S100A7/psoriasin expression in the human lung: unchanged in patients with COPD, but upregulated upon positive S. aureus detection.

BACKGROUND: Progressive airway inflammation and susceptibility to the airway colonisation and infection are characteristic for the pathophysiology of chronic obstructive pulmonary disease (COPD). Antimicrobial peptides (AMPs) are central to the function of the innate host immune response against microbial pathogens and are regulators of inflammation and immunity. S100A7/psoriasin, a recently described AMP, is an essential component of the human epithelia against invading pathogens and acts as an effector molecule of the host innate defence in the skin. We hypothesized that S100A7/psoriasin is involved in the airway mucosal immunity and differently regulated and expressed in the lung during progression of COPD. METHODS: S100A7/psoriasin gene expression was assessed in bronchial biopsies and bronchoalveolar lavage (BAL) fluid cells of healthy controls and COPD patients. Using confocal microscopy and immunohistochemistry, the protein expression of S100A7/psoriasin was investigated. RESULTS: Here, we report that S100A7/psoriasin, the major antimicrobial peptide of the human skin, is constitutively expressed in perinuclear granules of human bronchial epithelial cells and alveolar macrophages. Whereas typical activators of the innate immune response like TLR ligands and cytokines induced the upregulation of CXCL-8 mRNA and release of CXCL-8 by epithelial cells, S100A7/psoriasin mRNA expression was not modulated. To investigate a potential association of S100A7/psoriasin with COPD, S100A7/psoriasin mRNA expression was assessed in bronchial biopsies and BAL fluid cells of patients at different stages of COPD and controls. Overall, 10 healthy individuals and 34 COPD patients were enrolled in this study. We found an association of S100A7/psoriasin mRNA expression with bacterial detection in the tracheobronchial system (p = 0.0304), which was the strongest in individuals positive for with S. aureus (p = 0.0005). However, S100A7/psoriasin mRNA expression was not altered during the progression of COPD. CONCLUSIONS: S100A7/psoriasin gene expression is unchanged in the airways during COPD. The newly identified association of S100A7/psoriasin with S. aureus may provide new insights into the antimicrobial defence response of the human airways, leading to the induction of S100A7/psoriasin upon microbial challenge.

Analysis of risk factors in thoracic trauma patients with a comparison of a modern trauma centre: a mono-centre study.

OBJECTIVES: Thoracic trauma (TT) is the third most common cause of death after abdominal injury and head trauma in polytrauma patients. Its management is still a very challenging task. The purpose of this study was to analyse the risk factors affecting the outcome in a high-volume trauma centre and the efficacy of a specialised trauma team in level 1 trauma centres. PATIENTS AND METHODS: Between January 2003 and December 2012, data of all patients admitted to the accident and emergency (A&E) department were prospectively collected at the German Trauma Registry (GTR) and thereafter retrospectively analysed. Patients with chest trauma, an Injury Severity Score (ISS) ≥ 18 and an Abbreviated Injury Scale (AIS) > 2 in more than one body region were included. Patients were divided into two groups: group I included patients presenting with thoracic trauma between January 2003 and December 2007. The results of this group were compared with the results of another group (group II) in a later 5-year period (Jan. 2008-Dec. 2012). Univariate and multivariate analyses were performed, and differences with p < 0.05 were considered statistically significant. RESULTS: There were 630 patients (56%) with thoracic trauma. A total of 540 patients (48%) had associated extrathoracic injuries. Group I consisted of 285 patients (197 male, mean age 46 years). Group II consisted of 345 patients (251 male, mean age 49 years). Overall 90-day mortality was 17% (n = 48) in group I vs. 9% (n = 31) in group II (p = 0.024). Complication rates were higher in group I (p = 0.019). Higher Injury Severity Scores (ISSs) and higher Abbreviated Injury Acale (AIS) scores in the thoracic region yielded a higher rate of mortality (p < 0.0001). Young patients (< 40 years) were frequently exposed to severe thoracic injury but showed lower mortality rates (p = 0.014). Patients with severe lung contusions (n = 94) (15%) had higher morbidity and mortality (p < 0.001). Twenty-three (8%) patients underwent emergency thoracotomy in group I vs. 14 patients (4%) in group II (p = 0.041). Organ replacement procedures were needed in 18% of patients in group I vs. 31% of patients in group II (p = 0.038). CONCLUSIONS: The presence of severe lung contusion, a higher ISS and AISthoracic score and advanced age are independent risk factors that are directly related to a higher mortality rate. Management of blunt chest trauma with corrective chest tube insertion, optimal pain control and chest physiotherapy results in good outcomes in the majority of patients. Optimal management with better survival rates is achievable in specialised centres with multidisciplinary teamwork and the presence of thoracic surgical experience.

