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Cell survival and apoptosis, ASM-activity, Ceramide and Sphingomyelin levels after siRNA-mediated ASM depletion in NSCLC cell lines.

Decreased NFATc1 in T cells in the tumoural region of NSCLC patients with advanced disease stages.Cytokines secreted from NFATc1Î"CD4 total lung cells did not influence lung tumour cell growth. Representative dot plots of NFATc1 levels in T cells of lung tumor-bearing B6 WT mice in vivo treated and in vitro re-challenged with αPD-1antibodies.

The lipid hydrolase enzyme acid sphingomyelinase (ASM) is required for the conversion of the lipid cell membrane component sphingomyelin into ceramide. In cancer cells, ASM-mediated ceramide production is important for apoptosis, cell proliferation, and immune modulation, highlighting ASM as a potential multimodal therapeutic target. In this study, we demonstrate elevated ASM activity in the lung tumor environment and blood serum of patients with non–small cell lung cancer (NSCLC). RNAi-mediated attenuation of SMPD1 in human NSCLC cells rendered them resistant to serum starvation–induced apoptosis. In a murine model of lung adenocarcinoma, ASM deficiency reduced tumor development in a manner associated with significant enhancement of Th1-mediated and cytotoxic T-cell–mediated antitumor immunity. Our findings indicate that targeting ASM in NSCLC can act by tumor cell–intrinsic and –extrinsic mechanisms to suppress tumor cell growth, most notably by enabling an effective antitumor immune response by the host. Cancer Res; 77(21); 5963–76. ©2017 AACR.

Regulation of T cell phenotype and serum Ceramide/Sphingomyelin levels in wild type and ASM KO naïve and lung tumour bearing mice

Supplementary Figure Legends

Supplementary Figure 3: Representative dot plots of NFATc1 levels in T cells of lung tumor-bearing B6 WT mice in vivo treated and in vitro re-challenged with αPD-1antibodies.

Nuclear factor of activated T cells 1 (NFATc1) is a transcription factor activated by T-cell receptor (TCR) and Ca2+ signaling that affects T-cell activation and effector function. Upon tumor antigen challenge, TCR and calcium-release–activated channels are induced, promoting NFAT dephosphorylation and translocation into the nucleus. In this study, we report a progressive decrease of NFATc1 in lung tumor tissue and in tumor-infiltrating lymphocytes (TIL) of patients suffering from advanced-stage non–small cell lung cancer (NSCLC). Mice harboring conditionally inactivated NFATc1 in T cells (NFATc1ΔCD4) showed increased lung tumor growth associated with impaired T-cell activation and function. Furthermore, in the absence of NFATc1, reduced IL2 influenced the development of memory CD8+ T cells. We found a reduction of effector memory and CD103+ tissue-resident memory (TRM) T cells in the lung of tumor-bearing NFATc1ΔCD4 mice, underlining an impaired cytotoxic T-cell response and a reduced TRM tissue-homing capacity. In CD4+ICOS+ T cells, programmed cell death 1 (PD-1) was induced in the draining lymph nodes of these mice and associated with lung tumor cell growth. Targeting PD-1 resulted in NFATc1 induction in CD4+ and CD8+ T cells in tumor-bearing mice and was associated with increased antitumor cytotoxic functions. This study reveals a role of NFATc1 in the activation and cytotoxic functions of T cells, in the development of memory CD8+ T-cell subsets, and in the regulation of T-cell exhaustion. These data underline the indispensability of NFATc1 for successful antitumor immune responses in patients with NSCLC.Significance: The multifaceted role of NFATc1 in the activation and function of T cells during lung cancer development makes it a critical participant in antitumor immune responses in patients with NSCLC. Cancer Res; 78(13); 3619–33. ©2018 AACR.

