A connectivity mapping approach predicted acetylsalicylic acid (aspirin) to induce osteo/odontogenic differentiation of dental pulp cells.

AIM: To use connectivity mapping, a bioinformatics approach, to identify compounds that could induce odontogenic differentiation of dental pulp cells (DPCs) and to experimentally validate this effect. A subsidiary aim was to investigate the anti-inflammatory effect of any identified compound. METHODOLOGY: The Gene Expression Omnibus (GEO) database was searched for microarray data sets assessing odontogenic differentiation of human DPCs. An odontogenic gene expression signature was generated by differential expression analysis. The statistical significant connectivity map (ssCMap) method was used to identify compounds with a highly correlating gene expression pattern. DPCs were treated with the compound identified, and osteo/odontogenic differentiation was assessed by Alizarin red staining, alkaline phosphatase activity and expression of osteo/odontogenic genes ALPL, RUNX2, COL1A1, DSPP, DMP1 and SPP1 by RT-PCR. The anti-inflammatory effect of the compound was assessed using an ex vivo pulpitis model, and cytokine levels were measured with multiplex assay. Means were compared using the t-test or ANOVA followed by a Bonferroni post hoc test with the level of significance set at P ≤ 0.05. RESULTS: The GEO database search identified a specific gene expression signature for osteo/odontogenic differentiation. Analysis using ssCMap found that acetylsalicylic acid [(ASA)/aspirin] was the drug with the strongest correlation with that gene signature. The treatment of DPCs with 0.05 mmol L-1 ASA showed increased alkaline phosphatase activity (P < 0.001), mineralization (P < 0.05), and increased the expression of the osteo/odontogenic genes, DMP1 and DSPP (P < 0.05). Low concentration (0.05 mmol L-1 ) ASA reduced inflammatory cytokines IL-6 (P < 0.001), CCL21 (P < 0.05) and MMP-9 (P < 0.05) in an ex vivo pulpitis model. CONCLUSIONS: Connectivity mapping, a web-based informatics method, was successfully used to identify aspirin as a candidate drug that could modulate the differentiation of DPCs. Aspirin was shown to induce odontogenic differentiation in DPCs in vitro and this, together with its anti-inflammatory effects, makes it a potential candidate for vital pulp therapies.

Infection with Prevotella nigrescens induces TLR2 signalling and low levels of p65 mediated inflammation in Cystic Fibrosis bronchial epithelial cells.

Prevotella spp. are frequently identified in Cystic Fibrosis sputum. This study examined whether infection with Prevotella nigrescens, a frequently identified member of this species, contributes to inflammation in CF bronchial epithelial cells through activation of TLR- and NF-κB signalling pathways. CFBE41o- cells were infected with either P.nigrescens or Pseudomonas aeruginosa and incubated under anaerobic conditions for 4h. P.nigrescens activated TLR2 signalling but not TLR4 signalling while P.aeruginosa activated TLR4 signalling with a lesser effect on TLR2. P.aeruginosa induced significant IκBα phosphorylation 10min post infection with a return to control levels by 30min post infection. A significant induction in nuclear p65 DNA binding was observed at 2h post infection. In contrast, infection with P.nigrescens induced phosphorylation of IκBα 120min post infection, with significant induction in nuclear p65 DNA binding at 4h post infection only. Cytokine gene and protein responses were lower for P.nigrescens compared to P.aeruginosa. This study demonstrates the ability of a clinical P.nigrescens isolate to provoke a delayed NF-κB(p65) driven response through induction in TLR2 signalling and activation of sustained levels of IKKα.

Regulators of A20 (TNFAIP3): new drug-able targets in inflammation.

Persistent activation of the transcription factor Nuclear factor-κB (NF-κB) is central to the pathogenesis of many inflammatory disorders, including those of the lung such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD). Despite recent advances in treatment, management of the inflammatory component of these diseases still remains suboptimal. A20 is an endogenous negative regulator of NF-κB signaling, which has been widely described in several autoimmune and inflammatory disorders and more recently in terms of chronic lung disorders. However, the underlying mechanism for the apparent lack of A20 in CF, COPD, and asthma has not been investigated. Transcriptional regulation of A20 is complex and requires coordination of different transcription factors. In this review we examine the existing body of research evidence on the regulation of A20, concentrating on pulmonary inflammation. Special focus is given to the repressor downstream regulatory element antagonist modulator (DREAM) and its nuclear and cytosolic action to regulate inflammation. We provide evidence that would suggest the A20-DREAM axis to be an important player in (airway) inflammatory responses and point to DREAM as a potential future therapeutic target for the modification of phenotypic changes in airway inflammatory disorders. A schematic summary describing the role of DREAM in inflammation with a focus on chronic lung diseases as well as the possible consequences of altered DREAM expression on immune responses is provided.

