Caspase-11 Plays a Protective Role in Pulmonary Acinetobacter baumannii Infection.

Activation of caspase-11 by some Gram-negative bacteria triggers the caspase-1/interleukin 1ß (IL-1ß) pathway, independent of canonical inflammasomes. Acinetobacter baumannii is a Gram-negative, conditionally pathogenic bacterium that can cause severe pulmonary infection in hospitalized patients. A. baumannii was revealed to activate canonical and noncanonical inflammasome pathways in bone marrow-derived macrophages (BMDMs). Pulmonary infection of caspase-11-/- mice with A. baumannii showed that caspase-11 deficiency impaired A. baumannii clearance, exacerbated pulmonary pathological changes, and enhanced susceptibility to A. baumannii These data indicate that the caspase-11-mediated innate immune response plays a crucial role in defending against A. baumannii.

Lipoprotein-associated phospholipase A2, myeloperoxidase and vascular endothelial growth factor - predictors of high vascular risk in respiratory bacterial infections.

Objective. Respiratory bacterial infections are associated with important coagulation disturbances that amplify the pulmonary lesions and determine a more severe course of the disease. The aim of our study was to investigate the correlation between the evolution of the general clinical parameters and the occurrence of thrombotic events on one side, and plasma levels of selected proteins involved in inflammation and coagulation on the other side, with the intent to establish and to validate a laboratory test panel for the assessment of the vascular risk in patients with bacterial respiratory infections. Methods. The study included 111 patients (divided into two groups, 61 without thrombosis and 50 with thrombosis) with bacterial respiratory infections and 30 healthy controls, age and gender-matched. The baseline evaluation of the patients included clinical, biological, and respiratory examination. LpPLA2 and MPO activities were measured by the spectrophotometric method. VEGF was quantified with an ELISA kit. Results. The collected data showed a correlation between the occurrence of superimposed thrombosis in respiratory infection patients, and the intensity of the inflammatory process, reflected by the increased MPO activity, and the dynamics of LpPLA2 and VEGF. Conclusion. Bacterial respiratory infections associate thrombotic vascular events of various degrees of severity, which correlate with the intensity of the inflammatory process, and the severity of endothelium dysfunction at the level of microcirculation. Starting from the recorded data, and based on the established severity scales in use, it is possible to compute a vascular risk score that takes into consideration the values of the three biomarkers under investigation. Abbreviations: COPD = chronic obstructive pulmonary disease,hsCRP = high sensitivity C reactive protein,EC = endothelial cells, ICAM-1 = intercellular adhesion molecule1, LpPLA2 = lipoprotein-associated phospholipase A2, MPO = myeloperoxidase,NK cells = natural killer cells,VEGF = vascular endothelial growth factor, VCAM-1 = vascular cell adhesion molecule 1.

Attenuated heme oxygenase-1 responses predispose the elderly to pulmonary nontuberculous mycobacterial infections.

Pulmonary infections with nontuberculous mycobacteria (P-NTM), such as by Mycobacterium avium complex (M. avium), are increasingly found in the elderly, but the underlying mechanisms are unclear. Recent studies suggest that adaptive immunity is necessary, but not sufficient, for host defense against mycobacteria. Heme oxygenase-1 (HO-1) has been recognized as a critical modulator of granuloma formation and programmed cell death in mycobacterial infections. Old mice (18-21 mo) infected with M. avium had attenuated HO-1 response with diffuse inflammation, high burden of mycobacteria, poor granuloma formation, and decreased survival (45%), while young mice (4-6 mo) showed tight, well-defined granuloma, increased HO-1 expression, and increased survival (95%). To further test the role of HO-1 in increased susceptibility to P-NTM infections in the elderly, we used old and young HO-1+/+ and HO-1-/- mice. The transcriptional modulation of the JAK/STAT signaling pathway in HO-1-/- mice due to M. avium infection demonstrated similarities to infected wild-type old mice with upregulation of SOCS3 and inhibition of Bcl2. Higher expression of SOCS3 with downregulation of Bcl2 resulted in higher macrophage death via cellular necrosis. Finally, peripheral blood monocytes (PBMCs) from elderly patients with P-NTM also demonstrated attenuated HO-1 responses after M. avium stimulation and increased cell death due to cellular necrosis (9.69% ± 2.02) compared with apoptosis (4.75% ± 0.98). The augmented risk for P-NTM in the elderly is due, in part, to attenuated HO-1 responses, subsequent upregulation of SOCS3, and inhibition of Bcl2, leading to programmed cell death of macrophages, and sustained infection.

