kg diet) significantly inhibited the aforementioned immunological responses prior to the adverse effects on body growth and hematocrit. The data strongly indicate that wortmannin is an immunotoxic substance. The possibility that macrophage is the primary target cell type is discussed.