Induction of the inflammatory regulator A20 by gibberellic acid in airway epithelial cells.

BACKGROUND AND PURPOSE: NF-κB-driven inflammation is negatively regulated by the zinc finger protein A20. Gibberellic acid (GA3 ) is a plant-derived diterpenoid with documented anti-inflammatory activity, which is reported to induce A20-like zinc finger proteins in plants. Here, we sought to investigate the anti-inflammatory effect of GA3 in airway epithelial cells and determine if the anti-inflammatory action relates to A20 induction. EXPERIMENTAL APPROACH: Primary nasal epithelial cells and a human bronchial epithelial cell line (16HBE14o-) were used. Cells were pre-incubated with GA3 , stimulated with Pseudomonas aeruginosa LPS; IL-6 and IL-8 release, A20, NF-κB and IκBα expression were then evaluated. To determine if any observed anti-inflammatory effect occurred via an A20-dependent mechanism, A20 was silenced using siRNA. KEY RESULTS: Cells pre-incubated with GA3 had significantly increased levels of A20 mRNA (4 h) and protein (24 h), resulting in a significant reduction in IL-6 and IL-8 release. This effect was mediated via reduced IκBα degradation and reduced NF-κB (p65) expression. Furthermore, the anti-inflammatory action of GA3 was abolished in A20-silenced cells. CONCLUSIONS AND IMPLICATIONS: We showed that A20 induction by GA3 attenuates inflammation in airway epithelial cells, at least in part through its effect on NF-κB and IκBα. GA3 or gibberellin-derived derivatives could potentially be developed into anti-inflammatory drugs for the treatment of chronic inflammatory diseases associated with A20 dysfunction.

Increased concentrations of the oxidative DNA adduct 7,8-dihydro-8-oxo-2-deoxyguanosine in the germ-line of men with type 1 diabetes.

The effects of diabetes mellitus on male reproductive health have not been clearly defined. A previous publication from this group reported significantly higher levels of nuclear DNA fragmentation and mitochondrial DNA deletions in spermatozoa from men with type 1 diabetes. This study compared semen profiles, sperm DNA fragmentation and levels of oxidative DNA modification in spermatozoa of diabetic and non-diabetic men. Semen samples from 12 non-diabetic, fertile men and 11 type 1 diabetics were obtained and subjected to conventional light microscopic semen analysis. Nuclear DNA fragmentation was assessed using an alkaline Comet assay and concentrations of 7,8-dihydro-8-oxo-2-deoxyguanosine (8-OHdG), an oxidative adduct of the purine guanosine, were assessed by high-performance liquid chromatography. Conventional semen profiles were similar in both groups, whilst spermatozoa from type 1 diabetics showed significantly higher levels of DNA fragmentation (44% versus 27%; P < 0.05) and concentrations of 8-OHdG (3.6 versus 2.0 molecules of 8-OHdG per 10(5) molecules of deoxyguanosine; P < 0.05). Furthermore, a positive correlation was observed between DNA fragmentation and concentrations of 8-OHdG per 10(5) molecules of deoxyguanosine (rs = 0.7, P < 0.05). The genomic damage evident in spermatozoa of type 1 diabetics may have important implications for their fertility and the outcome of pregnancies fathered by these individuals.

Neutrophils and eosinophils: clinical implications of their appearance, presence and disappearance in asthma and COPD.

Asthma and chronic obstructive pulmonary disease (COPD) are common chronic disorders. Traditionally, asthma has been associated with an eosinophilic inflammation and COPD with neutrophilic inflammation. In this review we will highlight the maturation, recruitment, activation, action and apoptosis of these cells. In addition we will focus on the evidence for their presence in disease and suggest potential new therapeutic interventions.

Oxidative stress in lavage fluid of preterm infants at risk of chronic lung disease.

