Nontypeable Haemophilus influenzae induces COX-2 and PGE2 expression in lung epithelial cells via activation of p38 MAPK and NF-kappa B

<p>Abstract</p> <p>Background</p> <p>Nontypeable <it>Haemophilus influenzae </it>(NTHi) is an important respiratory pathogen implicated as an infectious trigger in chronic obstructive pulmonary disease, but its molecular interaction with human lung epithelial cells remains unclear. Herein, we tested that the hypothesis that NTHi induces the expression of cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) via activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappa B in pulmonary alveolar epithelial cells.</p> <p>Methods</p> <p>Human alveolar epithelial A549 cells were infected with different concentrations of NTHi. The phosphorylation of p38 MAPK was detected by Western blot analysis, the DNA binding activity of NF-kappa B was assessed by electrophoretic mobility shift assay (EMSA), and the expressions of COX-1 and 2 mRNA and PGE2 protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The roles of Toll-like receptor (TLR) 2 and TLR4, well known NTHi recognizing receptor in lung epithelial cell and gram-negative bacteria receptor, respectively, on the NTHi-induced COX-2 expression were investigated in the HEK293 cells overexpressing TLR2 and TLR4 <it>in vitro </it>and in the mouse model of NTHi-induced pneumonia by using TLR2 and TLR4 knock-out mice <it>in vivo</it>. In addition, the role of p38 MAPK and NF-kappa B on the NTHi-induced COX-2 and PGE2 expression was investigated by using their specific chemical inhibitors.</p> <p>Results</p> <p>NTHi induced COX-2 mRNA expression in a dose-dependent manner, but not COX-1 mRNA expression in A549 cells. The enhanced expression of PGE2 by NTHi infection was significantly decreased by pre-treatment of COX-2 specific inhibitor, but not by COX-1 inhibitor. NTHi induced COX-2 expression was mediated by TLR2 in the epithelial cell <it>in vitro </it>and in the lungs of mice <it>in vivo</it>. NTHi induced phosphorylation of p38 MAPK and up-regulated DNA binding activity of NF-kappa B. Moreover, the expressions of COX-2 and PGE2 were significantly inhibited by specific inhibitors of p38 MAPK and NF-kappa B. However, NTHi-induced DNA binding activity of NF-kappa B was not affected by the inhibition of p38 MAPK.</p> <p>Conclusion</p> <p>NTHi induces COX-2 and PGE2 expression in a p38 MAPK and NF-kappa B-dependent manner through TLR2 in lung epithelial cells <it>in vitro </it>and lung tissues <it>in vivo</it>. The full understanding of the role of endogenous anti-inflammatory PGE2 and its regulation will bring new insight to the resolution of inflammation in pulmonary bacterial infections.</p

Stress degradation studies and development of stability-indicating TLC-densitometry method for determination of prednisolone acetate and chloramphenicol in their individual and combined pharmaceutical formulations

A rapid and reproducible stability indicating TLC method was developed for the determination of prednisolone acetate and chloramphenicol in presence of their degraded products. Uniform degradation conditions were maintained by refluxing sixteen reaction mixtures for two hours at 80°C using parallel synthesizer including acidic, alkaline and neutral hydrolysis, oxidation and wet heating degradation. Oxidation at room temperature, photochemical and dry heating degradation studies were also carried out. Separation was done on TLC glass plates, pre-coated with silica gel 60F-254 using chloroform: methanol (14:1 v/v). Spots at Rf 0.21 ± 0.02 and Rf 0.41 ± 0.03 were recognized as chloramphenicol and prednisolone acetate, respectively. Quantitative analysis was done through densitometric measurements at multiwavelength (243 nm, λmax of prednisolone acetate and 278 nm, λmax of chloramphenicol), simultaneously. The developed method was optimized and validated as per ICH guidelines. Method was found linear over the concentration range of 200-6000 ng/spot with the correlation coefficient (r2 ± S.D.) of 0.9976 ± 3.5 and 0.9920 ± 2.5 for prednisolone acetate and chloramphenicol, respectively. The developed TLC method can be applied for routine analysis of prednisolone acetate and chloramphenicol in presence of their degraded products in their individual and combined pharmaceutical formulations

Whole breast and regional nodal irradiation in prone versus supine position in left sided breast cancer