α1-Antitrypsin reduces rhinovirus infection in primary human airway epithelial cells exposed to cigarette smoke.

Human rhinovirus (HRV) infections target airway epithelium and are the leading cause of acute exacerbations of COPD. Cigarette smoke (CS) increases the severity of viral infections, but there is no effective therapy for HRV infection. We determined whether α1-antitrypsin (A1AT) reduces HRV-16 infection in CS-exposed primary human airway epithelial cells. Brushed bronchial epithelial cells from normal subjects and patients diagnosed with COPD were cultured at air-liquid interface to induce mucociliary differentiation. These cells were treated with A1AT or bovine serum albumin for 2 hours and then exposed to air or whole cigarette smoke (WCS) with or without HRV-16 (5×10(4) 50% Tissue Culture Infective Dose [TCID50]/transwell) infection for 24 hours. WCS exposure significantly increased viral load by an average of fivefold and decreased the expression of antiviral genes interferon-λ1, OAS1, and MX1. When A1AT was added to WCS-exposed cells, viral load significantly decreased by an average of 29-fold. HRV-16 infection significantly increased HRV-16 receptor intercellular adhesion molecule-1 messenger RNA expression in air-exposed cells, which was decreased by A1AT. A1AT-mediated reduction of viral load was not accompanied by increased epithelial antiviral gene expression or by inhibiting the activity of 3C protease involved in viral replication or maturation. Our findings demonstrate that A1AT treatment prevents a WCS-induced increase in viral load and for the first time suggest a therapeutic effect of A1AT on HRV infection.

Increased ß-glucuronidase activity in bronchoalveolar lavage fluid of children with bacterial lung infection: A case-control study.

BACKGROUND AND OBJECTIVE: ß-Glucuronidase is a lysosomal enzyme released into the extracellular fluid during inflammation. Increased ß-glucuronidase activity in the cerebrospinal and peritoneal fluid has been shown to be a useful marker of bacterial inflammation. We explored the role of ß-glucuronidase in the detection of bacterial infection in bronchoalveolar lavage fluid (BALF) of paediatric patients. METHODS: In this case-control study, % polymorphonuclear cell count (PMN%), ß-glucuronidase activity, interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α) and elastase were measured in culture-positive (≥10(4) cfu/mL, C+) and -negative (C-) BALF samples obtained from children. RESULTS: A total of 92 BALF samples were analysed. The median ß-glucuronidase activity (measured in nanomoles of 4-methylumbelliferone (4-MU)/mL BALF/h) was 246.4 in C+ (interquartile range: 71.2-751) and 21.9 in C- (4.0-40.8) (P < 0.001). The levels of TNF-α and IL-8 were increased in C+ as compared with C- (5.4 (1.7-12.6) vs 0.7 (0.2-6.2) pg/mL, P < 0.001 and 288 (76-4300) vs 287 (89-1566) pg/mL, P = 0.042, respectively). Elastase level and PMN% did not differ significantly (50 (21-149) vs 26 (15-59) ng/mL, P = 0.051 and 20 (9-40) vs 18 (9-34) %, P = 0.674, respectively). The area under the curve of ß-glucuronidase activity (0.856, 95% confidence interval (CI): 0.767-0.920) was higher than that of TNF-α (0.718; 95% CI: 0.614-0.806; P = 0.040), IL-8 (0.623; 95% CI: 0.516-0.722; P = 0.001), elastase (0.645; 95% CI: 0.514-0.761; P = 0.008) and PMN% (0.526; 95 % CI: 0.418-0.632; P < 0.001). CONCLUSIONS: This study demonstrates a significant increase of ß-glucuronidase activity in BALF of children with culture-positive bacterial inflammation. In our population ß-glucuronidase activity showed superior predictive ability for bacterial lung infection than other markers of inflammation.

Acute exacerbations of COPD are associated with significant activation of matrix metalloproteinase 9 irrespectively of airway obstruction, emphysema and infection.

BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are associated with accelerated aggravation of clinical symptoms and deterioration of pulmonary function. The mechanisms by which exacerbations may contribute to airway remodeling and declined lung function are poorly understood. In this study, we investigated if AE-COPD are associated with differential expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in bronchoalveolar lavage (BAL). METHODS: COPD patients undergoing diagnostic bronchoscopy, with either stable disease (n = 53) or AE-COPD (n = 44), matched for their demographics and lung function parameters were included in this study. Protein levels of MMP-2,-9,-12 and of TIMP-1 and -2 in BAL were measured by ELISA. Enzymatic activity of MMP-2 and -9 was assessed by gelatin zymography. RESULTS: We observed that MMP-9, TIMP-1 and TIMP-2 were significantly increased in BAL during AE-COPD. Furthermore, there was a significant negative correlation of MMP-9, TIMP-1 and TIMP-2 with FEV1% predicted and a significant positive correlation of TIMP-1 and TIMP-2 with RV% predicted in AE-COPD. None of MMPs and TIMPs correlated with DLCO% predicted, indicating that they are associated with airway remodeling leading to obstruction rather than emphysema. In AE-COPD the gelatinolytic activity of MMP-2 was increased and furthermore, MMP-9 activation was significantly up-regulated irrespective of lung function, bacterial or viral infections and smoking. CONCLUSIONS: The results of this study indicate that during AE-COPD increased expression of TIMP-1, TIMP-2, and MMP-9 and activation of MMP-9 may be persistent aggravating factors associated with airway remodeling and obstruction, suggesting a pathway connecting frequent exacerbations to lung function decline.

Effects of an anti-inflammatory VAP-1/SSAO inhibitor, PXS-4728A, on pulmonary neutrophil migration.

BACKGROUND AND PURPOSE: The persistent influx of neutrophils into the lung and subsequent tissue damage are characteristics of COPD, cystic fibrosis and acute lung inflammation. VAP-1/SSAO is an endothelial bound adhesion molecule with amine oxidase activity that is reported to be involved in neutrophil egress from the microvasculature during inflammation. This study explored the role of VAP-1/SSAO in neutrophilic lung mediated diseases and examined the therapeutic potential of the selective inhibitor PXS-4728A. METHODS: Mice treated with PXS-4728A underwent intra-vital microscopy visualization of the cremaster muscle upon CXCL1/KC stimulation. LPS inflammation, Klebsiella pneumoniae infection, cecal ligation and puncture as well as rhinovirus exacerbated asthma models were also assessed using PXS-4728A. RESULTS: Selective VAP-1/SSAO inhibition by PXS-4728A diminished leukocyte rolling and adherence induced by CXCL1/KC. Inhibition of VAP-1/SSAO also dampened the migration of neutrophils to the lungs in response to LPS, Klebsiella pneumoniae lung infection and CLP induced sepsis; whilst still allowing for normal neutrophil defense function, resulting in increased survival. The functional effects of this inhibition were demonstrated in the RV exacerbated asthma model, with a reduction in cellular infiltrate correlating with a reduction in airways hyperractivity. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that the endothelial cell ligand VAP-1/SSAO contributes to the migration of neutrophils during acute lung inflammation, pulmonary infection and airway hyperractivity. These results highlight the potential of inhibiting of VAP-1/SSAO enzymatic function, by PXS-4728A, as a novel therapeutic approach in lung diseases that are characterized by neutrophilic pattern of inflammation.

Neutrophil elastase-mediated increase in airway temperature during inflammation.

BACKGROUND: How elevated temperature is generated during airway infections represents a hitherto unresolved physiological question. We hypothesized that innate immune defence mechanisms would increase luminal airway temperature during pulmonary infection. METHODS: We determined the temperature in the exhaled air of cystic fibrosis (CF) patients. To further test our hypothesis, a pouch inflammatory model using neutrophil elastase-deficient mice was employed. Next, the impact of temperature changes on the dominant CF pathogen Pseudomonas aeruginosa growth was tested by plating method and RNAseq. RESULTS: Here we show a temperature of ~38°C in neutrophil-dominated mucus plugs of chronically infected CF patients and implicate neutrophil elastase:α1-proteinase inhibitor complex formation as a relevant mechanism for the local temperature rise. Gene expression of the main pathogen in CF, P. aeruginosa, under anaerobic conditions at 38°C vs 30°C revealed increased virulence traits and characteristic cell wall changes. CONCLUSION: Neutrophil elastase mediates increase in airway temperature, which may contribute to P. aeruginosa selection during the course of chronic infection in CF.

Ciliated cultures from patients with primary ciliary dyskinesia do not produce nitric oxide or inducible nitric oxide synthase during early infection.