In contrast to lung fibroblasts--no signs of senescence in skin fibroblasts of patients with emphysema.

Smoking is known to be linked to skin ageing and there is evidence for premature senescence of parenchymal lung fibroblasts in emphysema. To reveal whether the emphysema-related changes in cellular phenotype extend beyond the lung, we compared the proliferation characteristics of lung and skin fibroblasts between patients with and without emphysema. Parenchymal lung fibroblasts and skin fibroblasts from the upper torso (thus limiting sun exposure bias) were obtained from patients without, or with mild, or with moderate to severe emphysema undergoing lung surgery. We analysed proliferation rate, population doublings (PD), staining for senescence-associated beta-galactosidase (beta-gal) and gene expression of IGFBP-3 and IGFBP-rP1. Population doubling time of lung fibroblasts differed between control, mild, and moderate to severe emphysema (median (IQR) 29.7(10.0), 33.4(6.1), 44.4(21.2) h; p=0.012) and staining for beta-gal was elevated in moderate to severe emphysema. Compared to control subjects, skin fibroblasts from patients with emphysema did not differ with respect to proliferation rate, PD and beta-gal staining, and showed a lower abundance of mRNA for IGFBP-3 and -rP1 (p<0.05, each). These results suggest that the induction of a senescent fibroblast phenotype by cigarette smoke, as observed in emphysema, primarily occurs in the lung.

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.

Osteosarcoma relapse after combined modality therapy: an analysis of unselected patients in the Cooperative Osteosarcoma Study Group (COSS).

PURPOSE: To evaluate the impact of patient, tumor, and treatment-related factors on outcome in unselected patients with recurrent osteosarcoma. PATIENTS AND METHODS: Five hundred seventy-six consecutive patients who had achieved a first complete surgical remission (CR) during combined-modality therapy on neoadjuvant Cooperative Osteosarcoma Study Group (COSS) protocols and then developed recurrent osteosarcoma were analyzed (median time from biopsy to relapse, 1.6 years; range, 0.1 to 14.3 years). There were 501 patients with metastases, 44 with local recurrences, and 31 with both. Metastases involved lungs (469 patients), bones (90 patients), and/or other sites (54 patients). RESULTS: After a median follow-up of 1.2 years for all patients and 4.2 years for survivors, actuarial overall survival (OS) rates at 2, 5, and 10 years were 0.38, 0.23, and 0.18, respectively. Five-year OS was 0.39 for 339 patients with and 0.00 for 229 patients without a second surgical CR (P < .0001). A long time to relapse, a solitary lesion, and, in the case of pulmonary metastases, unilateral disease and the absence of pleural disruption, were of positive prognostic value in uni- and multivariate analyses, as were a second surgical CR and the use of second-line chemotherapy. Radiotherapy was associated with moderately prolonged survival in patients without a second CR. The very limited prognostic differences associated with the use of second-line chemotherapy appeared to be more pronounced with polychemotherapy. CONCLUSION: Time to relapse and tumor burden correlate with postrelapse outcome in osteosarcoma. Complete surgery is an essential component of curative second-line therapy. Chemotherapy, particularly chemotherapy with more than one agent, may contribute to limited improvements in outcome.

A new computer-controlled air-liquid interface cultivation system for the generation of differentiated cell cultures of the airway epithelium.

The increased application of in vitro systems in pharmacology and toxicology requires cell culture systems that facilitate the cultivation process and ensure stable, reproducible and controllable cultivation conditions. Up to now, some devices have been developed for the cultivation of cells under submersed conditions. However, systems meeting the requirements of an air-liquid interface (ALI) cultivation for the special needs of bronchial epithelial cells for example are still lacking. In order to obtain in vivo like organization and differentiation of these cells they need to be cultivated under ALI conditions on microporous membranes in direct contact with the environmental atmosphere. For this purpose, a Long-Term-Cultivation system was developed (CULTEX(®) LTC-C system) for the computer-controlled cultivation of such cells. The transwell inserts are placed in an incubator module (24 inserts), which can be adjusted for the medium level (ultrasonic pulse-echosensor), time and volume-dependent medium exchange, and frequency for mixing the medium with a rotating disc for homogeneous distribution of medium and secretion components. Normal primary freshly isolated bronchial epithelial cells were cultivated for up to 38 days to show the efficiency of such a cultivation procedure for generating 3D cultures exhibiting in vivo-like pseudostratified organization of the cells as well as differentiation characteristics like mucus-producing and cilia-forming cells.