Background: Prone whole breast irradiation (WBI) leads to reduced heart and lung doses in breast cancer patients receiving adjuvant radiotherapy. In this feasibility trial, we investigated the prone position for whole breast + lymph node irradiation (WB + LNI). Methods: A new support device was developed for optimal target coverage, on which patients are positioned in a position resembling a phase from the crawl swimming technique (prone crawl position). Five left sided breast cancer patients were included and simulated in supine and prone position. For each patient, a treatment plan was made in prone and supine position for WB + LNI to the whole axilla and the unoperated part of the axilla. Patients served as their own controls for comparing dosimetry of target volumes and organs at risk (OAR) in prone versus in supine position. Results: Target volume coverage differed only slightly between prone and supine position. Doses were significantly reduced (P < 0.05) in prone position for ipsilateral lung (Dmean, D2, V5, V10, V20, V30), contralateral lung (Dmean, D2), contralateral breast (Dmean, D2 and for total axillary WB + LNI also V5), thyroid (Dmean, D2, V5, V10, V20, V30), oesophagus (Dmean and for partial axillary WB + LNI also D2 and V5), skin (D2 and for partial axillary WB + LNI V105 and V107). There were no significant differences for heart and humeral head doses. Conclusions: Prone crawl position in WB + LNI allows for good breast and nodal target coverage with better sparing of ipsilateral lung, thyroid, contralateral breast, contralateral lung and oesophagus when compared to supine position. There is no difference in heart and humeral head doses

Disordered microbial communities in asthmatic airways.

A rich microbial environment in infancy protects against asthma [1], [2] and infections precipitate asthma exacerbations [3]. We compared the airway microbiota at three levels in adult patients with asthma, the related condition of COPD, and controls. We also studied bronchial lavage from asthmatic children and controls.We identified 5,054 16S rRNA bacterial sequences from 43 subjects, detecting >70% of species present. The bronchial tree was not sterile, and contained a mean of 2,000 bacterial genomes per cm(2) surface sampled. Pathogenic Proteobacteria, particularly Haemophilus spp., were much more frequent in bronchi of adult asthmatics or patients with COPD than controls. We found similar highly significant increases in Proteobacteria in asthmatic children. Conversely, Bacteroidetes, particularly Prevotella spp., were more frequent in controls than adult or child asthmatics or COPD patients.The results show the bronchial tree to contain a characteristic microbiota, and suggest that this microbiota is disturbed in asthmatic airways

Genetic Signatures in the Envelope Glycoproteins of HIV-1 that Associate with Broadly Neutralizing Antibodies

A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for this region in the elicitation of broadly neutralizing antibody responses against HIV-1

Local Inflammation Induces Complement Crosstalk Which Amplifies the Antimicrobial Response

By eliciting inflammatory responses, the human immunosurveillance system notably combats invading pathogens, during which acute phase proteins (CRP and cytokines) are elevated markedly. However, the Pseudomonas aeruginosa is a persistent opportunistic pathogen prevalent at the site of local inflammation, and its acquisition of multiple antibiotic-resistance factors poses grave challenges to patient healthcare management. Using blood samples from infected patients, we demonstrate that P. aeruginosa is effectively killed in the plasma under defined local infection-inflammation condition, where slight acidosis and reduced calcium levels (pH 6.5, 2 mM calcium) typically prevail. We showed that this powerful antimicrobial activity is provoked by crosstalk between two plasma proteins; CRP∶L-ficolin interaction led to communication between the complement classical and lectin pathways from which two amplification events emerged. Assays for C4 deposition, phagocytosis, and protein competition consistently proved the functional significance of the amplification pathways in boosting complement-mediated antimicrobial activity. The infection-inflammation condition induced a 100-fold increase in CRP∶L-ficolin interaction in a pH- and calcium-sensitive manner. We conclude that the infection-induced local inflammatory conditions trigger a strong interaction between CRP∶L-ficolin, eliciting complement-amplification pathways which are autonomous and which co-exist with and reinforce the classical and lectin pathways. Our findings provide new insights into the host immune response to P. aeruginosa infection under pathological conditions and the potential development of new therapeutic strategies against bacterial infection

Multiple Changes in Peptide and Lipid Expression Associated with Regeneration in the Nervous System of the Medicinal Leech

peer reviewe

ICAR: endoscopic skull‐base surgery

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