Toll-Like Receptor 7/8 Ligand, S28463, Suppresses Ascaris suum-induced Allergic Asthma in Nonhuman Primates.

S28463 (S28), a ligand for Toll-like receptor 7/8, has been shown to have antiinflammatory properties in rodent models of allergic asthma. The principle goal of this study was to assess whether these antiinflammatory effects can also be observed in a nonhuman primate (NHP) model of allergic asthma. NHPs were sensitized then challenged with natural allergen, Ascaris suum extract. The animals were treated with S28 orally before each allergen challenge. The protective effect of S28 in NHPs was assessed by measuring various asthma-related phenotypes. We also characterized the metabolomic and proteomic signatures of the lung environment and plasma to identify markers associated with the disease and treatment. Our data demonstrate that clinically relevant parameters, such as wheal and flare response, blood IgE levels, recruitment of white blood cells to the bronchoalveolar space, and lung responsiveness, are decreased in the S28-treated allergic NHPs compared with nontreated allergic NHPs. Furthermore, we also identified markers that can distinguish allergic from nonallergic or allergic and drug-treated NHPs, such as metabolites, phosphocreatine and glutathione, in the plasma and BAL fluid, respectively; and inflammatory cytokines, IL-5 and IL-13, in the bronchoalveolar lavage fluid. Our preclinical study demonstrates that S28 has potential as a treatment for allergic asthma in primate species closely related to humans. Combined with our previous findings, we demonstrate that S28 is effective in different models of asthma and in different species, and has the antiinflammatory properties clinically relevant for the treatment of allergic asthma.

Dapsone treatment is efficient against persistent cutaneous and gastrointestinal symptoms in children with Henoch-Schönlein purpura.

BACKGROUND: Henoch-Schönlein purpura (HSP) is a systemic disorder characterized by leukocytoclastic vasculitis involving the capillaries and by the deposition of IgA immune complexes. An association between HSP and atypical bacteria is uncommon in children. METHODS AND RESULTS: Here we report three cases of children, aged 5, 4 and 16 years, who were diagnosed with HSP associated with Mycoplasma pneumoniae or Chlamydia pneumoniae infection. In all presented cases, persistent cutaneous manifestations and abdominal pain were resistant to antibiotics and corticosteroids, but resolved during 48 h after the introduction of dapsone. No adverse effects of treatment were observed. CONCLUSION: Dapsone, a sulphone with an anti-inflammatory activity, showed remarkable therapeutic efficacy against rash and gastrointestinal symptoms in children with HSP. Its administration should be considered particularly in persistent cutaneous form of HSP.

NAS agar is more suitable than McKay agar for primary culture of Streptococcus milleri group (SMG) fastidious bacteria, S. intermedius in particular.

Streptococcus milleri group (SMG) is a group of three streptococcal species (S. anginosus, intermedius and constellatus) that act as opportunist pathogens, among others in cystic fibrosis. Due to their fastidious character, they are both difficult to cultivate and to differentiate from less pathogenic streptococcal species, therefore being most probably underdiagnosed. Semi-selective McKay agar and NAS agar were developed to facilitate SMG recovery from clinical samples; however, direct comparison of recovery rates has not been published yet. We tested the performance of both media on 123 patient samples and demonstrated general superiority of NAS agar for SMG recovery during primary cultivation convincingly. This observation was also confirmed by quantitative drop tests during subculture. Despite the undisputed overall superiority of NAS agar over McKay agar, a smaller fraction of strains grew better on McKay agar. Inter-strain differences were the most probable explanation. Therefore, when economic conditions are not limiting and maximum recovery rate is desirable, both plates are advised to be used in parallel for primary cultivation of clinical samples.

[Prolonged culture in a humid chamber increases the yields of pathogenic bacteria from the respiratory tract samples of patients suffering from cystic fibrosis].

Current standards of care for cystic fibrosis (CF) patients lack unequivocal recommendations concerning the duration of primary culture of bacteriological samples. With the exception of Burkholderia cepacia (5 days), the minimum recommended duration of primary culture varies between 48 and 72 hours. Our aim was to evaluate the effect of an extended 10-day period of primary culture in a humid chamber in samples acquired from the respiratory tract of patients suffering from CF. Compared to standard culture, prolonged culture in a humid chamber yielded 1.85 times more isolates of pathogenic species in pharyngeal swabs (76 versus 41 isolates) and 1.4 times more isolates in sputum samples (116 versus 82), but only 1.14 times more isolates in nasal swabs (25 versus 22). Prolonged culture was most beneficial for Achromobacter spp. (6 versus 0), Stenotrophomonas maltophilia (16 versus 5) and Pseudomonas aeruginosa (69 versus 49), whereas there was little or no benefit at all for Staphylococcus aureus (87 versus 73) and Moraxella catarrhalis (10 versus 10). Therefore, prolonged culture in a humid chamber may definitely be recommended for pharyngeal swabs and sputum samples obtained from patients suffering from CF to achieve the maximum recovery rate of pathogenic bacteria, in particular non-fermenting Gram-negative rods.

