E-cigarettes and cigarettes worsen peripheral and central hemodynamics as well as arterial stiffness: A randomized, double-blinded pilot study.

The introduction of electronic cigarettes has led to widespread discussion on the cardiovascular risks compared to conventional smoking. We therefore conducted a randomized cross-over study of the acute use of three tobacco products, including a control group using a nicotine-free liquid. Fifteen active smokers were studied during and after smoking either a cigarette or an electronic cigarette with or without nicotine (eGo-T CE4 vaporizer). Subjects were blinded to the nicotine content of the electronic cigarette and were followed up for 2 hours after smoking a cigarette or vaping an electronic cigarette. Peripheral and central blood pressures as well as parameters of arterial stiffness were measured by a Mobil-O-Graph® device. The peripheral systolic blood pressure rose significantly for approximately 45 minutes after vaping nicotine-containing liquid ( p<0.05) and for approximately 15 minutes after smoking a conventional cigarette ( p<0.01), whereas nicotine-free liquids did not lead to significant changes during the first hour of follow-up. Likewise, heart rate remained elevated approximately 45 minutes after vaping an electronic cigarette with nicotine-containing liquid and over the first 30 minutes after smoking a cigarette in contrast to controls. Elevation of pulse wave velocity was independent from mean arterial pressure as well as heart rate in the electronic cigarette and cigarette groups. In this first of its kind trial, we observed changes in peripheral and central blood pressure and also in pulse wave velocity after smoking a cigarette as well as after vaping a nicotine-containing electronic cigarette. These findings may be associated with an increased long-term cardiovascular risk.

Modulation of the inflammatory response to Streptococcus pneumoniae in a model of acute lung tissue infection.

Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia and is a main cause of infectious deaths. However, little is known about host-pathogen interaction in human lung tissue. We tested the hypothesis that human alveolar macrophages (AMs) and alveolar epithelial cells (AECs) are important for initiating the host response against S. pneumoniae, and we evaluated the role of Toll-like receptor (TLR) 2, TLR4, and p38 mitogen-activated protein kinase (MAPK) signaling in the inflammatory response after pneumococcal infection. We established a novel model of acute S. pneumoniae infection using vital human lung specimens. In situ hybridization analysis showed that S. pneumoniae DNA was detected in 80 to 90% of AMs and 15 to 30% of AECs after in vitro infection accompanied by increased expression of inflammatory cytokines. Enhanced phosphorylation of p38 MAPK and increased TLR2 and 4 mRNA expression were observed in infected lung tissue. Thirty to fifty percent of AMs and 10 to 20% of AECs showed evidence of apoptosis 24 hours after pneumococcal infection. After macrophage deactivation with Clodronate/liposomes, infected lung tissue exhibited a significantly decreased release of inflammatory mediators. Inhibition of p38 MAPK signaling markedly reduced inflammatory cytokine release from human lungs, whereas TLR2 blockade revealed only minor effects. AMs are central resident immune cells during S. pneumoniae infection and are the main source of early proinflammatory cytokine release. p38 MAPK holds a major role in pathogen-induced pulmonary cytokine release and is a potential molecular target to modulate overwhelming lung inflammation.

Hospital acquired pneumonia with high-risk bacteria is associated with increased pulmonary matrix metalloproteinase activity.

BACKGROUND: Neutrophil products like matrix metalloproteinases (MMP), involved in bacterial defence mechanisms, possibly induce lung damage and are elevated locally during hospital- acquired pneumonia (HAP). In HAP the virulence of bacterial species is known to be different. The aim of this study was to investigate the influence of high-risk bacteria like S. aureus and pseudomonas species on pulmonary MMP concentration in human pneumonia. METHODS: In 37 patients with HAP and 16 controls, MMP-8, MMP-9 and tissue inhibitors of MMP (TIMP) were analysed by ELISA and MMP-9 activity using zymography in bronchoalveolar lavage (BAL). RESULTS: MMP-9 activity in mini-BAL was increased in HAP patients versus controls (149 +/- 41 vs. 34 +/- 11, p < 0.0001). In subgroup analysis, the highest MMP concentrations and activity were seen in patients with high-risk bacteria: patients with high-risk bacteria MMP-9 1168 +/- 266 vs. patients with low-risk bacteria 224 +/- 119 ng/ml p < 0.0001, MMP-9 gelatinolytic activity 325 +/- 106 vs. 67 +/- 14, p < 0.0002. In addition, the MMP-8 and MMP-9 concentration was associated with the state of ventilation and systemic inflammatory marker like CRP. CONCLUSION: Pulmonary MMP concentrations and MMP activity are elevated in patients with HAP. This effect is most pronounced in patients with high-risk bacteria. Artificial ventilation may play an additional role in protease activation.