Levels of different Ceramide and Sphingomyelin molecular species measured in the blood serum of patients with ADC and SCC as well as in healthy donors

Objectives The physiological number and distribution of mast cells (MCs) in the pediatric gastrointestinal (GI) tract is not well defined and reference values of normality are missing. To define a physiological and disease defining cut-off, a systematic histological exploration of MC distribution from the esophagus to the rectum in healthy as well as in patients with gastrointestinal food allergies (GFA) was performed. Methods Nine pediatric subjects that exhibited unremarkable histopathological evaluations or underwent endoscopy for surveillance reasons after a previous polypectomy of single colonic juvenile polyps served as reference cohort. In all of these subjects a chronic inflammatory disease (e.g. inflammatory bowel disease, celiac disease) or allergy was excluded. In addition, a group of 15 patients with gastrointestinal complaints suspected to be caused by a GFA were investigated. Immunohistochemistry was performed from all biopsies using CD117 (c-Kit) as a reliable marker to identify MCs in the lamina propria. Results There were distinct differences of MC counts in all parts of the pediatric GI tract. The highest counts of MCs in both symptomatic patients and control cohort, were found in the duodenum, terminal ileum, cecum and ascending colon. The lowest counts were found in the esophagus. Significant disparities between GFA and healthy subjects were found in the gastric corpus (22.1 ± 4.0 /HPF vs. 32.0 ± 10.1 /HPF; p = 0.034) and ascending colon (44.8 ± 10.4 /HPF vs. 60.4 ± 24.3 /HPF; p = 0.047). Conclusions Mucosal MC counts in the pediatric GI tract are higher than previously reported, with a considerable overlap between healthy and GFA patients. These results provide detailed information on distribution and numbers of MCs in pediatric allergic patients while allowing estimates of physiological values in childhood for the first time. With regard to diagnostic procedures in GFA further laboratory parameters have to be integrated.

The hypereosinophilic syndrome (HES) is a rare disorder characterized by hypereosinophilia and infiltration of various organs with eosinophils. Eosinophilic cystitis (EC), mimicking bladder cancer clinically but also in ultrasound and in radiographic imaging, is one potential manifestation of the HES occurring in adults as well as in children. This case report describes the course of disease in a 57-year-old male presenting with severe gait disorders and symptoms of a low compliance bladder caused by a large retropubic tumor. After extensive urine and serologic examination and histologic confirmation of EC the patient was subjected to medical treatment with cetirizine and prednisolone for 5 weeks. While gait disorders rapidly resolved, micturition normalized only 10 months after initiation of therapy. Based upon this course the authors recommend patience and reluctance concerning radical surgical intervention in EC. Key Points • Eosinophilic cystitis is a rare condition with app. 200 cases reported, so far. • Etiology of eosinophilic cystitis is obscure, but allergies and parasitic infections may trigger the disease. • Genetic alterations (e.g., BRAF mutations) may predispose for the disease • Corticosteroids and antihistamines are the backbone of therapy and may be complemented by antibiotics and non-steroidal anti-inflammatory drugs in case of concomitant (underlying) infections. • As recovery can occur even after a long time, radical surgery should be restricted to highly selected cases.

Gastrointestinally-mediated food allergy (GMA) incidentally may cause non-erosive oligoarthritis. A 56-year-old woman with seronegative spondyloarthritis observed arthritic flares following ingestion of certain foods. Blood analyses were unremarkable. Segmental gut lavage revealed plasmacellular infiltrates and eosinophilia in terminal ileum. Food allergen-specific IgE-analysis of lavage fluid showed significant polyvalent intestinal IgE-sensitization (>0.35kU/mg protein) towards wheat, rye, egg, soybean, pork, beef, nuts. Repeated exposure to the aforementioned foods caused arthritis within 48 hours; elimination diet engendered long-term remission. Seronegative local IgE-mediated GMA (type I) was diagnosed. Especially seronegative rheumatoid arthritis and undifferentiated oligoarthritis should be scrutinized for food-related muscoloskeletal symptoms before initiating immunosuppression.

International journal of molecular sciences 23(23), 15126 (2022). doi:10.3390/ijms232315126 special issue: "Special Issue "Neutrophil Extracellular Traps (NETs) in Immunity and Diseases" / Special Issue Editor: Małgorzata Wachowska, Guest Editor", Published by Molecular Diversity Preservation International, Basel