The Streptococcus milleri group in chronic obstructive pulmonary disease.

BACKGROUND AND AIMS: S. anginosus, constellatus and intermedius, also known as the Streptococcus milleri group (SMG) are three streptococcal species more frequently detected in cases of invasive disease, abscesses and empyema in particular. Recent research suggests they play a role in exacerbations of cystic fibrosis (CF). Owing to poor recovery on standard culture media and difficult differentiation from non-pathogenic streptococci, SMG may be underdiagnosed in routine settings. We aimed to establish the incidence of SMG in chronic obstructive pulmonary disease (COPD) patients compared to CF patients and to examine possible links of SMG to exacerbations that plays a key role in progression of COPD. METHODS: Altogether, 90 respiratory tract samples of patients suffering from CF or COPD were examined during the period from July 2012 to December 2013. Semi-selective McKay agar was used for primary cultivation of SMG and MALDI TOF MS was used for species identification that was confirmed by biochemical profiling and specific PCR. RESULTS: We confirmed the presence of SMG in CF (17.6% incidence in adult patients) and newly established its presence in COPD (10.3% incidence). In COPD, SMG was detected in 4 cases of acute exacerbations, where no other bacterial pathogen was detected. In 3/4 cases, increased CRP level indicated bacterial infection as a cause of the exacerbation and in all 3 cases, patients recovered during antibiotic treatment. CONCLUSIONS: Our data indicate SMG may act as opportunist pathogens able to cause exacerbations in COPD.

Recurrent membranous tracheal stenosis with a diverticulum in a 9-year-old boy. Not all that wheezes is asthma.

BACKGROUND: Benign tracheobronchial stenosis of the proximal airways may result from a variety of conditions and can cause dyspnea, cough, wheeze or stridor. METHODS AND RESULTS: In this report, we describe the case of a 9-year-old boy who was admitted to hospital with wheezing and progressive dyspnea lasting for six months. These abnormalities were diagnosed by multislice CT with 3-dimensional reconstruction of the trachea and by videobronchoscopy which demonstrated severe tracheal stenosis and an orifice of a tracheal diverticulum on the right side of the upper trachea. The stenosis was dilated with a balloon and vaporized with an Nd:YAG laser. Due to recurrent stenosis, the laser procedure had to be repeated several times in an approximately 4-6-month interval. CONCLUSION: Association between recurrent membranous tracheal stenosis and a tracheal diverticulum is a rare medical condition.

Performance of pyrosequencing versus MALDI-TOF MS in bacteria identification in chronic lung disease.

Rapid identification of the etiological agent in bacterial infection is necessary for correct diagnosis and appropriate therapy. In general, identification of pure cultures of bacteria using conventional phenotyping techniques requires 4-24 hours. Recently available new molecular technologies offer the potential of same day species identification once pure culture is available. Our aim was to evaluate the performance of rDNA V1 hypervariable region pyrosequencing, and the whole cell MALDI-TOF MS protein profiling in routine species identification. During the period from June 2012 to June 2014, 1.140 pure culture isolates were recovered from 402 samples from 126 patients suffering cystic fibrosis, chronic obstructive pulmonary disease or bronchiectasis. All the isolates were subjected to species identification by both techniques. Unfortunately, pyrosequencing was able to reach the species level in 43.2% of isolates only, whereas MALDI-TOF was clearly superior with 96.8% respectively. The overall sensitivity values also clearly underlined the superiority of MALDI-TOF MS with 96.8% compared to 85.1% achieved by pyrosequencing. Generally, MALDI-TOF MS turned out to be the best suitable technique in routine bacterial identification, whereas pyrosequencing could be recommended as the method of choice particularly in situations where MALDI-TOF MS fails to identify rare species.

Possible control of paternal imprinting of polymorphisms of the ADAM33 gene by epigenetic mechanisms and association with level of airway hyperresponsiveness in asthmatic children.