BACKGROUND: The mechanism behind why patients with primary ciliary dyskinesia (PCD) exhibit low nasal and exhaled nitric oxide (NO) remains unknown. One hypothesis is that reduced NO biosynthesis is caused by a defect in one or more NO synthases (NOSs). In healthy cells, the biosynthesis of NO is increased following exposure to respiratory pathogens. Here, we aimed to investigate whether ciliated epithelial cells from patients with PCD increase NO production following pneumococcal infection. METHODS: Human respiratory epithelium was cultured to a basal or ciliated cell phenotype using submerged or air-liquid interface cultures, respectively. Cells were exposed to media or pneumococci until cells became damaged (< 4 h). Apical fluids were collected prior and following infection, and NO production was determined using chemiluminescence. NOS gene expression was determined using real-time quantitative polymerase chain reaction. RESULTS: Levels of NO and NOS2 gene expression increased significantly following infection of healthy ciliated epithelial cells but not basal cells. No increase in NO was seen in ciliated cell cultures from patients with PCD, and NOS2 gene expression remained unchanged from baseline. CONCLUSIONS: These results suggest that the biosynthesis of NO in ciliated cells from patients with PCD is abnormal following early bacterial challenge, suggesting an abnormality in the function of inducible NOS in PCD.

Muramyl dipeptide mediated activation of human bronchial epithelial cells interacting with basophils: a novel mechanism of airway inflammation.

Respiratory tract bacterial infection can amplify and sustain airway inflammation. Intracytosolic nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is one member of the nucleotide binding and oligomerization domain (NOD)-like receptor (NLR) family, which senses the conserved structural peptidoglycan component muramyl dipeptide (MDP) in almost all bacteria. In the present study, activation of the NOD2 ligand MDP on primary human bronchial epithelial cells (HBE) co-cultured with human basophils was investigated. Cytokines, NOD2, adhesion molecules and intracellular signalling molecules were assayed by enzyme-linked immunosorbent assay or flow cytometry. The protein expression of NOD2 was confirmed in basophils/KU812 cells and HBE/human bronchial epithelial cell line (BEAS-2B) cells. MDP was found to up-regulate significantly the cell surface expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 on basophils and HBE in the co-culture system with or without basophil priming by interleukin (IL)-33 (all P < 0·05). MDP could further enhance the release of inflammatory cytokine IL-6 and chemokine CXCL8, and epithelium-derived anti-microbial peptide ß-defensin 2 in the co-culture. HBE cells were the major source for the release of IL-6, CXCL8 and ß-defensin2 upon stimulation by MDP in the co-culture system. The expression of ICAM-1 and VCAM-1 and release of IL-6 and CXCL8 were suppressed by various signalling molecule inhibitors, implying that the interaction between basophils and primary human bronchial epithelial cells could be regulated differentially by the mitogen-activated protein kinase pathways and nuclear transcription factors. The results therefore provide a new insight into the functional role of basophils in innate immunity, and the link between respiratory bacteria-mediated innate immunity and subsequent amplification of allergic inflammation in the airway.

Varespladib inhibits secretory phospholipase A2 in bronchoalveolar lavage of different types of neonatal lung injury.

Secretory phospholipase A2 (sPLA2), which links surfactant catabolism and lung inflammation, is associated with lung stiffness, surfactant dysfunction, and degree of respiratory support in acute respiratory distress syndrome and in some forms of neonatal lung injury. Varespladib potently inhibits sPLA2 in animal models. The authors investigate varespladib ex vivo efficacy in different forms of neonatal lung injury. Bronchoalveolar lavage fluid was obtained from 40 neonates affected by hyaline membrane disease, infections, or meconium aspiration and divided in 4 aliquots added with increasing varespladib or saline. sPLA2 activity, proteins, and albumin were measured. Dilution was corrected with the urea ratio. Varespladib was also tested in vitro against pancreatic sPLA2 mixed with different albumin concentration. Varespladib was able to inhibit sPLA2 in the types of neonatal lung injury investigated. sPLA2 activity was reduced in hyaline membrane disease (P < .0001), infections (P = .003), and meconium aspiration (P = .04) using 40 µM varespladib; 10 µM was able to lower enzyme activity (P = .001), with an IC(50) of 87 µM. An inverse relationship existed between protein level and activity reduction (r = 0.5; P = .029). The activity reduction/protein ratio tended to be higher in hyaline membrane disease. Varespladib efficacy was higher in vitro than in lavage fluids obtained from neonates (P < .001).