Identification of novel target genes in human lung tissue involved in chronic obstructive pulmonary disease.

Introduction: As part of a study aimed at illuminating at least some of the complex molecular events taking place in COPD, we screened tissues by means of transcriptome analyses. Materials and methods: Tissues were subjected to transcriptome analysis. Candidate genes were identified and validated by immunohistochemistry. Primary human lung cells were subjected to stimulation with cigarette smoke extract for further validation by real time PCR. Results: Six candidate genes were selected for further investigations: Aquaporin 3 (AQP3), extracellular matrix protein 1 (ECM1), four and a half LIM domain 1 (FHL1), milk fat globule epidermal growth factor 8 (MFGE8, lactadherin), phosphodiesterase 4D-interacting protein (PDE4DIP), and creatine transporter SLC6A8. All six proteins were allocated to distinct cell types by immunohistochemistry. Upon stimulation with cigarette smoke extract, human type II pneumocytes showed a dose-dependent down-regulation of MFGE8, while ECM1 and FHL1 also tended to be down-regulated. Although present, none of the candidates was regulated by cigarette smoke extract in primary human macrophages. Discussion: MFGE8 turned out to be an interesting new candidate gene in COPD deserving further studies.

A novel human ex vivo model for the analysis of molecular events during lung cancer chemotherapy.

BACKGROUND: Non-small cell lung cancer (NSCLC) causes most of cancer related deaths in humans and is characterized by poor prognosis regarding efficiency of chemotherapeutical treatment and long-term survival of the patients. The purpose of the present study was the development of a human ex vivo tissue culture model and the analysis of the effects of conventional chemotherapy, which then can serve as a tool to test new chemotherapeutical regimens in NSCLC. METHODS: In a short-term tissue culture model designated STST (Short-Term Stimulation of Tissues) in combination with the novel *HOPE-fixation and paraffin embedding method we examined the responsiveness of 41 human NSCLC tissue specimens to the individual cytotoxic drugs carboplatin, vinorelbine or gemcitabine. Viability was analyzed by LIFE/DEAD assay, TUNEL-staining and colorimetric MTT assay. Expression of Ki-67 protein and of BrdU (bromodeoxyuridine) uptake as markers for proliferation and of cleaved (activated) effector caspase-3 as indicator of late phase apoptosis were assessed by immunohistochemistry. Transcription of caspase-3 was analyzed by RT-PCR. Flow cytometry was utilized to determine caspase-3 in human cancer cell lines. RESULTS: Viability, proliferation and apoptosis of the tissues were moderately affected by cultivation. In human breast cancer, small-cell lung cancer (SCLC) and human cell lines (CPC-N, HEK) proliferative capacity was clearly reduced by all 3 chemotherapeutic agents in a very similar manner. Cleavage of caspase-3 was induced in the chemo-sensitive types of cancer (breast cancer, SCLC). Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas. CONCLUSION: Although there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously. Thus, STST provides a useful human model to study numerous aspects of mechanisms underlying tumor responsiveness towards improved anticancer treatment. The results presented here shall serve as a base for multiple functional tests of novel chemotherapeutic approaches to NSCLC in the future.

Coupling killing to neutralization: combined therapy with ceftriaxone/Pep19-2.5 counteracts sepsis in rabbits.

