Persistent and compartmentalised disruption of dendritic cell subpopulations in the lung following influenza A virus infection.

Immunological homeostasis in the respiratory tract is thought to require balanced interactions between networks of dendritic cell (DC) subsets in lung microenvironments in order to regulate tolerance or immunity to inhaled antigens and pathogens. Influenza A virus (IAV) poses a serious threat of long-term disruption to this balance through its potent pro-inflammatory activities. In this study, we have used a BALB/c mouse model of A/PR8/34 H1N1 Influenza Type A Virus infection to examine the effects of IAV on respiratory tissue DC subsets during the recovery phase following clearance of the virus. In adult mice, we found differences in the kinetics and activation states of DC residing in the airway mucosa (AMDC) compared to those in the parenchymal lung (PLDC) compartments. A significant depletion in the percentage of AMDC was observed at day 4 post-infection that was associated with a change in steady-state CD11b+ and CD11b- AMDC subset frequencies and significantly elevated CD40 and CD80 expression and that returned to baseline by day 14 post-infection. In contrast, percentages and total numbers of PLDC were significantly elevated at day 14 and remained so until day 21 post-infection. Accompanying this was a change in CD11b+and CD11b- PLDC subset frequencies and significant increase in CD40 and CD80 expression at these time points. Furthermore, mice infected with IAV at 4 weeks of age showed a significant increase in total numbers of PLDC, and increased CD40 expression on both AMDC and PLDC, when analysed as adults 35 days later. These data suggest that the rate of recovery of DC populations following IAV infection differs in the mucosal and parenchymal compartments of the lung and that DC populations can remain disrupted and activated for a prolonged period following viral clearance, into adulthood if infection occurred early in life.

Definitive intensity modulated radiotherapy in locally advanced hypopharygeal and laryngeal squamous cell carcinoma: mature treatment results and patterns of locoregional failure.

PurposeTo assess clinical outcomes and patterns of loco-regional failure (LRF) in relation to clinical target volumes (CTV) in patients with locally advanced hypopharyngeal and laryngeal squamous cell carcinoma (HL-SCC) treated with definitive intensity modulated radiotherapy (IMRT) and concurrent systemic therapy.MethodsData from HL-SCC patients treated from 2007 to 2010 were retrospectively evaluated. Primary endpoint was loco-regional control (LRC). Secondary endpoints included local (LC) and regional (RC) controls, distant metastasis free survival (DMFS), laryngectomy free survival (LFS), overall survival (OS), and acute and late toxicities. Time-to-event endpoints were estimated using Kaplan-Meier method, and univariate and multivariate analyses were performed using Cox proportional hazards models. Recurrent gross tumor volume (RTV) on post-treatment diagnostic imaging was analyzed in relation to corresponding CTV (in-volume, > 95% of RTV inside CTV; marginal, 20¿95% inside CTV; out-volume, < 20% inside CTV).ResultsFifty patients (stage III: 14, IVa: 33, IVb: 3) completed treatment and were included in the analysis (median follow-up of 4.2 years). Three-year LRC, DMFS and overall survival (OS) were 77%, 96% and 63%, respectively. Grade 2 and 3 acute toxicity were 38% and 62%, respectively; grade 2 and 3 late toxicity were 23% and 15%, respectively. We identified 10 patients with LRF (8 local, 1 regional, 1 local¿+¿regional). Six out of 10 RTVs were fully included in both elective and high-dose CTVs, and 4 RTVs were marginal to the high-dose CTVs.ConclusionThe treatment of locally advanced HL-SCC with definitive IMRT and concurrent systemic therapy provides good LRC rates with acceptable toxicity profile. Nevertheless, the analysis of LRFs in relation to CTVs showed in-volume relapses to be the major mode of recurrence indicating that novel strategies to overcome radioresistance are required.

Prognostic value of matrix metalloproteinases in oral squamous cell carcinoma

The aim of this study was to investigate whether there is a correlation between the expressions of four matrix metalloproteinases (MMPs): MMP-2, MMP-7, MMP-9 and MMP-13, and the TNM (tumour-node-metastasis) stages of oral squamous cell carcinoma (OSCC); and to explore the implication of these MMPs in OSCC dissemination. Samples from 61 patients diagnosed with oropharyngeal tumour were studied by immunohistochemistry against MMP-2, MMP-7, MMP-9 and MMP-13. The assessment of immunoreactivity was semi-quantitative. The results showed that MMP-2 and MMP-9 had similar expression patterns in the tumour cells with no changes in the immunoreactivity during tumour progression. MMP-9 always had the highest expression, whereas that of MMP-2 was moderate. MMP-7 showed a significant decrease in expression levels during tumour evolution. MMP-13 had constant expression levels within stage T2 and T3, but showed a remarkable decline in immunoreactivity in stage T4. No significant differences in the MMPs immunoreactivity between tumour cells and stroma were observed. Although strong evidence for the application of MMPs as reliable predictive markers for node metastasis was not acquired, we believe that combining patients' MMPs expression intensity and clinical features may improve the diagnosis and prognosis. Strong evidence for the application of MMPs as reliable predictive markers for node metastasis was not acquired. Application of MMPs as prognostic indicators for the malignancy potential of OSCC might be considered in every case of tumour examination. We believe that combining patients' MMPs expression intensity and clinical features may improve the process of making diagnosis and prognosis.