CFTR negatively regulates cyclooxygenase-2-PGE(2) positive feedback loop in inflammation.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent anion channel mostly expressed in epithelia. Accumulating evidence suggests that CF airway epithelia are overwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over-inflammatory condition observed in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increased cyclooxygenase-2 (COX-2) expression and over-production of prostaglandin E(2) (PGE(2)) in human CF bronchial epithelia cell line (CFBE41o--) with elevated NF-κB activity compared to a wild-type airway epithelial cell line (16HBE14o--). Moreover, we demonstrated that CFTR knockout mice had inherently higher levels of COX-2 and NF-κB activity, supporting the notion that lack of CFTR results in hyper-inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE(2) involving PKA and transcription factor, CREB. More importantly, overexpression of wild-type CFTR significantly suppressed COX-2 expression in CFBE41o- cells, and wild-type CFTR protein expression was significantly increased when 16HBE14o-- cells were challenged with LPS as well as PGE(2), indicating possible involvement of CFTR in negative regulation of COX-2/PGE(2). In conclusion, CFTR is a negative regulator of PGE(2)-mediated inflammatory response, defect of which may result in excessive activation of NF-κB, leading to over production of PGE(2) as seen in inflammatory CF tissues.

Identifying peroxidases and their oxidants in the early pathology of cystic fibrosis.

We aimed to determine whether myeloperoxidase (MPO) is the main peroxidase present in the airways of children with cystic fibrosis (CF) and to assess which oxidants it produces and whether they are associated with clinical features of CF. Children with CF (n=54) and without CF (n=16) underwent bronchoscopy and bronchoalveolar lavage (BAL) for assessment of pulmonary infection and inflammation. BAL fluid was analyzed for MPO, halogenated tyrosines as markers of hypohalous acids, thiocyanate, and protein carbonyls. MPO was the only peroxidase detected in BAL samples from children with CF and its concentration was markedly higher than in controls. Levels of 3-chlorotyrosine and 3-bromotyrosine in proteins were higher in the CF group. They correlated with neutrophils and MPO. The concentration of thiocyanate in BAL samples was below 1µM. Protein carbonyl levels correlated with MPO and halogenated tyrosines in patients with CF. Levels of MPO and halogenated tyrosines were higher in children with infections, especially Pseudomonas aeruginosa, and in the presence of respiratory symptoms. They also correlated with the Kanga clinical score. Our findings suggest that MPO produces hypobromous acid as well as hypochlorous acid in the airways of children with CF and that these oxidants are involved in the early pathogenesis of CF.

Acid sphingomyelinase inhibitors normalize pulmonary ceramide and inflammation in cystic fibrosis.

Employing genetic mouse models we have recently shown that ceramide accumulation is critically involved in the pathogenesis of cystic fibrosis (CF) lung disease. Genetic or systemic inhibition of the acid sphingomyelinase (Asm) is not feasible for treatment of patients or might cause adverse effects. Thus, a manipulation of ceramide specifically in lungs of CF mice must be developed. We tested whether inhalation of different acid sphingomyelinase inhibitors does reduce Asm activity and ceramide accumulation in lungs of CF mice. The efficacy and specificity of the drugs was determined. Ceramide was determined by mass spectrometry, DAG-kinase assays, and fluorescence microscopy. We determined pulmonary and systemic Asm activity, neutral sphingomyelinase (Nsm), ceramide, cytokines, and infection susceptibility. Mass spectroscopy, DAG-kinase assays, and semiquantitative immune fluorescence microscopy revealed that a standard diet did not influence ceramide in bronchial respiratory epithelial cells, while a diet with Peptamen severely affected the concentration of sphingolipids in CF lungs. Inhalation of the Asm inhibitors amitriptyline, trimipramine, desipramine, chlorprothixene, fluoxetine, amlodipine, or sertraline restored normal ceramide concentrations in murine bronchial epithelial cells, reduced inflammation in the lung of CF mice and prevented infection with Pseudomonas aeruginosa. All drugs showed very similar efficacy. Inhalation of the drugs was without systemic effects and did not inhibit Nsm. These findings employing several structurally different Asm inhibitors identify Asm as primary target in the lung to reduce ceramide concentrations. Inhaling an Asm inhibitor may be a beneficial treatment for CF, with minimal adverse systemic effects.

