CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens.

BACKGROUND: Antimicrobial peptides are important components of the host defence with a broad range of functions including direct antimicrobial activity and modulation of inflammation. Lack of cathelin-related antimicrobial peptide (CRAMP) was associated with higher mortality and bacterial burden and impaired neutrophil granulocyte infiltration in a model of pneumococcal meningitis. The present study was designed to characterize the effects of CRAMP deficiency on glial response and phagocytosis after exposure to bacterial stimuli. METHODS: CRAMP-knock out and wildtype glial cells were exposed to bacterial supernatants from Streptococcus pneumoniae and Neisseria meningitides or the bacterial cell wall components lipopolysaccharide and peptidoglycan. Cell viability, expression of pro- and anti-inflammatory mediators and activation of signal transduction pathways, phagocytosis rate and glial cell phenotype were investigated by means of cell viability assays, immunohistochemistry, real-time RT-PCR and Western blot. RESULTS: CRAMP-deficiency was associated with stronger expression of pro-inflammatory and weakened expression of anti-inflammatory cytokines indicating a higher degree of glial cell activation even under resting-state conditions. Furthermore, increased translocation of nuclear factor 'kappa-light-chain-enhancer' of activated B-cells was observed and phagocytosis of S. pneumoniae was reduced in CRAMP-deficient microglia indicating impaired antimicrobial activity. CONCLUSIONS: In conclusion, the present study detected severe alterations of the glial immune response due to lack of CRAMP. The results indicate the importance of CRAMP to maintain and regulate the delicate balance between beneficial and harmful immune response in the brain.

Lack of Proinflammatory Cytokine Interleukin-6 or Tumor Necrosis Factor Receptor-1 Results in a Failure of the Innate Immune Response after Bacterial Meningitis.

The most frequent pathogen that causes bacterial meningitis is the Gram-positive bacterium Streptococcus pneumoniae. By entering the brain, host cells will be activated and proinflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are released. The goal of the current study was to examine the interaction between IL-6 and TNFR1 as receptor for TNF-α and the innate immune response in vivo in a model of Streptococcus pneumoniae-induced meningitis. For the experiments IL-6(-/-), TNFR1(-/-), and TNFR1-IL-6(-/-) KO mice were used. Our results revealed higher mortality rates and bacterial burden after infection in TNFR1(-/-), IL-6(-/-), and TNFR1-IL-6(-/-) mice and a decreased immune response including lower neutrophil infiltration in the meninges of TNFR1(-/-) and TNFR1-IL-6(-/-) mice in contrast to IL-6(-/-) and wild type mice. Furthermore, the increased mortality of TNFR1(-/-) and TNFR1-IL-6(-/-) mice correlated with decreased glial cell activation compared to IL-6(-/-) or wild type mice after pneumococcal meningitis. Altogether, the results show the importance of TNFR1 and IL-6 in the regulation of the innate immune response. The lack of TNFR1 and IL-6 results in higher mortality by weakened immune defence, whereas the lack of TNFR1 results in more severe impairment of the innate immune response than the lack of IL-6 alone.

Patients' satisfaction with local and general anaesthesia for video-assisted thoracoscopic surgery-results of the first randomized controlled trial PASSAT.