Benzyl alcohol is a widely used preservative, solvent and fragrance material. According to published data, it is a rare sensitizer in humans.To identify characteristics and sensitization patterns of patients with positive patch test reactions to benzyl alcohol and to check the reliability of the patch test preparation benzyl alcohol 1% pet.Retrospective analysis of data from the Information Network of Departments of Dermatology (IVDK), 2010-2019.Of 70 867 patients patch tested with benzyl alcohol 1% pet., 146 (0.21%) showed a positive reaction, most of them (89%) only weakly positive. The number of doubtful and irritant reactions significantly exceeded the number of positive reactions. Reproducibility of positive test reactions was low. Among benzyl alcohol-positive patients, compared to benzyl alcohol-negative patients, there were significantly more patients with leg dermatitis (17.8% vs. 8.6%), more patients aged 40 years or more (81.5% vs. 70.5%) and more patients who were tested because of a suspected intolerance reaction to topical medications (34.9% vs. 16.6%). Concomitant positive reactions were mainly seen to fragrances, preservatives and ointment bases.Sensitization to benzyl alcohol occurs very rarely, mainly in patients with stasis dermatitis. In view of our results, benzyl alcohol cannot be regarded as a significant contact allergen, and therefore marking it as skin sensitizer 1B and labelling it with H 317 is not helpful.

Background Major pathologic response (MPR) determines favorable outcome in locally advanced non-small cell lung cancer after induction therapy (IT) followed by lung resection. The aim of this retrospective study was to identify the prognostic relevance of MPR in long-term interval. Methods In 55 patients, the survival rate according to MPR and non-MPR was estimated by Kaplan–Meier method and compared using log-rank, Breslow, and Tarone–Ware tests. Results The IT included chemoradiation with 50.4 Gy (range: 45–56.4 Gy) combined with platinum-based chemotherapy in 52 patients (94.5%) and platinum-based chemotherapy in 3 patients (5.5%). Perioperative morbidity and 30-day mortality were 36 and 3.6%, respectively. The estimated 5-year postoperative and progressive-free survivals were statistically significantly improved in MPR versus non-MPR with 53.5 versus 18% and 49.4 versus 18.5%, respectively. According to the log-rank, Breslow, and Tarone–Ware tests, the MPR demonstrates prognostic significance in early, long-term, and whole postoperative interval. Conclusion MPR is associated with a robust correlation to long-term postoperative and recurrence-free survival improvement, and can potentially simplify the multidisciplinary debate and allow further stratification of adjuvant treatment in multimodality therapy.

Tumor suppressor SMARCA4 (BRG1), a key SWI/SNF chromatin remodeling gene, is frequently inactivated in cancers and is not directly druggable. We recently uncovered that SMARCA4 loss in an ovarian cancer subtype causes cyclin D1 deficiency leading to susceptibility to CDK4/6 inhibition. Here, we show that this vulnerability is conserved in non-small cell lung cancer (NSCLC), where SMARCA4 loss also results in reduced cyclin D1 expression and selective sensitivity to CDK4/6 inhibitors. In addition, SMARCA2, another SWI/SNF subunit lost in a subset of NSCLCs, also regulates cyclin D1 and drug response when SMARCA4 is absent. Mechanistically, SMARCA4/2 loss reduces cyclin D1 expression by a combination of restricting CCND1 chromatin accessibility and suppressing c-Jun, a transcription activator of CCND1. Furthermore, SMARCA4 loss is synthetic lethal with CDK4/6 inhibition both in vitro and in vivo, suggesting that FDA-approved CDK4/6 inhibitors could be effective to treat this significant subgroup of NSCLCs., Non-small cell lung cancers with inactivating SMARCA4 mutations are currently undruggable. Here, the authors show that the absence of SMARCA4/2 reduces chromatin accessibility at the CCND1 locus, leading to a subsequent reduction in cyclin D1 expression, which promotes vulnerability of these cancers to CDK4/6 inhibition.

Although lung cancer is the leading cause of cancer death worldwide, the mechanisms how lung cancer cells evade the immune system remain incompletely understood. Here, we discovered IL-9-dependent signaling mechanisms that drive immune evasion in non-small cell lung cancer (NSCLC). We found increased IL-9 and IL-21 production by T cells in the tumoral region of the lung of patients with NSCLC, suggesting the presence of Th9 cells in the lung tumor microenvironment. Moreover, we noted IL-9 producing Tregs in NSCLC. IL-9 target cells in NSCLC consisted of IL-9R+ tumor cells and tumor-infiltrating lymphocytes. In two murine experimental models of NSCLC, and in vitro , IL-9 prevented cell death and controlled growth of lung adenocarcinoma cells. Targeted deletion of IL-9 resulted in successful lung tumor rejection in vivo associated with an induction of IL-21 and reduction of lung pSTAT3, Th17 and Treg cells. Finally, anti-IL-9 antibody immunotherapy resulted in suppression of tumor development even in established experimental NSCLC and was associated with reduced IL-10 production and induction of IL-21 in the lung. In conclusion, our findings indicate that IL-9 drives immune escape of lung tumor cells via effects on tumor cell survival and tumor infiltrating T cells. Thus, strategies blocking IL-9 emerge as a new approach for clinical therapy of lung cancer. Funding: This work was supported by DFG grant FI817-5-1,2 and by a DFG FI817- 5-3 granted to SF. M.T.C. work is supported by a DFG CH 1428/2-1. MHK was supported by NIH grant AI057459. Declaration of Interest: None to declare. Ethical Approval: Our human study was performed at the Friedrich-Alexander-University Erlangen-Nurnberg, Germany, after being approved by the ethics review board of the University of Erlangen (Re-No: 56_12B; DRKS-ID: DRKS00005376).