Towards personalized medicine: Chemosensitivity assays of patient lung cancer cell spheroids in a perfused microfluidic platform

Cancer is responsible for millions of deaths worldwide and the variability in disease patterns calls for patient-specific treatment. Therefore, personalized treatment is expected to become a daily routine in prospective clinical tests. In addition to genetic mutation analysis, predictive chemosensitive assays using patient's cells will be carried out as a decision making tool. However, prior to their widespread application in clinics, several challenges linked to the establishment of such assays need to be addressed. To best predict the drug response in a patient, the cellular environment needs to resemble that of the tumor. Furthermore, the formation of homogeneous replicates from a scarce amount of patient's cells is essential to compare the responses under various conditions (compound and concentration). Here, we present a microfluidic device for homogeneous spheroid formation in eight replicates in a perfused microenvironment. Spheroid replicates from either a cell line or primary cells from adenocarcinoma patients were successfully created. To further mimic the tumor microenvironment, spheroid co-culture of primary lung cancer epithelial cells and primary pericytes were tested. A higher chemoresistance in primary co-culture spheroids compared to primary monoculture spheroids was found when both were constantly perfused with cisplatin. This result is thought to be due to the barrier created by the pericytes around the tumor spheroids. Thus, this device can be used for additional chemosensitivity assays (e.g. sequential treatment) of patient material to further approach the personalized oncology field.

Tall cell papillary thyroid carcinoma: new diagnostic criteria and mutations in BRAF and TERT

The tall cell (TC) variant of papillary thyroid carcinoma (PTC) has an unfavorable prognosis. The diagnostic criteria remain inconsistent, and the role of a minor TC component is unclear. Molecular diagnostic markers are not available; however, there are two potential candidates: BRAF V600E and telomerase reverse transcriptase (TERT) promoter mutations. Using a novel approach, we enriched a collective with PTCs that harbored an adverse outcome, which overcame the limited statistical power of most studies. This enabled us to review 125 PTC patients, 57 of which had an adverse outcome. The proportion of TCs that constituted a poor prognosis was assessed. All of the tumors underwent sequencing for TERT promoter and BRAF V600E mutational status and were stained with an antibody to detect the BRAF V600E mutation. A 10% cutoff for TCs was significantly associated with advanced tumor stage and lymph node metastasis. Multivariate analysis showed that TCs above 10% were the only significant factor for overall, tumor-specific, and relapse-free survival. Seven percent of the cases had a TERT promoter mutation, whereas 61% demonstrated a BRAF mutation. The presence of TC was significantly associated with TERT promoter and BRAF mutations. TERT predicted highly significant tumor relapse (P<0.001). PTCs comprised of at least 10% TCs are associated with an adverse clinical outcome and should be reported accordingly. BRAF did not influence patient outcome. Nevertheless, a positive status should encourage the search for TCs. TERT promoter mutations are a strong predictor of tumor relapse, but their role as a surrogate marker for TCs is limited.

Cigarette Smoke Exposure Increases Myeloid Cell Production and Lung Bacterial Clearance in Mice

Pneumonia is a leading cause of hospitalization in patients with chronic obstructive pulmonary disease (COPD). Although most COPD patients are smokers, the effects of cigarette smoke exposure on clearance of lung bacterial pathogens and on immune and inflammatory responses are incompletely defined. Here, clearance of Streptococcus pneumoniae and Pseudomonas aeruginosa and associated immune responses were examined in mice exposed to cigarette smoke or following smoking cessation. Mice exposed to cigarette smoke for 6 weeks or 4 months demonstrated decreased lung bacterial burden compared to air-exposed mice when infected 16-24 hours post-exposure. When infection was performed after smoke cessation, bacterial clearance kinetics of mice previously exposed to smoke reversed to comparable levels as those of control mice suggesting that the observed defects were not dependent on adaptive immunological memory to bacterial determinants found in smoke. Comparing cytokine levels and myeloid cell production prior to infection in mice exposed to cigarette smoke relative to mice never exposed or following smoke cessation revealed that reduced bacterial burden was most strongly associated with higher levels of IL-1β and GM-CSF in the lungs and with increased neutrophil reserve and monocyte turnover in the bone marrow. Using serpinb1a-deficient mice with reduced neutrophil numbers and treatment with G-CSF showed that increased neutrophil numbers contribute only in part to the effect of smoke on infection. Our findings indicate that cigarette smoke induces a temporary and reversible increase in clearance of lung pathogens, which correlates with local inflammation and increased myeloid cell output from the bone marrow.

