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

Skills training in minimally invasive surgery in Dutch obstetrics and gynecology residency curriculum

The complexity of acquiring minimally invasive surgical (MIS) skills, combined with smaller case volumes for residents have pushed the development of skills training facilities on simulators outside the operating room (OR). Medico-legal and financial constraints have stimulated this development even more. However, the implementation of simulator training into a residency curriculum is shown to be troublesome. MIS skills training is organized in a uniform and easily applicable way in the Dutch obstetrics and gynecology residency curriculum. Every resident is obliged to attend the same basic surgical skills course, named Cobra-alpha course, intentionally during postgraduate year (PGY) 1 or 2. Furthermore, surgical skills are trained, evaluated and expanded on simulators in teaching hospitals. Additional to the Cobra-alpha course, residents may attend advanced training courses and congresses focusing on laparoscopy and hysteroscopy. This organization guarantees a uniform introduction to MIS skills training for every resident. However, preconditions for continuous training and evaluation after this introduction have to be optimized

Retention of basic laparoscopic skills after a structured training program

The purpose of this study was to test the retention of basic laparoscopic skills on a box trainer 1 year after a short training program. For a prior study, eight medical students without prior experience (novices) underwent baseline testing, followed by five weekly training sessions and a final test. During each of seven sessions, they performed five tasks on an inanimate box trainer. Scores were calculated by adding up the time to completion of the task with penalty points, consequently rewarding speed and precision. The sum score was the sum of the five scores. One year later, seven of them underwent retention testing for the current study. The final test results were compared with retention test results as a measure of durability of acquired skills. Novices’ scores did not worsen significantly for four out of five tasks (i.e., placing a pipe cleaner p = 0.46, placing beads p = 0.24, cutting a circle p = 0.31, and knot tying p = 0.13). However, deterioration was observed in the performance on stretching a rubber band (p < 0.05), as well as in the sum score (p < 0.05). Nevertheless, all retention scores remained better than the baseline results. In conclusion, basic laparoscopic skills acquired during a short training program merely sustain over time. However, ongoing practice is advisable, especially to preserve tissue-handling skills, since these may be the first to deteriorate

Neutrophil elastase reduces secretion of secretory leukoproteinase inhibitor (SLPI) by lung epithelial cells: role of charge of the proteinase-inhibitor complex

<p>Abstract</p> <p>Background</p> <p>Secretory leukoproteinase inhibitor (SLPI) is an important inhibitor of neutrophil elastase (NE), a proteinase implicated in the pathogenesis of lung diseases such as COPD. SLPI also has antimicrobial and anti-inflammatory properties, but the concentration of SLPI in lung secretions in COPD varies inversely with infection and the concentration of NE. A fall in SLPI concentration is also seen in culture supernatants of respiratory cells exposed to NE, for unknown reasons. We investigated the hypothesis that SLPI complexed with NE associates with cell membranes <it>in vitro</it>.</p> <p>Methods</p> <p>Respiratory epithelial cells were cultured in the presence of SLPI, varying doses of proteinases over time, and in different experimental conditions. The likely predicted charge of the complex between SLPI and proteinases was assessed by theoretical molecular modelling.</p> <p>Results</p> <p>We observed a rapid, linear decrease in SLPI concentration in culture supernatants with increasing concentration of NE and cathepsin G, but not with other serine proteinases. The effect of NE was inhibited fully by a synthetic NE inhibitor only when added at the same time as NE. Direct contact between NE and SLPI was required for a fall in SLPI concentration. Passive binding to cell culture plate materials was able to remove a substantial amount of SLPI both with and without NE. Theoretical molecular modelling of the structure of SLPI in complex with various proteinases showed a greater positive charge for the complex with NE and cathepsin G than for other proteinases, such as trypsin and mast cell tryptase, that also bind SLPI but without reducing its concentration.</p> <p>Conclusion</p> <p>These data suggest that NE-mediated decrease in SLPI is a passive, charge-dependent phenomenon <it>in vitro</it>, which may correlate with changes observed <it>in vivo</it>.</p

HSPVdb—the Human Short Peptide Variation Database for improved mass spectrometry-based detection of polymorphic HLA-ligands