Sepsis, which is induced by severe bacterial infections, is a major cause of death worldwide, and therapies combating the disease are urgently needed. Because many drugs have failed in clinical trials despite their efficacy in mouse models, the development of reliable animal models of sepsis is in great demand. Several studies have suggested that rabbits reflect sepsis-related symptoms more accurately than mice. In this study, we evaluated a rabbit model of acute sepsis caused by the intravenous inoculation of Salmonella enterica. The model reproduces numerous symptoms characteristic of human sepsis including hyperlactatemia, hyperglycemia, leukopenia, hypothermia and the hyperproduction of several pro-inflammatory cytokines. Hence, it was chosen to investigate the proposed ability of Pep19-2.5-an anti-endotoxic peptide with high affinity to lipopolysaccharide and lipoprotein-to attenuate sepsis-associated pathologies in combination with an antibiotic (ceftriaxone). We demonstrate that a combination of Pep19-2.5 and ceftriaxone administered intravenously to the rabbits (1) kills bacteria and eliminates bacteremia 30 min post challenge; (2) inhibits Toll-like receptor 4 agonists in serum 90 min post challenge; (3) reduces serum levels of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor α); and (4) reverts to hypothermia and gives rise to temperature values indistinguishable from basal levels 330 min post challenge. The two components of the combination displayed synergism in some of these activities, and Pep19-2.5 notably counteracted the endotoxin-inducing potential of ceftriaxone. Thus, the combination therapy of Pep19-2.5 and ceftriaxone holds promise as a candidate for human sepsis therapy.

Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients.

BACKGROUND: Cigarette smoke exposure including biologically active lipopolysaccharide (LPS) in the particulate phase of cigarette smoke induces activation of alveolar macrophages (AM) and alveolar epithelial cells leading to production of inflammatory mediators. This represents a crucial mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Respiratory pathogens are a major cause of exacerbations leading to recurrent cycles of injury and repair. The interaction between pathogen-associated molecular patterns and the host is mediated by pattern recognition receptors (PRR's). In the present study we characterized the expression of Toll-like receptor (TLR)- 2, TLR4 and CD14 on human AM compared to autologous monocytes obtained from patients with COPD, healthy smokers and non-smokers. METHODS: The study population consisted of 14 COPD patients without evidence for acute exacerbation, 10 healthy smokers and 17 healthy non-smokers stratified according to age. The expression of TLR2, TLR4 and CD14 surface molecules on human AM compared to autologous monocytes was assessed ex vivo using FACS analysis. In situ hybridization was performed on bronchoalveolar lavage (BAL) cells by application of the new developed HOPE-fixative. RESULTS: The expression of TLR2, TLR4 and CD14 on AM from COPD patients, smokers and non-smokers was reduced as compared to autologous monocytes. Comparing AM we detected a reduced expression of TLR2 in COPD patients and smokers. In addition TLR2 mRNA and protein expression was increased after LPS stimulation on non-smokers AM in contrast to smokers and COPD patients. CONCLUSION: Our data suggest a smoke related change in the phenotype of AM's and the cellular response to microbial stimulation which may be associated with impairment of host defenses in the lower respiratory tract.

Human lung cancer cells express functionally active Toll-like receptor 9.

BACKGROUND: CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology. METHODS: The expression of TLR9 in HOPE-fixed non-small lung cancer, non-malignant tissue and tumor cell lines was assessed using immunohistochemistry, confocal microscopy, in situ hybridization, RT-PCR and DNA-sequencing. Apoptosis and chemokine expression was detected by FACS analysis and the Bio-Plex system. RESULTS: We found high TLR9 signal intensities in the cytoplasm of tumor cells in the majority of lung cancer specimens as well as in all tested tumor cell lines. In contrast to this non-malignant lung tissues showed only sporadically weak expression. Stimulation of HeLa and A549 cells with CpG-ODN induced secretion of monocyte chemoattractant protein-1 and reduction of spontaneous and tumor necrosis factor-alpha induced apoptosis. CONCLUSIONS: Here we show that TLR9 is expressed in a selection of human lung cancer tissues and various tumor cell lines. The expression of functionally active TLR9 in human malignant tumors might affect treatment approaches using CpG-ODN and shows that malignant cells can be regarded as active players in tumor-immunology.

Long-term mortality and quality of life in intensive care patients treated for pneumonia and/or sepsis: Predictors of mortality and quality of life in patients with sepsis/pneumonia.