INTRODUCTION: ADAM33 is the candidate gene most commonly associated with asthma and airway hyperreactivity (AHR). AIM: The aim of this study was to determine whether level of AHR is associated with certain alleles or haplotypes of the ADAM33 gene in asthmatic children. METHODS: One hundred and nine asthmatic children and 46 controls from the general population were examined with spirometry before and after histamine and methacholine inhalation. All subjects were genotyped for single-nucleotide polymorphisms (SNPs) of the ADAM33 gene. Haplotypes were determined according to genotypes of the patient's parents. RESULTS: We found the three most frequent ADAM33 haplotypes (a1-3) were associated with the highest level of AHR to methacholine and histamine in 66% of asthmatic children. The paternally transmitted GGGCTTTCGCA haplotype was seen in 73.3% asthmatic children with serious AHR to methacholine challenge (paternal and maternal origin of haplotype 73.3% to 37.5, P=0.046) Significant differences in the relative frequency of paternal haplotypes with high levels of AHR to histamine were found (P=0.013). CONCLUSION: ADAM33 haplotypes (a1, a2, a3) are associated with severity of AHR and are significantly more often transmitted in the paternal line.

Association of STAT6 and ADAM33 single nucleotide polymorphisms with asthma bronchiale and IgE level and its possible epigenetic background.

BACKGROUND: ADAM33 and STAT6 belong to the candidate genes that have been commonly associated with asthma, bronchial hyperresponsiveness or IgE levels. Our objective was to assess the association of 11 SNPs of the ADAM33 and 6 of the STAT6 and their haplotypes with IgE levels and asthma. We also evaluated the possible role of parental origin of haplotypes on IgE levels. METHODS: We enrolled 109 children with asthma and 45 healthy controls. Genotyping was performed by TaqMan probes and confirmed by sequencing. Haplotype construction was based on the knowledge of parental genotypes and also inferred by using the EM algorithm and Bayes' theorem. RESULTS: None of the SNPs were associated with elevated IgE level or asthma. We found that the most frequent STAT6 haplotype ATTCAA (built from rs324012, rs324011, rs841718, rs3024974, rs3024974, rs4559 SNPs, respectively) was associated with elevated total IgE levels (P=0.01) and this haplotype was predominantly transmitted paternally (P<0.001). We compared our results with those of studies performed on German and Australian Caucasian populations and found that rs324011, rs3024974 and rs4559 SNPs in STAT6 should have a major effect on IgE levels. Therefore, we suggest the TCA haplotype alone (built from rs324011, rs3024974 and rs4559 SNPs, respectively) in STAT6 is associated with total IgE elevation. CONCLUSIONS: The influence of paternal origin of the STAT6 haplotype on IgE levels is surprising but the exact role of possible paternal imprinting in STAT6 regulation should be investigated and confirmed in future studies.

The anti-inflammatory effects of inhaled corticosteroids versus anti-leukotrienes on the lymphocyte P-glycoprotein (PGP) expression in asthmatic children.

BACKGROUND: Inhaled corticosteroids (ICS) are used in asthma therapy for their anti-inflammatory effects. P-glycoprotein (PGP) is a transmembrane efflux pump for many drugs, including corticosteroids. Expression of PGP is associated with therapy resistant disease. OBJECTIVE: The purpose of this study was to compare expression levels of PGP in blood lymphocytes of pediatric asthma patients either on ICS or on the leukotriene inhibitor montelukast medication. PATIENTS AND METHODS: The evaluation of lymphocyte PGP expression was performed on a sample of 99 children (66 boys and 33 girls) aged 2-18 (median 12) years with intermittent or persisting mild asthma, (as defined by GINY 2002). The asthmatic children were divided into 3 groups: (1) treated by ICS budesonide 200-400 microg per day (n = 27) more than 1 year; (2) treated by oral montelukast 4 or 5 mg at an age-based dose (n = 16); and (3) treated by inhaled corticosteroids and montelukast (n = 45). Reference PGP values were obtained from a group of 64 healthy children (23 boys and 29 girls) aged 2-15 years (median, 10 years). The expression of lymphocyte PGP was determined on fixed and permeabilized blood mononuclear cells using indirect immunofluorescence staining technique by flow cytometry modified according to Boer et al., 1997. RESULTS: Based on the weighted medians for PGP expression in peripheral blood lymphocytes, we found a significant difference (p < 10(-3)) between the group of asthma patients (n = 99), (619 +/- 5.3) and healthy controls (n = 64) (446.9 +/- 4.4). Second, there was a lower level of PGP expression in the group treated by ICS (548.6 +/- 9) than the group treated by montelukast and montelukast with budesonide (643.2 +/- 6.3) (p < 10(-6)). CONCLUSIONS: The anti-inflammatory activity of ICS is more effective in decreasing the production of pro-inflammatory mediators and results in reduced multidrug resistence (MDR-1) gene activity and expression of lymphocyte PGP in asthmatic children.