There is evidence that oxidative stress plays a role in the development of chronic lung disease (CLD), with immature lungs being particularly sensitive to the injurious effect of oxygen and mechanical ventilation. We analyzed total ascorbate, urate, and protein carbonyls in 102 bronchoalveolar lavage fluid samples from 38 babies (33 preterm, 24-36 wk gestation; 5 term, 37-39 wk gestation). Preterm babies had significantly decreasing concentrations of ascorbate, urate, and protein carbonyls during the first 9 days of life (days 1-3, 4-6, and 7-9, Kruskal-Wallis ANOVA: P = 0.016, P < 0.0001, and P = 0.010, respectively). Preterm babies had significantly higher protein carbonyl concentrations at days 1-3 and 4-6 (P = 0.005 and P = 0.044) compared with term babies. Very preterm babies (24-28 wk gestation) had increased concentrations of protein carbonyls at days 4-6 (P = 0.056) and significantly decreased ascorbate concentrations at days 4-6 (P = 0.004) compared with preterm babies (29-36 wk gestation). Urate concentrations were significantly elevated at days 1-3 (P = 0.023) in preterm babies who subsequently developed CLD. This study has shown the presence of oxidative stress in the lungs of preterm babies during ventilation, especially in those who subsequently developed CLD.

Oxidative stress and increased type-IV collagenase levels in bronchoalveolar lavage fluid from newborn babies.

Oxidative stress may increase lung permeability by up-regulation of matrix-metalloproteinase-9 (MMP-9), a type-IV collagenase that can disrupt alveolar basement membranes. We have compared a marker of oxidative stress (protein carbonyl residues) with levels of MMP-9 and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), in bronchoalveolar lavage samples from newborn babies. Bronchoalveolar lavage samples (n = 87, two from each time point) were taken in the first 6 postnatal days from 41 ventilated babies: 18 of <29 wk gestation, 10 of 29-36 wk, 9 term with persistent fetal circulation, and 4 term without lung disease. Respiratory disease severity at the time of bronchoalveolar lavage was assessed using the arterial-alveolar oxygen tension ratio. One sample from each time point was used for the measurement of MMP-9 by zymography and TIMP-1 by ELISA. The second sample was used to measure carbonyl group concentrations, also using an ELISA. Correlations were calculated between protein carbonyls, arterial-alveolar oxygen tension ratio, and MMP-9 and TIMP-1 concentrations. Significant correlations were found between carbonyl concentrations and arterial-alveolar oxygen tension ratio (r = -0.325, p = 0.0031, n = 81), MMP-9 (r = 0.331, p < 0.0029, n = 79), and TIMP-1 (r = 0.436, p < 0.0001, n = 87). Worsening respiratory disease in newborn babies is associated with increased carbonyl concentrations in neonatal bronchoalveolar lavage fluid, and these correlated with MMP-9 and TIMP-1 levels. Increased oxidative stress may damage the lung by increasing type-IV collagenase activity, causing disruption of the extracellular matrix.

Antioxidants and protein carbonyls in bronchoalveolar lavage fluid of children: normal data.

Antioxidant-oxidant imbalances in bronchoalveolar lavage fluid (BAL) are thought to contribute to oxidative stress in respiratory disease. However, normal reference ranges for BAL antioxidants and oxidized proteins in children are not available. In this study, we recruited 124 children attending for elective surgery for a noninflammatory condition; 83 were nonasthmatic, nonatopic (N) and 41 were nonasthmatic, atopic (NA). A nonbronchoscopic lavage was performed and ascorbate, uric acid, alpha-tocopherol, and protein carbonyl (as a measure of oxidative damage) concentrations were determined in BAL fluid. The 95% reference range was 0.112-1.897 micromol/L for ascorbate, 0.149-2.163 micromol/L for urate, 0.0029-0.066 micromol/L for alpha-tocopherol, and 0.280-4.529 nmol/mg for protein carbonyls in BAL fluid. Age, gender, and exposure to environmental tobacco smoke did not affect the concentration of ascorbate, urate, alpha-tocopherol, or protein carbonyls. However, in multiple linear regression analyses, the type of home heating (glass-fronted fires or oil-fired central heating) was found to influence ascorbate and urate concentrations in the BAL fluid (ss-coefficient for ascorbate: 0.445, p = 0.031; for urate: 0.114, p = 0.001). There was no significant difference between the N and NA group in BAL fluid concentrations of ascorbate, urate, or protein carbonyls. The alpha-tocopherol concentration was significantly increased in the NA group (p = 0.037). Uric acid and alpha-tocopherol concentrations in BAL fluid and serum were not correlated. Intriguingly, serum and BAL ascorbate concentrations were significantly correlated (r = 0.297, p = 0.018, n = 63), which may offer an explanation for why supplementing the diet with vitamin C can improve asthma symptoms. Further studies will investigate the role of BAL antioxidant concentrations in children with inflammatory respiratory diseases.

Chronic cough in children: bronchoalveolar lavage findings.