Primary metastatic osteosarcoma: presentation and outcome of patients treated on neoadjuvant Cooperative Osteosarcoma Study Group protocols.

PURPOSE: To determine demographic data and define prognostic factors for long-term outcome in patients presenting with high-grade osteosarcoma of bone with clinically detectable metastases at initial presentation. PATIENTS AND METHODS: Of 1,765 patients with newly diagnosed, previously untreated high-grade osteosarcomas of bone registered in the neoadjuvant Cooperative Osteosarcoma Study Group studies before 1999, 202 patients (11.4%) had proven metastases at diagnosis and therefore were enrolled onto an analysis of demographic-, tumor-, and treatment-related variables, response, and survival. The intended therapeutic strategy included pre- and postoperative multiagent chemotherapy as well as aggressive surgery of all resectable lesions. RESULTS: With a median follow-up of 1.9 years (5.5 years for survivors), 60 patients were alive, 37 of whom were in continuously complete surgical remission. Actuarial overall survival rates at 5 and 10 (same value for 15) years were 29% (SE = 3%) and 24% (SE = 4%), respectively. In univariate analysis, survival was significantly correlated with patient age, site of the primary tumor, number and location of metastases, number of involved organ systems, histologic response of the primary tumor to preoperative chemotherapy, and completeness and time point of surgical resection of all tumor sites. However, after multivariate Cox regression analysis, only multiple metastases at diagnosis (relative hazard rate [RHR] = 2.3) and macroscopically incomplete surgical resection (RHR = 2.4) remained significantly associated with inferior outcomes. CONCLUSION: The number of metastases at diagnosis and the completeness of surgical resection of all clinically detected tumor sites are of independent prognostic value in patients with proven primary metastatic osteosarcoma.

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.

Tissue microarrays from HOPE-fixed specimens allow for enhanced high throughput molecular analyses in paraffin-embedded material.

In this study, we describe a technique that allows for the production and the use of tissue microarrays (TMA) from HOPE-fixed, paraffin-embedded specimens. The combination of these two technologies unites the advantages of the high throughput aspects in TMA, with good preservation of nucleic acids, proteins, and morphology of HOPE-fixed tissues, thus substantially widening the capabilities presently available for molecular studies in paraffin-embedded tissues.

Evaluation of E-cigarette liquid vapor and mainstream cigarette smoke after direct exposure of primary human bronchial epithelial cells.

E-cigarettes are emerging products, often described as "reduced-risk" nicotine products or alternatives to combustible cigarettes. Many smokers switch to e-cigarettes to quit or significantly reduce smoking. However, no regulations for e-cigarettes are currently into force, so that the quality and safety of e-liquids is not necessarily guaranteed. We exposed primary human bronchial epithelial cells of two different donors to vapor of e-cigarette liquid with or without nicotine, vapor of the carrier substances propylene glycol and glycerol as well as to mainstream smoke of K3R4F research cigarettes. The exposure was done in a CULTEX® RFS compact  module, allowing the exposure of the cells at the air-liquid interface. 24 h post-exposure, cell viability and oxidative stress levels in the cells were analyzed. We found toxicological effects of e-cigarette vapor and the pure carrier substances, whereas the nicotine concentration did not have an effect on the cell viability. The viability of mainstream smoke cigarette exposed cells was 4.5-8 times lower and the oxidative stress levels 4.5-5 times higher than those of e-cigarette vapor exposed cells, depending on the donor. Our experimental setup delivered reproducible data and thus provides the opportunity for routine testing of e-cigarette liquids to ensure safety and quality for the user.

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.

The role of intercostal nerve preservation in pain control after thoracotomy.