Montelukast decreases plasma endothelin-1 and serum eosinophil cationic protein levels in paediatric atopic asthma.

BACKGROUND AND OBJECTIVE: Endothelin-1 (ET-1) is produced by vascular endothelial cells and epithelial cells, T-lymphocytes and phagocytes. Increased ET-1 levels have been demonstrated in the bronchial epithelium of asthma patients. In vitro, ET-1 stimulates mucus secretion, activates proinflammatory cells - macrophages and mast cells - and serves as a mitogenic stimulus for fibroblasts and smooth muscle. In addition, ET-1 activates phospholipase 2. Compared with healthy individuals, asthma patients have increased ET-1 levels during an attack and following stabilisation. Our study was designed to examine plasma ET-1 (P-ET) levels in paediatric atopic patients newly diagnosed with persistent mild bronchial asthma and 1 month after initiation of montelukast therapy. METHODS: Patients' histories were examined, and their blood eosinophil leucocyte count and levels of total serum immunoglobulin E (S-IgE), serum eosinophil cationic protein (S-ECP) and P-ET were determined. Thirty-six patients with persistent mild bronchial asthma were treated with the leukotriene receptor antagonist montelukast, administered once a day for 4 weeks. Second P-ET and S-ECP level determinations were made 4 weeks later with all the children included in the study. P-ET levels were also determined in a group of 27 healthy children who had no atopy in their medical histories and were taking no drugs (including montelukast), and who served as controls. RESULTS: The mean +/- SD pretreatment P-ET level in the 36 study children was 11.542 +/- 6.408 pg/L, and this decreased to 5.636 +/- 4.419 pg/L after 1 month's therapy with montelukast (statistically significant difference; p < 0.0001). Both of these values were significantly higher (p < 0.0001 and p < 0.031, respectively) than the mean level in the control group of 27 children (3.543 +/- 2.497 pg/L). The mean pretreatment S-ECP level was 35.78 +/- 19.58 microg/L, and this decreased to 19.54 +/- 13.86 microg/L after 1 month's therapy (p < 0.001). CONCLUSIONS: This study demonstrated a decrease in P-ET levels in children with mild asthma receiving montelukast. This indicates a reduction in the severity of the inflammatory response and, hence, provides evidence for the anti-inflammatory effect of montelukast. Monitoring both ET-1 and ECP levels at regular follow-up may be useful in assessing these two facets of activity of chronic inflammation in bronchial asthma.

Percentage of eosinophils with surface CD44 and levels of eosinophil cationic protein in the peripheral blood in children with bronchial asthma and their changes after montelukast therapy.

Bronchial asthma (BA) is chronic inflammation of the respiratory tract with a role played by a variety of cells, particularly mast cells, eosinophils (Eo), and T lymphocytes. The serum levels of Eo cationic protein (S-ECP) reflect the severity of bronchial inflammation and the level of bronchial hyperreactivity in asthma patients. One of the most important adhesion molecules is CD44. We examined S-ECP, the percentage of Eo with surface CD44 expression (EoCD44), and Eo count in the peripheral blood of newly diagnosed pediatric atopic patients with intermittent and persistent mild BA according to the Global Iniative for Asthma 2002, in a proportion of patients 3 months after initiation of montelukast therapy. Ninety-seven children with BA had their medical history taken, and S-ECP, with the percentage of EoCD44 determined by direct fluorescence from whole blood using flow cytometry with a Coulter EPICS XL cytometer, and Eo count, total serum immunoglobulin E levels (S-IgE) were determined. Therapy with montelukast (5 mg daily) was started in 23 children. Three months after the first collection, a second S-ECP level and EoCD44 count determinations were made. An inverse correlation between S-ECP and EoCD44 (-0.602; p < 0.0001) was found in the 97 children with BA. In the 23 children receiving montelukast we documented inverse correlation of fluctuation on S-ECP and EoCD44 after 3 months. These results were not significant. An inverse correlation between S-ECP and percent of EoCD44 was established in the 97 children with asthma before therapy initiation. The lower percentage of EoCD44 in peripheral blood in asthmatic children is due to Eo inflammation activity and attests to massive Eo invasion into the airways. Determination of the percentage proportion of EoCD44 is another potential indirect marker of the multiple features of Eo inflammation.