Isolated chronic cough in childhood is a common complaint. Although the symptom cough is included in the definition of clildhood asthma, there is debate as to whether the majoritv of these children have asthma. The authors studied children with isolated chronic cough looking for evidence of airway inflammation typical of asthma, with increased numbers of airway eosinophils as assessed from bronchoalveolar lavage (BAL). The investigations were carried out on 23 children (median age: 6.7 yrs; range: 1.7-12.75 yrs), attending the Royal Belfast Hospital for Sick Children for elective surgery, who also had a chronic unexplained cough. Written informed consent was obtained from the parent(s) and a nonbronchoscopic BAL was performed. BAL samples were analysed for total and differential white cell counts and also for the inflammatory mediators, eosinophil cationic protein (ECP) and histamine. Results were compared with a group of normal nonatopic children and also a group of atopic asthmatic children, who had been recruited for other studies on airway inflammation. There was a small but statistically significant increase in BAL percentage eosinophils in the children with chronic cough compared with nonasthmatic controls (0.28% versus 0.10%, p=0.03). However, the children with cough had lower percentage eosinophils than the atopic asthmatic controls (0.28% versus 0.66%, p=0.01). Three out of 23 children with chronic cough had BAL eosinophils greater than the normal upper 95% reference interval in BAL. There was a small but statistically significant increase in percentage neutrophils in the children with cough compared with the nonasthmatic controls (5.85% versus 3.21%, p=0.03). Four out of the 23 children had BAL neutrophils greater than the normal upper 95% reference interval in BAL. The authors conclude that only a minority of children with chronic unexplained cough have asthmatic-type airway inflammation. It is speculated that the increased percentage neutrophils in bronchoalveolar lavage from children with cough could relate to underlying persistent airways infection.

Serum eosinophil cationic protein (ECP): reference values in healthy nonatopic children.

BACKGROUND: Although serum ECP concentrations have been reported in normal children, there are currently no published upper cutoff reference limits for serum ECP in normal, nonatopic, nonasthmatic children aged 1-15 years. METHODS: We recruited 123 nonatopic, nonasthmatic normal children attending the Royal Belfast Hospital for Sick Children for elective surgery and measured serum ECP concentrations. The effects of age and exposure to environmental tobacco smoke (ETS) on the upper reference limits were studied by multiple regression and fractional polynomials. RESULTS: The median serum ECP concentration was 6.5 microg/l and the 95th and 97.5 th percentiles were 18.8 and 19.9 microg/l. The median and 95th percentile did not vary with age. Exposure to ETS was not associated with altered serum ECP concentrations (P = 0.14). CONCLUSIONS: The 95th and 97.5 th percentiles for serum ECP for normal, nonatopic, nonasthmatic children (aged 1-15 years) were 19 and 20 microg/l, respectively. Age and exposure to parental ETS did not significantly alter serum ECP concentrations or the normal upper reference limits. Our data provide cutoff upper reference limits for normal children for use of serum ECP in a clinical or research setting.

Serum eosinophilic cationic protein and blood eosinophil counts for the prediction of the presence of airways inflammation in children with wheezing.

BACKGROUND: Serum eosinophilic cationic protein (ECP) concentrations may be useful noninvasive markers of airways inflammation in atopic asthma. However, the usefulness of serum ECP measurement for the prediction of airways inflammation in children with a history of wheezing is unknown. OBJECTIVE: To determine the test accuracy of serum ECP and blood eosinophil percentage as noninvasive markers of eosinophilic airways inflammation. METHODS: Bronchoalveolar lavage (BAL) fluid and peripheral blood samples for eosinophil percentages and serum ECP were obtained from children undergoing elective surgery and who gave a history of wheezing in the previous year. Sensitivity, specificity and likelihood ratios (LH) and the area under the curve (AUC) for the receiver operator characteristic (ROC) curve were calculated for each blood marker for the prediction of airways inflammation defined by a BAL eosinophil percentage > 0.86. Data were analysed on the basis of how recently symptoms had occurred. RESULTS: Seventy-seven children (median age 6.75 years) were studied. An AUC of 0.75 (log serum ECP concentration) and 0.76 (log blood eosinophil percentage) was obtained for predicting airways inflammation. A serum ECP > 13 microg/L yielded a LH of 4.4, whereas using a cutoff blood eosinophils > 4% yielded a LH of 1.9, for the prediction of elevated eosinophils in BAL. Serum ECP and eosinophil percentages in BAL and blood were lowest (not statistically significant) when last symptoms had occurred more than 12 weeks previously. CONCLUSIONS: Serum ECP and blood eosinophil percentages are useful markers for predicting eosinophilic airways inflammation in wheezing children.

Inflammatory mediators in bronchoalveolar lavage samples from children with and without asthma.