OBJECTIVES: Pain control after thoracotomy is an important issue that affects the outcome in thoracic surgery. Intercostal nerve preservation (ICNP) has increased interest in the outcomes of conventional thoracotomy. The current study critically evaluates the role of preservation of the intercostal nerve in early and late pain control and its benefit in patients undergoing thoracotomy. METHODS: Data obtained prospectively between January 2006 and December 2010 by a study colleague at our division of General Thoracic Surgery were retrospectively analysed. There were 491 patients who underwent thoracotomy. Eighty-one patients were excluded from the study due to incompatible data. Patients were divided into two groups according to the intercostal nerve state: Group I consisted of patients with ICNP and Group II consisted of patients with intercostal nerve sacrifice. RESULTS: Group I consisted of 288 patients [206 male (71%), P < 0.001, mean age 66 years]. Group II consisted of 122 patients [79 male (64%), P = 0.001, mean age 66 years]. There was less use of opiate in Group I (P = 0.019). Early mobilization of the patients was significantly higher in Group I (P = 0.031). The rate of pneumonia and re-admission to the intensive care unit was higher in Group II (P = 0.017 and 0.023, respectively). The rate of pain-free patients at discharge was significantly higher in Group I (P = 0.028). A 2-week follow-up after hospital discharge showed parasternal hypoesthesia to be more in Group II (P = 0.034). Significant patient contentment in Group I was noticed (P = 0.014). Chronic post-thoracotomy pain (CPTP) was higher in Group II (P = 0.016). CONCLUSIONS: ICNP without harvesting an intercostal muscle flap achieves excellent outcomes in controlling acute post-thoracotomy pain and CPTP. ICNP is an effective, simple method to perform, and it should be considered as standard in performing thoracotomy.

A compartment-specific transcriptome analysis reveals survival-relevant marker genes in the stroma fraction of squamous non-small cell lung cancer.

Although it is increasingly recognized that the tumor biology is influenced by the tumor stroma, prognostic gene signatures are usually derived from tissue consisting of tumor cells and surrounding stroma. This study presents a compartment-specific transcriptome analysis of lung squamous cell carcinoma (SCC) samples microdissected into tumor parenchyma and stroma fractions. Typical tumor and stroma genes were identified based on the expression ratios between the two compartments. Our results indicate that in SCC many markers related to longer survival are predominantly expressed in the stroma, particularly genes of the MHC-II complex. Stromal upregulation of MHC-II genes seems crucial for a clinically relevant antitumor immune response in SCC.

Proteomics out of the archive: Two-dimensional electrophoresis and mass spectrometry using HOPE-fixed, paraffin-embedded tissues.

Proteome analyses provide diagnostic information which can be essential for therapeutic predictions. The application of such techniques for analyzing paraffin-embedded tissue samples is widely hampered by the use of formalin fixation requiring antigen retrieval procedures in molecular pathology. In prior studies, the HEPES-glutamic acid buffer-mediated organic solvent protection effect (HOPE) technique of tissue fixation has been shown to provide a broad array of biochemical investigations with excellent preservation of morphological structures, DNA, RNA, and proteins, thus supporting the multimethod analysis of archived specimens. Here we show that HOPE fixation is also useful in proteomic investigations by allowing two-dimensional electrophoresis (2DE) and mass spectrometry, using lung cancer tissues. Two-dimensional gels of two-protein extraction protocols derived from HOPE-fixed material displayed characteristic spot patterns with high reproducibility. For comparison, 2DE analysis of ethanol-fixed, formalin-fixed, and frozen samples from the same tissues was performed. Western blotting confirmed immunoreactivity of 2DE-separated proteins from HOPE-fixed tissue samples. Additionally, distinct spots were excised from HOPE-derived 2D gels and successfully subjected to peptide mass fingerprinting. In conclusion, paraffin archives containing HOPE-fixed tissues are applicable to a wide spectrum of molecular investigations including common biochemical methods for proteome analyses and therefore represent a unique source for molecular investigations in the rapidly growing field of molecular pathology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Expression of the acute phase protein haptoglobin in human lung cancer and tumor-free lung tissues.