OBJECTIVES: The objective of this single-centre, open, randomized control trial was to compare the patients' satisfaction with local anaesthesia (LA) or general anaesthesia (GA) for video-assisted thoracoscopy. METHODS: Patients with indication for video-assisted thoracoscopy pleural management, mediastinal biopsies or lung wedge resections were randomized for LA or GA. LA was administered along with no or mild sedation and no airway devices maintaining spontaneous breathing, and GA was administered along with double-lumen tube and one-lung ventilation. The primary end point was anaesthesia-related satisfaction according to psychometrically validated questionnaires. Patients not willing to be randomized could attend based on their desired anaesthesia, forming the preference arm. RESULTS: Fifty patients were allocated to LA and 57 patients to GA. Age, smoking habits and lung function were similarly distributed in both groups. There was no significant difference between the 2 groups with regard to patient satisfaction with anaesthesiology care (median 2.75 vs 2.75, P = 0.74), general perioperative care (2.50 vs 2.50, P = 0.57), recovery after surgery (2.00 vs 2.00, P = 0.16, 3-point Likert scales). Surgeons and anaesthesiologists alike were less satisfied with feasibility (P < 0.01 each) with patients in the LA group. Operation time, postoperative pain scales, delirium and complication rate were similar in both groups. LA patients had a significantly shorter stay in hospital (mean 3.9 vs 6.0 days, P < 0.01). Of 18 patients in the preference arm, 17 chose LA, resulting in similar satisfaction. CONCLUSIONS: Patients were equally satisfied with both types of anaesthesia, regardless of whether the type of anaesthesia was randomized or deliberately chosen. LA is as safe as GA but correlated with shorter length of stay. Almost all patients of the preference arm chose LA. Considering the benefits of LA, it should be offered to patients as an equivalent alternative to GA whenever medically appropriate and feasible.

Most patient conditions do not a priori debilitate the sensitivity of thoracic ultrasound in thoracic surgery-a prospective comparative study.

BACKGROUND: The few existing studies on the accuracy of lung ultrasound in the detection of a postoperative pneumothorax after thoracic surgery differ in the sonographic technique and the inclusion criteria. Several conditions are considered unfavourable in the sonographic examination of the lung. We aim to test these conditions for their impact on the diagnostic accuracy of lung ultrasound. METHODS: We compared lung ultrasound and chest roentgenograms for the detection of a pneumothorax after lung-resecting surgery in two prospective trials (register ID DRKS00014557 and DRKS00020216). The ultrasound examiners and radiologists were blinded towards the corresponding findings. We performed posthoc subgroup analyses to determine the influence of various patient or surgery related conditions on the sensitivity and specificity of ultrasound in the detection of pneumothorax. RESULTS: We performed 340 examinations in 208 patients. The covariates were age, gender, body mass index, smoking status, severity of chronic obstructive pulmonary disease, previous ipsilateral operation or irradiation, thoracotomy, postoperative skin emphysema, indwelling chest tube and X-ray in supine position. In univariate analysis, an indwelling chest-tube was associated with a higher sensitivity (58%, p = 0.04), and a postoperative subcutaneous emphysema with a lower specificity (73% vs. 88%, p = 0.02). None of the other subgroups differed in sensitivity or specificity from the total population . CONCLUSIONS: Most of the patient- or surgery related conditions usually considered unfavourable for lung ultrasound did not impair the sensitivity or specificity of lung ultrasound. Further studies should not excluce patients with these conditions, but test the accuracy under routine conditions. TRIAL REGISTRATION: DRKS, DRKS00014557, registered 06/09/2018, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00014557 and DRKS00020216, registered 03/12/2019, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00020216.

Role of the cathelicidin-related antimicrobial peptide in inflammation and mortality in a mouse model of bacterial meningitis.

Antimicrobial peptides (AP) are important components of the innate immune system, yet little is known about their expression and function in the brain. Our previous work revealed upregulated gene expression of cathelicidin-related AP (CRAMP) following bacterial meningitis in primary rat glial cells as well as bactericidal activity against frequent meningitis-causing bacteria. However, the effect of cathelicidin expression on the progression of inflammation and mortality in bacterial meningitis remains unknown. Therefore, we used CRAMP-deficient mice to investigate the effect of CRAMP on bacterial growth, inflammatory responses and mortality in meningitis. Meningitis was induced by intracerebral injection of type 3 Streptococcus pneumoniae. The degree of inflammation was analyzed in various brain regions by means of immunohistochemistry and real-time RT-PCR. CRAMP deficiency led to a higher mortality rate that was associated with increased bacterial titers in the cerebellum, blood and spleen as well as decreased meningeal neutrophil infiltration. CRAMP-deficient mice displayed a higher degree of glial cell activation that was accompanied by a more pronounced proinflammatory response. Taken together, this work provides insight into the important role of CRAMP as part of the innate immune defense against pathogens in bacterial CNS infections.