T cell epitopes derived from polymorphic proteins or from proteins encoded by alternative reading frames (ARFs) play an important role in (tumor) immunology. Identification of these peptides is successfully performed with mass spectrometry. In a mass spectrometry-based approach, the recorded tandem mass spectra are matched against hypothetical spectra generated from known protein sequence databases. Commonly used protein databases contain a minimal level of redundancy, and thus, are not suitable data sources for searching polymorphic T cell epitopes, either in normal or ARFs. At the same time, however, these databases contain much non-polymorphic sequence information, thereby complicating the matching of recorded and theoretical spectra, and increasing the potential for finding false positives. Therefore, we created a database with peptides from ARFs and peptide variation arising from single nucleotide polymorphisms (SNPs). It is based on the human mRNA sequences from the well-annotated reference sequence (RefSeq) database and associated variation information derived from the Single Nucleotide Polymorphism Database (dbSNP). In this process, we removed all non-polymorphic information. Investigation of the frequency of SNPs in the dbSNP revealed that many SNPs are non-polymorphic “SNPs”. Therefore, we removed those from our dedicated database, and this resulted in a comprehensive high quality database, which we coined the Human Short Peptide Variation Database (HSPVdb). The value of our HSPVdb is shown by identification of the majority of published polymorphic SNP- and/or ARF-derived epitopes from a mass spectrometry-based proteomics workflow, and by a large variety of polymorphic peptides identified as potential T cell epitopes in the HLA-ligandome presented by the Epstein–Barr virus cells

Interventions for renal vasculitis in adults. A systematic review

<p>Abstract</p> <p>Background</p> <p>Renal vasculitis presents as rapidly progressive glomerulonephritis and comprises of a group of conditions characterised by acute kidney failure, haematuria and proteinuria. Treatment of these conditions involves the use of steroid and non-steroid agents with or without adjunctive plasma exchange. Although immunosuppression has been successful, many questions remain unanswered in terms of dose and duration of therapy, the use of plasma exchange and the role of new therapies. This systematic review was conducted to determine the benefits and harms of any intervention for the treatment of renal vasculitis in adults.</p> <p>Methods</p> <p>We searched the Cochrane Central Register of Controlled Trials, the Cochrane Renal Group Specialised Register, MEDLINE and EMBASE to June 2009. Randomised controlled trials investigating any intervention for the treatment of adults were included. Two authors independently assessed study quality and extracted data. Statistical analyses were performed using a random effects model and results expressed as risk ratio with 95% confidence intervals for dichotomous outcomes or mean difference for continuous outcomes.</p> <p>Results</p> <p>Twenty two studies (1674 patients) were included. Plasma exchange as adjunctive therapy significantly reduces the risk of end-stage kidney disease at 12 months (five studies: RR 0.47, CI 0.30 to 0.75). Four studies compared the use of pulse and continuous administration of cyclophosphamide. Remission rates were equivalent but pulse treatment causes an increased risk of relapse (4 studies: RR 1.79, CI 1.11 to 2.87) compared with continuous cyclophosphamide. Azathioprine has equivalent efficacy as a maintenance agent to cyclophosphamide with fewer episodes of leukopenia. Mycophenolate mofetil may be equivalent to cyclophosphamide as an induction agent but resulted in a higher relapse rate when tested against Azathioprine in remission maintenance. Rituximab is an effective remission induction agent. Methotrexate or Leflunomide are potential choices in remission maintenance therapy. Oral co-trimoxazole did not reduce relapses significantly in Wegener's granulomatosis.</p> <p>Conclusions</p> <p>Plasma exchange is effective in patients with severe ARF secondary to vasculitis. Pulse cyclophosphamide results in an increased risk of relapse when compared to continuous oral use but a reduced total dose. Whilst cyclophosphamide is standard induction treatment, rituximab and mycophenolate mofetil are also effective. Azathioprine, methotrexate and leflunomide are effective as maintenance therapy. Further studies are required to more clearly delineate the appropriate place of newer agents within an evidence-based therapeutic strategy.</p

Mycophenolate mofetil versus cyclophosphamide for remission induction in ANCA-associated vasculitis: a randomised, non-inferiority trial