Lung mucosal immunity controls the entrance of infectious agents and allergens into the airways and thus must be tightly regulated. The anti-inflammatory function of Interleukin-10 (IL-10) has been demonstrated in spontaneous Th1-driven colitis in IL-10 deficient mice (IL-10-/-). Allergic asthma is characterized by pathological pulmonary Th2 responses to allergens. Here we investigated the role of IL-10 in lung homeostasis in allergic asthma by analyzing wild type and IL-10 deficient mice in a model of asthma induced by house dust mite (HDM). Targeted deletion of IL-10 resulted in increased lung inflammation, airway mucus production and serum IgE. In further cellular analysis of this lung inflammation we found that IL-10 deficient asthmatic mice had induction of CD4+GATA-3+Th2 cells and SiglecF+CD45.2+CD62L-CD101+inflammatory eosinophils(iEos) as demonstrated by FACS analysis in isolated lung cells of IL-10-/- and WT mice treated with or without HDM. Consistently, the Th2 cytokines IL-4 and IL-13 were found induced in the lung cell supernatants after anti-CD3 and anti-CD28 antibody treatment. Finally, Interleukin-9 (IL-9), a pleiotropic cytokine that influences different target cells involved in allergic asthma, is induced by IL-4 and TGF-beta. Consistently, IL-9 was found induced in the lung cell supernatants of IL-10 deficient mice stimulated with anti CD3 and anti CD28 antibodies. In conclusion, these data indicate that the anti-inflammatory effects of IL-10 in asthma relate to the inhibition of the Th2 transcription factor GATA-3 which results in inhibition of both IL-4 and IL-9. Thus IL-10 is involved in the resolution of allergic asthma by inhibiting GATA-3 expression.

Immunotherapy is a promising treatment for lung cancer by activation of cytotoxic CD4+ and CD8+ T cells in the tumour microenvironment and suppression of Tregs cells. We previously demonstrated that the recognition and elimination of lung tumour cells by T lymphocytes requires the presence of T-box-expressed in T cells (T-bet), a transcriptional regulator that induces Th1, cytolytic CD8+ T and NK cells and inhibits Treg cells. Moreover, recent studies show that IL-2 in the tumour microenvironment supports acquisition of cytotoxic T helper cells through the transcriptional repressor B lymphocytes-induced maturation protein-1 (Blimp-1). As Blimp-1 also affects the homeostasis and function of CD4+, CD8+ T cells as well as Treg cells, we hypothesized that this transcription factor might be important in lung cancer. We thus started to analyze the Blimp-1 protein expression by Western blot analysis using proteins isolated from the lung of a cohort of patients that underwent surgery because of non-small-cell lung carcinoma (NSCLC) and subsequent histological classification as lung adenocarcinoma (ADC). We found that patients with ADC had a significant induction of Blimp-1 protein expression in the tumoural region of ADC (TU, solid tumour) as compared control lung region (CTR, tumour-free control area). We further analyzed the expression of T-bet in these patients and found that T-bet, as opposed to Blimp-1, was mainly expressed in the control region of the lung and downregulated in the tumoural region. Thus, these data suggest that Blimp-1 might represent an additional new marker to be considered in the absence of T-bet as driver of anti-tumor immune responses in lung cancer.

