Peter Zabel

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

BackgroundCpG-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.MethodsThe 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.ResultsWe 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.ConclusionsHere 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.

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

BackroundCigarette 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 (PRRs). 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.MethodsThe 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.ResultsThe 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.ConclusionOur data suggest a smoke related change in the phenotype of AMs and the cellular response to microbial stimulation which may be associated with impairment of host defenses in the lower respiratory tract.

Spatial and temporal heterogeneity of regional lung ventilation determined by electrical impedance tomography during pulmonary function testing.

Electrical impedance tomography (EIT) is a functional imaging modality capable of tracing continuously regional pulmonary gas volume changes. The aim of our study was to determine if EIT was able to assess spatial and temporal heterogeneity of ventilation during pulmonary function testing in 14 young (37 ± 10 yr, mean age ± SD) and 12 elderly (71 ± 9 yr) subjects without lung disease and in 33 patients with chronic obstructive pulmonary disease (71 ± 9 yr). EIT and spirometry examinations were performed during tidal breathing and a forced vital capacity (FVC) maneuver preceded by full inspiration to total lung capacity. Regional inspiratory vital capacity (IVC); FVC; forced expiratory volume in 1 s (FEV(1)); FEV(1)/FVC; times required to expire 25%, 50%, 75%, and 90% of FVC (t(25), t(50), t(75), t(90)); and tidal volume (V(T)) were determined in 912 EIT image pixels in the chest cross section. Coefficients of variation (CV) were calculated from all pixel values of IVC, FVC, FEV(1), and V(T) to characterize the ventilation heterogeneity. The highest values were found in patients, and no differences existed between the healthy young and elderly subjects. Receiver-operating characteristics curves showed that CV of regional IVC, FVC, FEV(1), and V(T) discriminated the young and elderly subjects from the patients. Frequency distributions of pixel FEV(1)/FVC, t(25), t(50), t(75), and t(90) identified the highest ventilation heterogeneity in patients but distinguished also the healthy young from the elderly subjects. These results indicate that EIT may provide additional information during pulmonary function testing and identify pathologic and age-related spatial and temporal heterogeneity of regional lung function.

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.

Sepsis severity predicts outcome in community-acquired pneumococcal pneumonia

Easily performed prognostic rules are helpful for guiding the intensity of monitoring and treatment of patients. The aim of the present study was to compare the predictive value of the sepsis score and the Confusion, Respiratory rate (≥30 breaths·min−1), Blood pressure (systolic value <90 mmHg or diastolic value ≤60 mmHg) and age ≥65 yrs (CRB-65) score in 105 patients with community-acquired pneumococcal pneumonia. In addition, the influence of timing of the antimicrobial treatment on outcome was investigated. The sepsis and the CRB-65 scores were used to allocate patients to subgroups with low, intermediate and high risk. Comparable, highly predictive values for mortality were found for both scores (sepsis score versus CRB-65): 1) low-risk group, 0 versus 0%; 2) intermediate-risk group, 0 versus 8.6%; 3) high-risk group, 30.6 versus 40%, with an area under the curve of 0.867 versus 0.845. Patients with ambulatory antibiotic pre-treatment had less severe disease with a lower acute physiology score, lower white blood cell count and a faster decline of C-reactive protein levels. No pre-treated patient died. In summary, both scores performed equally well in predicting mortality. The prediction of survival in the intermediate-risk group might be more accurate with the sepsis score. Pre-hospital antibiotic treatment was associated with less severe disease.

Treatment and vaccines for severe acute respiratory syndrome

Summary The causative agent of severe acute respiratory syndrome (SARS), which affected over 8000 individuals worldwide and was responsible for over 700 deaths in the 2002–2003 outbreak, is a coronavirus that was unknown before the outbreak. Although many different treatments were used during the outbreak, none were implemented in a controlled fashion. Thus, the optimal treatment for SARS is unknown. Since the outbreak, much work has been done testing new agents against SARS using in-vitro methods and animal models. In addition, global research efforts have focused on the development of vaccines against SARS. Efforts should be made to evaluate the most promising treatments and vaccines in controlled clinical trials, should another SARS outbreak occur.

The TGF-beta-Pseudoreceptor BAMBI is strongly expressed in COPD lungs and regulated by nontypeable Haemophilus influenzae