Diagnostic utility of adenosine deaminase (ADA) activity in pleural fluid and serum of tuberculous and non-tuberculous respiratory disease patients.

Adenosine deaminase activity (ADA) was assayed in pleural fluid and serum of 42 subjects with pleural effusion. Twenty-nine of them had TB pleural effusion and the remaining 13 had pleural effusion due to non-TB respiratory diseases. Serum adenosine deaminase activity were also measured in 32 pulmonary tuberculosis patients without pleural effusion and equal numbers of healthy controls without systemic diseases for comparative analysis. The patients attending the medicine out-patient department (MOPD) of the B. P. Koirala Institute of Health Sciences, Dharan, Nepal were taken as study subjects. Serum and pleural fluid ADA activities were assayed spectrophotometrically by the method of Guisti and Gallanti. The mean serum ADA activity was significantly increased in patients with tubercular pleural effusion (34.53 +/- 10.27 IU/l) compared to pulmonary tuberculosis patients without pleural effusion (26.54 +/- 4.76 IU/l), (p = 0.004), those with non-TB respiratory disease (16.71 +/- 5.16 IU/l), (p = 0.0001) and healthy controls (15.53 +/- 4.4 IU/l) (p = 0.0001). The mean ADA in the pleural fluid of tubercular pleural effusion patients (90.29 +/- 54.80 IU/l) was significantly higher compared to those with non-TB respiratory disease (24.43 +/- 9.28 IU/l) (p = 0.0001). Using the lowest cutoff value for enzyme activity in the serum of patients with TB pleural effusion (25 IU/l), a test sensitivity of 72.41% and specificity of 81.53% were obtained. Using the lowest cutoff value for enzyme activity in pleural fluid of patients with TB pleural effusion (45 IU/l) the sensitivity and specificity for diagnosis were 76.10% and 100%, respectively. Therefore, the measurement of ADA in tubercular pleural effusion has a utility in the diagnosis of tuberculosis when other clinical and laboratory tests are negative.

Urease produced by Coccidioides posadasii contributes to the virulence of this respiratory pathogen.

Urease activity during in vitro growth in the saprobic and parasitic phases of Coccidioides spp. is partly responsible for production of intracellular ammonia released into the culture media and contributes to alkalinity of the external microenvironment. Although the amino acid sequence of the urease of Coccidioides posadasii lacks a predicted signal peptide, the protein is transported from the cytosol into vesicles and the central vacuole of parasitic cells (spherules). Enzymatically active urease is released from the contents of mature spherules during the parasitic cycle endosporulation stage. The endospores, together with the urease and additional material which escape from the ruptured parasitic cells, elicit an intense host inflammatory response. Ammonia production by the spherules of C. posadasii is markedly increased by the availability of exogenous urea found in relatively high concentrations at sites of coccidioidal infection in the lungs of mice. Direct measurement of the pH at these infection sites revealed an alkaline microenvironment. Disruption of the urease gene of C. posadasii resulted in a marked reduction in the amount of ammonia secreted in vitro by the fungal cells. BALB/c mice challenged intranasally with the mutant strain showed increased survival, a well-organized granulomatous response to infection, and better clearance of the pathogen than animals challenged with either the parental or the reconstituted (revertant) strain. We conclude that ammonia and enzymatically active urease released from spherules during the parasitic cycle of C. posadasii contribute to host tissue damage, which exacerbates the severity of coccidioidal infection and enhances the virulence of this human respiratory pathogen.

Transaminase levels in ventilated children with respiratory syncytial virus bronchiolitis.

OBJECTIVES: To compare disease severity as judged by duration of ventilation, inotrope use and mortality in children ventilated for respiratory syncytial virus (RSV)-positive lower respiratory tract infection (LRTI) with and without elevated transaminase levels and to determine the aetiology of elevated transaminase levels in this patient group. DESIGN: Prospective observational study. SETTING: Twenty-two-bed Paediatric Intensive Care Unit. PATIENTS: Forty-eight ventilated children with RSV-positive LRTI. MEASUREMENTS AND RESULTS: Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured daily. In patients with elevated transaminase levels infection with the following viruses was investigated: hepatitis A, B and C viruses, cytomegalovirus, Epstein Barr virus, adenovirus, influenza virus, and parainfluenza viruses (types I, II, and III). Elevated transaminase levels were detected in 22 (46%) patients. The duration of mechanical ventilation (geometric mean; 95% CI) was significantly ( P<0.05) longer in the group with elevated transaminase levels: 10.6 (9.4; 11.7) days versus 3.5 (2.8; 4.2) days. This difference remained significant in patients without congenital heart disease. Inotrope use was more common and all deaths occurred in the group with elevated transaminase levels ( P<0.05). All patients who died and all but two patients with inotrope requirements had underlying congenital heart disease. One patient with elevated transaminase levels had a simultaneous infection with influenza A virus. CONCLUSIONS: RSV disease in ventilated children was more severe if transaminase levels were elevated. Transaminase level elevation was due to hepatitis in the majority of patients. In patients with congenital heart disease we also detected myocardial involvement.