BACKGROUND Confocal laser endomicroscopy (CLE) enables "in vivo" microscopic tissue diagnosis based on tissue reflectance or tissue fluorescence upon application of fluorescence agents. The aim of the present study was to evaluate CLE as a new diagnostic approach for differentiation between malignant versus non-malignant pleural effusions. MATERIAL AND METHODS In 100 patients with pleural effusions, thoracentesis was performed. Cresyl violet and acriflavine were used as contrast agents for probe-based CLE of effusions. CLE video sequences were assessed by 4 independent investigators (2 experienced in this technique, 2 with only basic knowledge). In addition, all CLE samples were evaluated by an expert pathologist (p). Results were compared with conventional cytology of effusions and histology of cell blocks. RESULTS CLE reliably permitted identification of malignant cells in pleural effusions. Sensitivity for detection of malignant effusions was 87% (p: 87%) and 81% (p: 72%) for acriflavine and cresyl violet, respectively. With regard to specificity, acriflavine and cresyl violet yielded a mean value of 99% (p: 100%) and 92% (p: 100%). CONCLUSIONS In this pilot study, CLE permitted simple and rapid detection of malignant pleural effusions. Larger prospective studies are warranted to corroborate our findings.

Nuclear factor of activated T cells 1 (NFATc1) is a transcription factor activated by T-cell receptor (TCR) and Ca2+ signaling that affects T-cell activation and effector function. Upon tumor antigen challenge, TCR and calcium-release–activated channels are induced, promoting NFAT dephosphorylation and translocation into the nucleus. In this study, we report a progressive decrease of NFATc1 in lung tumor tissue and in tumor-infiltrating lymphocytes (TIL) of patients suffering from advanced-stage non–small cell lung cancer (NSCLC). Mice harboring conditionally inactivated NFATc1 in T cells (NFATc1ΔCD4) showed increased lung tumor growth associated with impaired T-cell activation and function. Furthermore, in the absence of NFATc1, reduced IL2 influenced the development of memory CD8+ T cells. We found a reduction of effector memory and CD103+ tissue-resident memory (TRM) T cells in the lung of tumor-bearing NFATc1ΔCD4 mice, underlining an impaired cytotoxic T-cell response and a reduced TRM tissue-homing capacity. In CD4+ICOS+ T cells, programmed cell death 1 (PD-1) was induced in the draining lymph nodes of these mice and associated with lung tumor cell growth. Targeting PD-1 resulted in NFATc1 induction in CD4+ and CD8+ T cells in tumor-bearing mice and was associated with increased antitumor cytotoxic functions. This study reveals a role of NFATc1 in the activation and cytotoxic functions of T cells, in the development of memory CD8+ T-cell subsets, and in the regulation of T-cell exhaustion. These data underline the indispensability of NFATc1 for successful antitumor immune responses in patients with NSCLC. Significance: The multifaceted role of NFATc1 in the activation and function of T cells during lung cancer development makes it a critical participant in antitumor immune responses in patients with NSCLC. Cancer Res; 78(13); 3619–33. ©2018 AACR.

Zusammenfassung Hintergrund Der prognostische Stellenwert von „Salvage“-Resektionen nach definitiver Radiochemotherapie im Vergleich zur kurativen Lungenresektion nach Induktionstherapie bei lokal fortgeschrittenem nicht kleinzelligem Lungenkarzinom im Stadium IIIA/B wurde bislang nur selten untersucht. Ziel unserer retrospektiven Datenanalyse war, die perioperativen Ergebnisse, das postoperative und rezidivfreie Überleben nach beiden Verfahren zu vergleichen und mögliche relevante Prognosefaktoren zu identifizieren. Patienten und Methoden Im Zeitraum von Juni 2008 bis Mai 2017 wurden 43 Patienten mit Resektion nach Induktionstherapie (Gruppe 1) und 14 nach definitiver Radiochemotherapie (Gruppe 2) identifiziert. Die retrospektive Datenanalyse erfasste demografische Faktoren, Tumorstadium und Lokalisation, das initiale Therapieregime, die präoperative Regression, die perioperative Mortalität und Letalität, das postoperative und rezidivfreie Überleben. Ergebnisse In Gruppe 2 wurde eine signifikant höhere Gesamtstrahlendosis appliziert (p = 0,00) und ein signifikant längeres Zeitintervall zwischen der Radiochemotherapie und Lungenresektion (p = 0,02) festgestellt. Zusätzlich wurde ein signifikant höherer perioperativer Blutverlust und häufigere Bluttransfusionen beobachtet (p = 0,003 bzw. p = 0,005). Die perioperative Morbidität und Letalität waren nach beiden Verfahren statistisch vergleichbar (p = 0,72 bzw. 0,395). Das 5-Jahres-Überleben postoperativ betrug in Gruppe 1 55% bzw. Gruppe 2 48% (log-rank p = 0,353). Das rezidivfreie Überleben nach 5 Jahren war in Gruppe 1 53% bzw. in Gruppe 2 42% (log-rank p = 0,180). In Gruppe 2 trat häufiger diffuse Metastasierung auf, während in Gruppe 1 Oligometastasierung beobachtet wurde. Schlussfolgerung Die Prognose nach Salvage-Resektion erscheint vergleichbar mit den Ergebnissen der kurativen Resektion nach Induktionstherapie. Diffuse Fernmetastasierung wird häufig beobachtet. Eine strenge Patientenselektion ist erforderlich.