Besides its main function, i.e., the binding of free hemoglobin and prevention of oxidative stress, the acute phase protein haptoglobin acts as a potent immunoreactive modulator. As part of an investigation that aimed at illuminating the role of acute phase proteins in the local defense of the lungs, this study is the first to describe the expression and synthesis of haptoglobin in human lung tissues and lung tumors. Prompted by the results obtained from a transcription array study, we analyzed 115 lung (cancer) specimens using immunohistochemistry. Thirty-seven specimens were subjected to mRNA-in situ hybridization. 40.4% of the adenocarcinomas showed distinct granular and perinuclear staining of the tumor cells. By contrast, only 4.8% of the squamous cell carcinomas showed haptoglobin within tumor cells, but 19% displayed haptoglobin expressing alveolar epithelial cells type II surrounding the tumor. One small cell lung cancer displayed haptoglobin expression. In tumor-free lungs, we located haptoglobin in alveolar macrophages, alveolar epithelial cells type II, and bronchiolar cells. In situ hybridization verified the results of immunohistochemistry. The results were further verified by RT-PCR and Western blot compared to liver tissues, which both showed comparable amounts of haptoglobin mRNA and protein in NSCLC and in liver, while tumor-free lung tissues showed lower expression. Due to the known immunomodulatory effects of haptoglobin, its broad expression and synthesis within human lung tissues strongly suggests a function as a fundamental pulmonary local defense element.

Second and subsequent recurrences of osteosarcoma: presentation, treatment, and outcomes of 249 consecutive cooperative osteosarcoma study group patients.

PURPOSE: To evaluate patient and tumor characteristics, treatment, and outcomes in a large cohort of unselected patients with second and subsequent recurrences of osteosarcoma. PATIENTS AND METHODS: Two hundred forty-nine consecutive patients who had originally received combined-modality therapy on neoadjuvant Cooperative Osteosarcoma Study Group protocols and went on to develop a total of 409 second and subsequent osteosarcoma recurrences were analyzed for patient-, tumor-, and treatment-related factors and outcomes. RESULTS: Five-year overall and event-free survival rates were 16% and 9% for 249 second, 14% and 0% for 93 third, 13% and 6% for 38 fourth, and 18% and 0% for 14 fifth recurrences, respectively. The proportion of recurrences confined to the lungs decreased and the proportion of those with chest wall involvement increased with increasing numbers of recurrences. The duration of relapse-free intervals and the number of lesions at recurrence correlated with outcomes. While only one of 205 patients with rerecurrence survived past 5 years without surgical remission, 5-year overall and event-free survival rates were 32% and 18% for 119 second, 26% and 0% for 45 third, 28% and 13% for 20 fourth, and 53% and 0% for five fifth recurrences, respectively, in which a renewed surgical remission was achieved. The use of chemotherapy correlated with longer survival in patients without surgical remissions. CONCLUSION: To our knowledge, this is the first report of survival estimates derived from large cohorts of unselected patients with second and subsequent osteosarcoma recurrences. It confirms the overwhelming importance of surgical clearance. Prognostic indicators after rerecurrences resemble those known from first recurrence. The exact role of re-treatment with chemotherapy, particularly in the adjuvant situation, remains to be defined.

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-dependent 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.

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.

Isolated bronchi of patients with COPD show decreased histamine responsiveness compared to smokers with normal lung function.

This study compared airway responsiveness in vitro, as measured in isolated bronchi, with responsiveness in vivo in patients with COPD and smokers with normal lung function. In 9 patients with COPD (mean (range) FEV, 55 (30-78) %predicted) and 8 smokers with normal lung function (FEV1 101 (89-117) %predicted), who underwent surgery for lung cancer, responses to inhaled histamine and salbutamol were assessed before surgery. Bronchial specimens of 1-4 mm internal diameter were studied in the organ bath and histamine concentration-response curves assessed. All patients with COPD and none of the control individuals were hyperresponsive to inhaled histamine. Five patients with COPD and no control patient showed a bronchodilator response to salbutamol. Opposite to these findings, bronchial rings in the organ bath demonstrated a rightward shift of histamine concentration-response curves in COPD compared to controls, (p < 0.005). Accordingly, pED50 but not Emax differed statistically (p = 0.0016) between groups, mean+/-SEM values of pED50 in COPD (controls) being 4.67+/-0.08 (5.29+/-0.15) and of Emax 672+/-86 (772+/-120) mg. Patients with COPD showing hyperresponsiveness to inhaled histamine demonstrated lower responsiveness of their isolated bronchi compared to smokers with normal lung function. This suggests that in vivo hyperresponsiveness is based on other mechanisms than alterations in smooth muscle physiology.