Interleukin-1beta, interleukin-6, tumour necrosis factor-alpha and interferon-gamma released by a viral infection and an aseptic inflammation reduce CYP1A1, 1A2 and 3A6 expression in rabbit hepatocytes.

Inflammation reduces activity and expression of hepatic cytochrome P450 (P450) and therefore diminishes drug biotransformation. This study aimed to identify the serum mediators triggered by a viral infection and an aseptic inflammation that downregulate P450 isoforms. Incubation of hepatocytes with serum from rabbits with a turpentine-induced inflammation or humans with a viral infection decreased the amount of cytochrome 1A1 (CYP1A1), 1A2 and 3A6 mRNA and apoproteins. By serum fractionation and immuno-neutralization, we showed that in the aseptic inflammation, interleukin-6 and, to a lesser degree, interleukin-1beta are involved in the downregulation of all three isoforms. In serum from humans with a viral infection, interleukin-1beta, interleukin-6, interferon-gamma and tumour necrosis factor-alpha contribute to the downregulation of P450 isoforms. CYP1A1 and 1A2 are regulated by serum mediators at the transcriptional level, while the expression of CYP3A6 appears to be under the control of pre- and posttranscriptional mechanisms.

Colistin stimulates the activity of neutrophil elastase and Pseudomonas aeruginosa elastase.

Nonantimicrobial effects of antibiotics may contribute to their activity in the treatment of infective airway disease. The aim of this study was to identify antibiotics used for the treatment of infection in cystic fibrosis that may alter the activity of human neutrophil elastase (HNE) and Pseudomonas aeruginosa elastase (PE). The effect of antibiotics on the activity of purified HNE and PE, and HNE in sputum was assessed using colourimetric and fluorescent substrate assays by kinetic measurements, and by examining the interaction of HNE with inhibitors. Ceftazidime, tobramycin, and gentamycin slightly inhibited purified HNE activity whereas erythromycin and colistin significantly stimulated purified HNE and PE (395 and 557%, respectively). However, only colistin increased HNE activity in sputum (+102%) and was therefore studied in more detail. This increase in activity was not due an interference with the specific inhibition of HNE by alpha1-antitrypsin but colistin was found to reverse the inhibitory effects of small molecular weight molecules like heparin. Colistin increases the activity of human neutrophil elastase and Pseudomonas aeruginosa elastase, two proteases that contribute to the pathogenesis of cystic fibrosis airway disease.

Elevated levels of myeloperoxidase, pro-inflammatory cytokines and chemokines in naturally acquired upper respiratory tract infections.

Upper respiratory tract infections (URTIs) are characterised by a neutrophilic mucosal infiltration. The purpose of this study was to investigate the time course of release of the cytokines/chemokines interleukins (IL) IL-1beta, IL-1ra, tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-8, interferon-gamma (IFN-gamma) and monocyte chemotactic protein (MCP-1), soluble intercellular adhesion molecule-1 (sICAM-1), myeloperoxidase (MPO) and bradykinin in nasal secretions of patients with a naturally acquired URTI. A total of 117 healthy adult volunteers were recruited for baseline nasal lavages, 39 of whom developed URTI symptoms within 6 months and returned to our centre within 48 h. Lavages were performed daily during the symptomatic period and 3 weeks thereafter, with symptoms no longer present. Compared to baseline, significantly elevated concentrations of total protein, bradykinin, IL-1beta, TNF-alpha, IL-6, IL-8, MCP-1, IFN-gamma, MPO and sICAM-1 were detected in nasal lavage fluids of symptomatic patients, whereas IL-1ra remained unaltered. All studied variables reached baseline 3 weeks after the URTI. Naturally acquired URTI represent a limited, neutrophilic inflammatory reaction, orchestrated by the release of pro-inflammatory cytokines and chemokines.