PURPOSE: The purpose of this study is to evaluate long-term mortality and quality of life (QoL) of intensive care patients with pneumonia and/or sepsis 1 year after discharge and to identify potential predictors for these outcome measures. METHODS: This retrospective cohort study analyzed all patients admitted to the intensive care unit (ICU) of a German university hospital with diagnosis of pneumonia and/or sepsis between 2008 and 2009. Quality of life was assessed by telephone interview or mail using the standardized EuroQol 5-dimension questionaire. RESULTS: Of 1406 patients treated in the ICU within the observational period, 217 met the inclusion criteria. Whereas in-hospital mortality differed significantly between pneumonia (17%) and sepsis (46%) (P < .001), 1-year mortality was not statistically significant (51% and 65%, P = .057). A high Simplified Acute Physiology Score (SAPS) II value was associated with high in-hospital mortality but failed to predict 1-year mortality. Quality of life, measured 1 year after discharge by visual analog scale (VAS), was 50% ± 25%, which was significantly lower than in a matched control group (70% ± 20%; P < .001). A high SAPS II score on admission did not correlate with VAS but was an independent predictor of a low EuroQol 5-dimension index. CONCLUSIONS: The high post-ICU mortality of patients with pneumonia and sepsis emphasizes the need to focus on long-term follow-up in ICU studies and demonstrates that even when sepsis signs are missing, critically ill patients due to pneumonia have high 1-year mortality. Simplified Acute Physiology Score II does not predict long-term mortality, but a low SAPS II on admission might be useful to identify patients with good physical status after 1 year. TAKE HOME MESSAGE: Hospital mortality of patients treated for pneumonia and/or sepsis is high and increases significantly within the first year after discharge. The SAPS II predicts in-hospital mortality and the physical components of QoL but not long-term mortality. TWEET: One-year mortality of ICU pneumonia patients is equally high as in sepsis patients. Simplified Acute Physiology Score II cannot predict long-term mortality but can predict QoL.

Alveolar epithelial cells type II are major target cells for C. pneumoniae in chronic but not in acute respiratory infection.

Pulmonary presence of Chlamydia pneumoniae is associated with acute and chronic infections. We show that unapparent chlamydial infection in four out of 31 chronic obstructive pulmonary disease (COPD) patients (12.9%) is characterized by a significant increase in infected alveolar epithelial cells type II (18.2 +/- 3.5% vs. 2.3 +/- 0.9; IHC/ISH) compared to a newly established model of acute chlamydial infection (ACIM) in vital lung specimens from pulmonary lobectomy. Expression of cHSP60 demonstrated pathogen viability and virulence in the ACIM. We conclude that target cells differ in acute and chronic chlamydial infection and suggest the ACIM as a novel tool to analyze the host-pathogen-interactions in acute respiratory infections.

Nontypeable Haemophilus Influenzae Infection Upregulates the NLRP3 Inflammasome and Leads to Caspase-1-Dependent Secretion of Interleukin-1ß - A Possible Pathway of Exacerbations in COPD.

RATIONALE: Nontypeable Haemophilus influenzae (NTHi) is the most common cause for bacterial exacerbations in chronic obstructive pulmonary disease (COPD). Recent investigations suggest the participation of the inflammasome in the pathomechanism of airway inflammation. The inflammasome is a cytosolic protein complex important for early inflammatory responses, by processing Interleukin-1ß (IL-1ß) to its active form. OBJECTIVES: Since inflammasome activation has been described for a variety of inflammatory diseases, we investigated whether this pathway plays a role in NTHi infection of the airways. METHODS: A murine macrophage cell line (RAW 264.7), human alveolar macrophages and human lung tissue (HLT) were stimulated with viable or non-viable NTHi and/or nigericin, a potassium ionophore. Secreted cytokines were measured with ELISA and participating proteins detected via Western Blot or immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: Western Blot analysis of cells and immunohistochemistry of lung tissue detected the inflammasome key components NLRP3 and caspase-1 after stimulation, leading to a significant induction of IL-1ß expression (RAW: control at the lower detection limit vs. NTHi 505±111pg/ml, p<0.01). Inhibition of caspase-1 in human lung tissue led to a significant reduction of IL-1ß and IL-18 levels (IL-1ß: NTHi 24 h 17423±3198pg/ml vs. NTHi+Z-YVAD-FMK 6961±1751pg/ml, p<0.01). CONCLUSION: Our data demonstrate the upregulation of the NRLP3-inflammasome during NTHi-induced inflammation in respiratory cells and tissues. Our findings concerning caspase-1 dependent IL-1ß release suggest a role for the inflammasome in respiratory tract infections with NTHi which may be relevant for the pathogenesis of bacterial exacerbations in COPD.

Mortality in human sepsis is associated with downregulation of Toll-like receptor 2 and CD14 expression on blood monocytes.