Bronchial asthma and Chlamydia pneumoniae antibodies in children aged 4-8 years in Olomouc district.

BACKGROUND: Although several studies have demonstrated an association between infection with Chlamydia pneumoniae and asthma, these were mainly limited to exacerbation of symptoms in adults with known asthma OBJECTIVE: We investigated the role of C. pneumoniae infection in 149 atopic children with chronic cough and asthma, comparing them with 241 control non-atopic subjects presenting at Olomouc hospital between 1999 and 2003 with non-specific symptoms (temperature above normal (subfebrile), abdominal pain, arthralgia, and other symptoms. METHODS: The levels of C. pneumoniae-specific antibodies were measured using Chlamydien-rELISA kits (Medac, Hamburg, Germany). RESULTS: In a group of 83 atopic children with chronic cough, IgM and IgG antibodies to C. pneumoniae were demonstrated in 20 children (24 %). Among children with bronchial asthma, positive antibody was present in 29 children (44 %; /p = 0,052/); of this number, 24 (36 %; /p = 0,06/) had IgM and IgG antibodies while 5 children (8 %) had IgA and IgG antibodies against C. pneumoniae. A group of non-atopic children with non-specific symptoms included 38 children (16 %) with antibody positivity; 27 children (11 %) with IgM and IgG antibodies and 11 children (5 %) with IgA and IgG antibodies against C. pneumoniae. CONCLUSIONS: Asthma in children was associated with elevated levels of IgM and IgG antibodies to C. pneumoniae.

Treatment of chronic cough in children with montelukast,a leukotriene receptor antagonist.

Twenty-two children (13 boys and 9 girls) with chronic cough were treated with the leukotriene receptor antagonist montelukast (Singulair tbl. 5 mg) administered once daily for four weeks. In 14 children (68%), the cough ceased during the third week of treatment. Children responding to montelukast were found to have higher blood levels of eosinophil cationic protein (S-ECP) in the pretreatment blood sample than children with no response (responders 14.88+/-2.651 microg/l versus nonresponders 6.62+/-0.948 microg/l; p<0.01). Blood S-ECP levels remained higher also in the post-treatment blood sample in responders (10.55+/-1.631 microg/l) compared to nonresponders (6.13+/-0.937 microg/l; p<0.05). The difference is statistically significant. There were also differences in absolute eosinophil blood count and IgE blood levels between the two groups in the pretreatment blood sample. Using 24-hour pH-metry, two children not responding to therapy were subsequently diagnosed to have gastroesophageal reflux. Judging from the results, one might deduct that patients with chronic cough who have increased levels of serum ECP and absolute eosinophil blood counts are likely to benefit from treatment with montelukast.

Dose-related efficacy and safety of formoterol (Oxis) Turbuhaler compared with salmeterol Diskhaler in children with asthma.

To compare the dose-related bronchodilator efficacy and tolerability of formoterol (Oxis) Turbuhaler with salmeterol Diskhaler and placebo in children with asthma. A single-dose, randomized, double-blind, incomplete crossover study of 68 children (7-17 years), with moderate-to-severe asthma, 82% receiving inhaled corticosteroids. Patients received four of six treatments [4.5, 9, 18, or 36 microg formoterol (6, 12, 24 or 48 microg metered doses), 50 microg salmeterol (metered dose) or placebo] at 12-h visits, separated by > or =3 days. Forced expiratory volume in 1 s (FEV1), pulse, blood pressure, electrocardiogram, adverse events and urine formoterol were assessed. The therapeutic ratio of formoterol vs. salmeterol was estimated from the efficacy and systemic effects results. All active treatments significantly improved FEV1 compared with placebo. Formoterol 9-36 microg provided dose-related increases over salmeterol in lung function: average 12-h FEV1 (increases of 4.9-8.7%, p < 0.001) and FEV1 at 12 h post-dose (7.0-12.2%, p < 0.001). The onset of effect of formoterol was also significantly faster than salmeterol for doses > or =9 microg. Salmeterol 50 microg was estimated to be equieffective to 3.3 microg formoterol for 12-h average FEV1 and the estimated equieffective dose for a variety of systemic effects was 7.8-13.5 microg formoterol. All treatments were well tolerated. Formoterol (Oxis) Turbuhaler 4.5-36 microg provided dose-related improvements in bronchodilator efficacy in children with asthma. Formoterol > or =9 microg provided superior bronchodilator efficacy over 12 h compared with salmeterol Diskhaler 50 microg with no increase in systemic effects.