OBJECTIVES: Cyclophosphamide induction regimens are effective for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), but are associated with infections, malignancies and infertility. Mycophenolate mofetil (MMF) has shown high remission rates in small studies of AAV. METHODS: We conducted a randomised controlled trial to investigate whether MMF was non-inferior to cyclophosphamide for remission induction in AAV. 140 newly diagnosed patients were randomly assigned to MMF or pulsed cyclophosphamide. All patients received the same oral glucocorticoid regimen and were switched to azathioprine following remission. The primary endpoint was remission by 6 months requiring compliance with the tapering glucocorticoid regimen. Patients with an eGFR <15 mL/min were excluded from the study. RESULTS: At baseline, ANCA subtype, disease activity and organ involvement were similar between groups. Non-inferiority was demonstrated for the primary remission endpoint, which occurred in 47 patients (67%) in the MMF group and 43 patients (61%) in the cyclophosphamide group (risk difference 5.7%, 90% CI -7.5% to 19%). Following remission, more relapses occurred in the MMF group (23 patients, 33%) compared with the cyclophosphamide group (13 patients, 19%) (incidence rate ratio 1.97, 95% CI 0.96 to 4.23, p=0.049). In MPO-ANCA patients, relapses occurred in 12% of the cyclophosphamide group and 15% of the MMF group. In PR3-ANCA patients, relapses occurred in 24% of the cyclophosphamide group and 48% of the MMF group. Serious infections were similar between groups (26% MMF group, 17% cyclophosphamide group) (OR 1.67, 95% CI 0.68 to 4.19, p=0.3). CONCLUSION: MMF was non-inferior to cyclophosphamide for remission induction in AAV, but resulted in higher relapse rate. TRIAL REGISTRATION NUMBER: NCT00414128

Severe congenital neutropenia in a multigenerational family with a novel neutrophil elastase (ELANE) mutation

We have analysed a family with nine congenital neutropenia patients in four generations, several of which we have studied in a long-term follow-up of over 25 years. The patients were mild to severe neutropenic and suffered from various recurrent bacterial infections. Mutations in the genes ELANE, CSF3R and GFI1 have been reported in patients with autosomal dominant congenital neutropenias. Using a small-scale linkage analysis with markers around the ELANE, CSF3R, CSF3 and GFI1 genes, we were able to determine that the disease segregated with markers around the ELANE gene. We identified a novel mutation in the ELANE gene in all of the affected family members that was not present in any of the healthy family members. The mutation leads to an A28S missense mutation in the mature protein. None of these patients developed leukaemia. This is the first truly multigenerational family with mutations in ELANE as unambiguous cause of severe congenital neutropenia SCN

Mycobacterium tuberculosis peptides presented by HLA-E molecules are targets for human CD8 T-cells with cytotoxic as well as regulatory activity

Tuberculosis (TB) is an escalating global health problem and improved vaccines against TB are urgently needed. HLA-E restricted responses may be of interest for vaccine development since HLA-E displays very limited polymorphism (only 2 coding variants exist), and is not down-regulated by HIV-infection. The peptides from Mycobacterium tuberculosis (Mtb) potentially presented by HLA-E molecules, however, are unknown. Here we describe human T-cell responses to Mtb-derived peptides containing predicted HLA-E binding motifs and binding-affinity for HLA-E. We observed CD8(+) T-cell proliferation to the majority of the 69 peptides tested in Mtb responsive adults as well as in BCG-vaccinated infants. CD8(+) T-cells were cytotoxic against target-cells transfected with HLA-E only in the presence of specific peptide. These T cells were also able to lyse M. bovis BCG infected, but not control monocytes, suggesting recognition of antigens during mycobacterial infection. In addition, peptide induced CD8(+) T-cells also displayed regulatory activity, since they inhibited T-cell proliferation. This regulatory activity was cell contact-dependent, and at least partly dependent on membrane-bound TGF-beta. Our results significantly increase our understanding of the human immune response to Mtb by identification of CD8(+) T-cell responses to novel HLA-E binding peptides of Mtb, which have cytotoxic as well as immunoregulatory activity

Corporate reputation in the spanish context: An interaction between reporting to stakeholders and industry.

ABSTRACT: The authors describe the intensity and orientation of the corporate social responsibility (CSR) reporting in four Spanish industries and explore the relationship that exists between both concepts and an independent measurement of reputation for CSR (CSRR). The results demonstrate that the CSR reporting is especially relevant and useful in the finance industry. Finance companies report significantly more CSR information than most industries in Spain, and this reporting is more closely linked to their CSRR than the CSR reporting of basic, consumer goods and services industries. Borra