BackgroundNontypeable Haemophilus influenzae (NTHI) may play a role as an infectious trigger in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available regarding the influence of acute and persistent infection on tissue remodelling and repair factors such as transforming growth factor (TGF)-β.MethodsNTHI infection in lung tissues obtained from COPD patients and controls was studied in vivo and using an in vitro model. Infection experiments were performed with two different clinical isolates. Detection of NTHI was done using in situ hybridization (ISH) in unstimulated and in in vitro infected lung tissue. For characterization of TGF-β signaling molecules a transcriptome array was performed. Expression of the TGF-pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) was analyzed using immunohistochemistry (IHC), ISH and PCR. CXC chemokine ligand (CXCL)-8, tumor necrosis factor (TNF)-α and TGF-β expression were evaluated in lung tissue and cell culture using ELISA.ResultsIn 38% of COPD patients infection with NTHI was detected in vivo in contrast to 0% of controls (p < 0.05). Transcriptome arrays showed no significant changes of TGF-β receptors 1 and 2 and Smad-3 expression, whereas a strong expression of BAMBI with upregulation after in vitro infection of COPD lung tissue was demonstrated. BAMBI was expressed ubiquitously on alveolar macrophages (AM) and to a lesser degree on alveolar epithelial cells (AEC). Measurement of cytokine concentrations in lung tissue supernatants revealed a decreased expression of TGF-β (p < 0.05) in combination with a strong proinflammatory response (p < 0.01).ConclusionsWe show for the first time the expression of the TGF pseudoreceptor BAMBI in the human lung, which is upregulated in response to NTHI infection in COPD lung tissue in vivo and in vitro. The combination of NTHI-mediated induction of proinflammatory cytokines and inhibition of TGF-β expression may influence inflammation induced tissue remodeling.

TKTL1 is overexpressed in a large portion of non-small cell lung cancer specimens

In several tumors the transketolase activity, controlled inter alia by enzymes of the pentose phosphate pathway which is an alternative, energy generating reaction-cascade to glycolysis, has been correlated with proliferation. The increase of thiamine-dependant transketolase enzyme reactions is induced especially through upregulated transketolase-like enzyme 1 (TKTL1)-activity; that shows TKTL1 to be a causative enzyme for tumors enhanced, anaerobic glucose degradation. We investigated TKTL1-expression in 88 human, formalin-fixed non-small cell lung cancer tissues and 24 carcinomas of the breast by immunohistochemical stainings applying a 0 to 3 staining-score system (3 = strongest expression). For means of validation we additionally stained 40 NSCLC fixed and paraffin-embedded utilizing the HOPE-technique; showing comparable results to the formalin-fixed, paraffin-embedded specimens (not shown). Potential correlations with age, sex, TNM-classification parameters and tumor grading as well as tumor transcription factor 1 (TTF1) and surfactant protein A (SPA) expression were investigated. 40.9% of the analyzed lung tumors expressed TKTL1 weakly (Score 1), 38.6% moderately (score 2) and 17.1% strongly (score 3). 3 tumors were diagnosed TKTL1-negative (3.4%; score 0). All Breast cancer specimen stainings were positive and scored 1: 32%; scored 2: 36%; scored 3: 32%. Alveolar macrophages and Alveolar Epithelial Cells Type II were also found to be TKTL1-positive.None of the listed clinical parameters could be found to show a significant correlation to TKTL1 signal appearance.Although we describe the expression of TKTL1 in lung cancers, we need to state that up till now there is no scientific indication for any treatment regimens based upon these findings.

Enhanced molecular analyses by combination of the HOPE-technique and laser microdissection

As part of an investigation aimed at illuminating the possibilities and limits of the HOPE-fixation and paraffin-embedding technique we here describe a novel procedure which was developed in order to combine the benefits of the HOPE-technique with the capabilities of laser microdissection. The presented procedure avoids the need for amplification of template-RNA thus facilitating reliable and reproducible results. The excellent preservation of nucleic acids, proteins, and morphology in HOPE-fixed, paraffin-embedded tissues enhances the molecular applications available to date with materials acquired by laser microdissection when compared to formalin fixed, paraffin-embedded tissues, thus substantially extending the methodological panel in tissue based research.

The role of tumor necrosis factor in the development of multiple organ failure in a murine model.

Objective To elucidate the role of tumor necrosis factor (TNF) in the development of multiple organ dysfunction syndrome (MODS) after zymosan-induced peritonitis in mice. Design Prospective controlled laboratory study on zymosan-induced generalized inflammation in mice. Over ≤28 days, a single intraperitoneal administration of zymosan induced a three-phase illness in C57BL mice, rendering them very ill with MODS-like symptoms from day 7 onward. Additionally, the same experiment was performed on C57BL/6 TNF-Rc-p55 knockout mice to elucidate the role of TNF and its receptor p55. Setting Animal research laboratory. Subjects Inbred C57BL mice and C57BL p55−/− mice received a single sterile intraperitoneal injection of zymosan suspended in paraffin oil (0.75 mg/g of body weight). Measurements and Main Results The animals were monitored for survival, condition, and body weight for ≤28 days. At 3, 7, 14, and 28 days after zymosan administration, bronchoalveolar lavage was performed and lungs and livers were extracted for isolation of RNA and histopathologic evaluation. Reverse-transcriptase-polymerase chain reaction was performed to quantify TNF-&agr; messenger RNA (mRNA) in the respective organs. Both animal strains went through initial shock with a high mortality rate during the first 3 days. The C57BL mice developed MODS with typical symptoms and histopathologic results correlating with excessive TNF-&agr; mRNA expression from day 7 onward. In contrast, no disease, histopathologic changes, nor TNF-&agr; mRNA expression in liver or lung was found within the TNF-Rc-p55−/− mice. Conclusion Organ-derived TNF-&agr; plays a crucial role in the development of MODS in this murine model.