Pattern recognition receptors are a key component of the first line host defense against infection, recognizing specific microbial products. We hypothesize that monocyte hyporesponsiveness in human sepsis is associated with a downregulation of the pattern recognition receptors Toll-like receptor (TLR)-2 and TLR4.Protein expression of CD14, TLR2 and TLR4 on blood monocytes was examined using flow cytometry from 29 patients with sepsis and 14 healthy controls. In addition LPS stimulated TNF-alpha and IL-10 production was studied in a 24 hour whole blood assay.We found an increased expression of CD14, TLR2 and TLR4 in patients with sepsis compared to controls (p < 0.01). In patients with sepsis, death was associated with significant lower CD14 and TLR2 expression at admission (CD14: 25.7 +- 19.1 vs 39.1 +- 17.3 mean fluorescence intensity [MFI], p = 0.02; TLR2: 21.8 +- 9.4 vs. 30.9 +- 9.6, p = 0.01). At 72 hours the TLR2 expression on monocytes was associated with the IL-10 inducibility after LPS stimulation (r = 0.52, p = 0.02) and the CD14 expression with the IL-6, IL-10 and TNF inducibility.We conclude that septic patients are characterized by an increased expression of CD14, TLR2 and TLR4 on monocytes compared to controls. Death is associated with downregulation of TLR2 and CD14 expression on monocytes correlating with reduced cytokine inducibility. We suggest that CD14 and TLR2 are a key factor in monocyte hyporesponsibility during severe sepsis.

Disparate innate immune responses to persistent and acute Chlamydia pneumoniae infection in chronic obstructive pulmonary disease.

RATIONALE: Chlamydia pneumoniae (Cpn) infection may play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available comparing persistent and acute infection of this pathogen in the human respiratory tract. OBJECTIVES: To study Cpn-induced innate immune responses in lung tissue from patients with COPD and control subjects ex vivo and in vitro. METHODS: Cpn detection was done by nested polymerase chain reaction, in situ hybridization, and immunohistochemistry ex vivo in unstimulated tissue and in vitro using an acute Cpn infection model. As main endpoints for the assessment of early cellular responses, nuclear factor (NF)-kappaB activation and CXC chemokine ligand (CXCL)-8 expression were evaluated. The role of Toll-like receptors (TLRs) as recognition molecules in Cpn-induced innate responses was tested by blocking experiments. MEASUREMENTS AND MAIN RESULTS: Fifteen percent of patients with COPD were chronically infected with Cpn in contrast to 0% of control subjects (p < 0.05). There were no differences in CXCL-8 and NF-kappaB expression between infected and noninfected COPD tissue ex vivo. In contrast, acute in vitro infection induced an intense innate immune response including up-regulation of TLR2. Blocking experiments demonstrated the predominant role of TLR2 in induction of the early immune response, whereas no influence on chlamydial infection rates was observed. CONCLUSIONS: Acute in vitro infection of human lung tissue with Cpn elicited a marked innate response via TLR2, whereas chronic chlamydial infection in patients with COPD was not associated with enhanced cellular activation. These findings suggest different roles of Cpn during acute and chronic stages of pulmonary infection.

Toll-like receptor 2 is expressed by alveolar epithelial cells type II and macrophages in the human lung.

The ability of the host to recognize pulmonary invasion by pathogenic organisms and establish an appropriate host response to infection requires innate immune defense mechanisms. Early bacterial clearance in the lung is mediated by alveolar macrophages (AM) and polymorphonuclear neutrophils. Additionally alveolar epithelial cells type II (AEC-II) may act as immunoregulatory cells. The toll-like receptors (TLR) are part of this innate immune defense, recognizing conserved patterns on microorganisms. Toll-like receptor 2 (TLR2) is crucial in detecting components of gram-positive bacteria and mycobacteria. Signals initiated by the interaction of TLR2 with bacterial components direct the subsequent inflammatory response. The detection of TLR2 mRNA in human lung tissue prompted us to localize the expression of mRNA and protein at the cellular level using a novel method for tissue fixation. We utilized HOPE-fixed lung specimen sections for targeting mRNA by in situ hybridization and protein by immunohistochemistry using the monoclonal antibody TL2.1. In normal lung areas the expression of TLR2 mRNA and protein was found to be located in cells resembling AEC-II and AM. Expression of mRNA was verified by RT-PCR and DNA sequencing. These results indicate a potential mechanism of increased immunosurveillance at the alveolar level controlling the localized infection.