BACKGROUND: We investigated whether eosinophils and mast cells, found in the airways of children with wheeze, were activated during relatively asymptomatic periods. METHODS: A nonbronchoscopic bronchoalveolar lavage (BAL) procedure was performed on children presenting for an elective surgical procedure. Eosinophil-derived (eosinophil cationic protein, ECP) and mast cell-derived (histamine/tryptase) mediator concentrations were measured in the BAL fluid. A detailed history and serum immunoglobulin E were used to classify the children into four groups: atopic with and without asthma, viral-associated wheeze and normal controls. RESULTS: The ECP concentrations in BAL from atopic asthmatic subjects were significantly higher than those measured in BAL from normal controls (P < 0.01), no other groups differed significantly. Histamine concentrations were elevated in both the atopic asthmatic and viral-associated wheeze groups compared with controls (P < 0.02) and additionally higher concentrations were obtained in atopics with asthma compared with atopics without asthma (P < 0.03). Tryptase concentrations did not differ between groups, although the tryptase and histamine concentrations correlated significantly (r = 0.78, P < 0.0001). CONCLUSIONS: Elevated histamine concentrations were found in children with wheeze regardless of the aetiology, whereas ECP was only elevated in those asthmatics with atopy. This suggests that even in relatively quiescent periods, there is some on going activation of airway eosinophils in children with atopic asthma.

In vitro effects of the pyrethroid S-bioallethrin on lymphocytes and basophils from atopic and nonatopic subjects.

Synthetic pyrethroids are increasingly used as insecticides and marketed as having relatively low human toxicity. The aim of this study was to examine the in vitro effects of the synthetic pyrethroid S-bioallethrin on human blood lymphocytes and basophils in atopic individuals and nonatopic control subjects. S-bioallethrin caused inhibition of lymphocyte proliferation after a 72-h culture period in a concentration-dependent manner. The inhibition of the lymphocyte proliferation by S-bioallethrin at the concentration 6.5 microM correlated well with the total serum IgE values (r = -0.89, P < 0.001). Samples from atopic subjects were more sensitive to this inhibition than those from nonatopic volunteers. The regulatory interleukin-4/interferon-gamma (JL-4/IFN-gamma) balance showed a significant difference between atopic and nonatopic subjects after a short-term culture period (24 h) in the presence of the same concentration range of S-bioallethrin (P < 0.001). Additionally, IFN-gamma secretion was consistently lower in cells from the atopic donors. Furthermore, S-bioallethrin induced histamine release from human basophils in a concentration-dependent manner. Although the effect was small compared to histamine liberators such as N-formyl-Met-Leu-Phe and anti-IgE, the response to S-bioallethrin was significantly different in atopic donors from nonatopic (P = 0.0431). These findings are the first demonstration of the immunotoxicologic properties of the synthetic pyrethroid S-bioallethrin by this combined in vitro approach with human lymphocytes and basophils. Further studies will investigate the responses of lymphocytes from patients who are sensitive to these agents.

Bronchoalveolar lavage findings suggest two different forms of childhood asthma.

BACKGROUND: It seems plausible that children with atopy and persistent asthma symptoms will, like their adult counterparts, have chronic airways inflammation. However, many young children with no other atopic features have episodic wheezing that is triggered solely by viral respiratory infections. Little is known as to whether airways inflammation occurs in these two asthma patterns during relatively asymptomatic periods. METHODS: Using a non-bronchoscopic bronchoalveolar lavage (BAL) procedure on children presenting for an elective surgical procedure, this study has investigated the cellular constituents of BAL fluid in children with a history of atopic asthma (AA) non-asthmatic atopic children (NAA) or viral associated wheeze (VAW). RESULTS: A total of 95 children was studied: 52 with atopic asthma (8.0 years, range 1.1-15.3, 36 male), 23 with non-asthmatic atopy (median age 8.3 years, range 1.7-13.6, 11 male) and 20 with VAW (3.1 years, range 1.0-8.2, 13 male). No complications were observed during the lavage procedure and no adverse events were noted post-operatively. Total lavage fluid recovered was similar in all groups and the total cell numbers were higher in the VAW group. Eosinophil (P < or = 0.005) and mast cell (P < or = 0.05) numbers were significantly elevated in the group with atopic asthma. CONCLUSIONS: During relatively asymptomatic periods there is on-going airways inflammation, as demonstrated by eosinophil and mast cell recruitment, in children with asthma and atopy but not in children with viral associated wheeze or atopy alone. This strongly suggests that there are different underlying pathophysiological mechanisms in these two groups of children who wheeze.