CpG oligodeoxynucleotides induce the expression of the antimicrobial peptide cathelicidin in glial cells.

During bacterial infections, antimicrobial peptides are synthesised as an important part of the innate immune system. However, expression and function in the central nervous system (CNS) need further investigations. The aim of this study was to examine the involvement of the pattern-recognition-receptor toll-like receptor 9 (TLR9) in the expression of the cathelin-related antimicrobial peptide (CRAMP) and to characterise the participating signal transduction pathways. In primary TLR9 deficient and wildtype mice astrocytes as well as microglia cells, the expression of CRAMP after treatment with the TLR9 agonist unmethylated cytosine-guanine oligodeoxynucleotide motifs (CpG-DNA) was examined in vitro. In vivo CRAMP expression after intraventricular infusion of CpG-DNA in TLR9 deficient and wildtype mice as well as in mice with pneumococcal meningitis localised in glial cells was determined. Furthermore, the regulation of different signal transduction pathways involved in CpG-DNA-induced CRAMP expression in glial cells was analysed. An in vitro and in vivo CpG-DNA-induced increase of CRAMP expression in astrocytes and microglia cells using real time RT-PCR and immunofluorescence was demonstrated. Different signal transduction pathways such as mitogen-activated protein kinases and inflammatory mediated pathways are involved in the expression of CRAMP in primary glial cells. Interestingly, TLR9-deficient glial cells showed a reduced but not completely abolished CRAMP mRNA expression and ERK1/2 phosphorylation in response to CpG-DNA treatment. On the other side in vivo, TLR9 deletion did not change CRAMP expression after bacterial infection. In conclusion, our results show that TLR9 can induce the expression of antimicrobial peptides such as CRAMP in response to bacterial DNA motifs in primary glial cells. Additional findings suggest also that CpG-DNA-induced effects are not only mediated by TLR9, but also mediated by other pattern recognition receptors.

Involvement of formyl peptide receptors in receptor for advanced glycation end products (RAGE)--and amyloid beta 1-42-induced signal transduction in glial cells.

BACKGROUND: Recent studies suggest that the chemotactic G-protein-coupled-receptor (GPCR) formyl-peptide-receptor-like-1 (FPRL1) and the receptor-for-advanced-glycation-end-products (RAGE) play an important role in the inflammatory response involved in neurodegenerative disorders such as Alzheimer's disease (AD).Therefore, the expression and co-localisation of mouse formyl peptide receptor (mFPR) 1 and 2 as well as RAGE in an APP/PS1 transgenic mouse model using immunofluorescence and real-time RT-PCR were analysed. The involvement of rat or human FPR1/FPRL1 (corresponds to mFPR1/2) and RAGE in amyloid-ß 1-42 (Aß1-42)-induced signalling were investigated by extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Furthermore, the cAMP level in primary rat glial cells (microglia and astrocytes) and transfected HEK 293 cells was measured. Formyl peptide receptors and RAGE were inhibited by a small synthetic antagonist WRW4 and an inactive receptor variant delta-RAGE, lacking the intracytoplasmatic domains. RESULTS: We demonstrated a strong increase of mFPR1/2 and RAGE expression in the cortex and hippocampus of APP/PS1 transgenic mice co-localised to the glial cells. In addition, the Aß1-42-induced signal transduction is dependant on FPRL1, but also on FPR1. For the first time, we have shown a functional interaction between FPRL1/FPR1 and RAGE in RAGE ligands S100B- or AGE-mediated signalling by ERK1/2 phosphorylation and cAMP level measurement. In addition a possible physical interaction between FPRL1 as well as FPR1 and RAGE was shown with co-immunoprecipitation and fluorescence microscopy. CONCLUSIONS: The results suggest that both formyl peptide receptors play an essential role in Aß1-42-induced signal transduction in glial cells. The interaction with RAGE could explain the broad ligand spectrum of formyl peptide receptors and their important role for inflammation and the host defence against infections.