Cell cycle reactivation of cochlear progenitor cells in neonatal FUCCI mice by a GSK3 small molecule inhibitor.

Due to the lack of regenerative capacity of the mammalian auditory epithelium, sensory hair cell loss results in permanent hearing deficit. Nevertheless, a population of tissue resident stem/progenitor cells has been recently described. Identification of methods to trigger their activity could lead to exploitation of their potential therapeutically. Here we validate the use of transgenic mice reporting cell cycle progression (FUCCI), and stemness (Lgr5-GFP), as a valuable tool to identify regulators of cell cycle re-entry of supporting cells within the auditory epithelium. The small molecule compound CHIR99021 was used to inhibit GSK3 activity. This led to a significant increase in the fraction of proliferating sphere-forming cells, labeled by the FUCCI markers and in the percentage of Lgr5-GFP + cells, as well as a selective increase in the fraction of S-G2-M cells in the Lgr5 + population. Using whole mount cultures of the organ of Corti we detected a statistically significant increment in the fraction of proliferating Sox2 supporting cells after CHIR99021 treatment, but only rarely appearance of novel MyoVIIa+/Edu + hair cells. In conclusion, these tools provide a robust mean to identify novel regulators of auditory organ regeneration and to clarify the contribution of stem cell activity.

Lung cancer in the Canton of St. Gallen, Eastern Switzerland: sex-associated differences in smoking habits, disease presentation and survival.

BACKGROUND The aim of this study was to assess sex-associated differences in lung cancer patients in Eastern Switzerland. METHODS All 670 lung cancer patients referred to the cancer center in St. Gallen between January 2000 and December 2005 were retrospectively analyzed. We investigated sex-associated differences in age, smoking habits, histology, stage, treatment and survival. RESULTS There were 474 (71%) men and 196 (29%) women with lung cancer. Mean age at the time of diagnosis was 64 years for women and 67 years for men (p = 0.01). Of the patients <55 years of age, 47 (24%) were women and only 65 (14%) were men. Men smoked significantly more than women (median pack-years: 50 vs. 30; p < 0.001). Of the heavy smokers (>40 pack-years), 278 (56%) were men and 68 (33%) were women. More men had squamous cell carcinoma (36%) than women (17%). Conversely, more women presented with adenocarcinoma (48%) than men (27%). No significant sex-associated differences were observed when analyzing first treatments received. Median overall survival was 10 months for both sexes. CONCLUSIONS In Eastern Switzerland, women with lung cancer were younger, more likely to have smoked significantly less and more likely to have adenocarcinoma, compared to men with lung cancer. These findings are consistent with those found in other western populations.

Human lung-derived mesenchymal stem cell-conditioned medium exerts in vitro antitumor effects in malignant pleural mesothelioma cell lines.

BACKGROUND The soluble factors secreted by mesenchymal stem cells are thought to either support or inhibit tumor growth. Herein, we investigated whether the human lung-derived mesenchymal stem cell-conditioned medium (hlMSC-CM) exerts antitumor activity in malignant pleural mesothelioma cell lines H28, H2052 and Meso4. METHODS hlMSC-CM was collected from the human lung-derived mesenchymal stem cells. Inhibition of tumor cell growth was based on the reduction of cell viability and inhibition of cell proliferation using the XTT and BrdU assays, respectively. Elimination of tumor spheroids was assessed by the anchorage-independent sphere formation assay. The cytokine profile of hlMSC-CM was determined by a chemiluminescence-based cytokine array. RESULTS Our data showed that hlMSC-CM contains a broad range of soluble factors which include: cytokines, chemokines, hormones, growth and angiogenic factors, matrix metalloproteinases, metalloproteinase inhibitors and cell-cell mediator proteins. The 48- and 72-hour hlMSC-CM treatments of H28, H2052 and Meso4 cell lines elicited significant decreases in cell viability and inhibited cell proliferation. The 72-hour hlMSC-CM incubation of H28 cells completely eliminated the drug-resistant sphere-forming cells, which is more potent than twice the half maximal inhibitory concentration of cisplatin. CONCLUSIONS Our findings indicate that the cell-free hlMSC-CM confers in vitro antitumor activities via soluble factors in the tested mesothelioma cells and, hence, may serve as a therapeutic tool to augment the current treatment strategies in malignant pleural mesothelioma.