Although still a matter of controversial discussion, skeletal myoblasts are one of the options for stem cell transplantation improving cardiac function after myocardial infarction, exhibiting several advantages including the availability, the ability of self-renewal and differentiation, and the lack of ethical and immunological problems. The aim of this study was to investigate the impact of stem cell therapy with skeletal myoblasts on experimental venous bypass grafts in a mouse model of vein graft disease. Forty C57BL/6J mice underwent bypass grafting interposing a venous bypass graft of the donor mouse into the carotid artery of the recipient mouse. Twenty mice received periadventitially treatment with 1 million fluorescence labeled skeletal myoblasts suspended in culture medium (treatment group), the other twenty mice received only culture medium without myoblasts (control group). Two weeks after bypass surgery, the vein grafts of all 40 mice were harvested, stained and histologically investigated under light and immunofluorescence microscope. Against our expectations, skeletal myoblasts stayed in place and were still located in the adventitia after bypass grafting. Additionally, vein grafts of the myoblast group revealed a 2fold increased neoneointima formation, a decreased media thickness, a slightly increased neovascularization, a higher percentage of reendothelialization and also a slightly higher percentage of PDGFR ɑ, PDGFR ß, MMP-7 and MMP-9 positive cells, suggesting a paracrine mechanism responsible for accelerated neointima formation. In conclusion, the results of our study do not support the use of skeletal myoblast for the treatment of vein graft disease after coronary artery bypass surgery.

// Djeda Belharazem 1 , Albert Grass 1 , Cornelia Paul 1 , Mario Vitacolonna 2 , Berthold Schalke 3 , Ralf J. Rieker 4, 5 , Daniel Korner 6 , Philipp Jungebluth 6 , Katja Simon-Keller 1 , Peter Hohenberger 2 , Eric M. Roessner 2 , Karsten Wiebe 7 , Thomas Grater 8 , Thomas Kyriss 9 , German Ott 10, 11 , Peter Geserick 12 , Martin Leverkus 12, 13, * , Philipp Strobel 14 and Alexander Marx 1 1 Institute of Pathology and Medical Research Center (ZMF), University Medical Centre Mannheim, University of Heidelberg, Mannheim, Germany 2 Department of Thoracic Surgery, University Medical Centre Mannheim, University of Heidelberg, Mannheim, Germany 3 Department of Neurology, University of Regensburg, Regensburg, Germany 4 Institute of Pathology, University Hospital Heidelberg, University of Heidelberg, Heidelberg, Germany 5 Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany 6 Department of Thoracic Surgery, Thorax Clinic, University of Heidelberg, Heidelberg, Germany 7 Department of Thoracic Surgery, University of Munster, Munster, Germany 8 Department of Thoracic Surgery, Clinic Lowenstein, Lowenstein, Germany 9 Department of Thoracic Surgery, Clinic Schillerhohe, Robert-Bosch-Hospital, Gerlingen, Germany 10 Department of Clinical Pathology, Robert-Bosch-Hospital, Stuttgart, Germany 11 Dr. Margarete Fischer-Bosch Institute for Clinical Pharmacology, Stuttgart, Germany 12 Department of Dermatology, Venereology, and Allergology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany 13 Department for Dermatology and Allergology, University Hospital Aachen, RWTH Aachen, Aachen, Germany 14 Institute of Pathology, University Medical Center Gottingen, University of Gottingen, Gottingen, Germany * Deceased Correspondence to: Djeda Belharazem, email: djeda.belharazem@medma.uni-heidelberg.de Keywords: thymic tumors, cFLIP, NF-κB, senescence, autophagy Received: September 01, 2016 Accepted: December 26, 2016 Published: February 06, 2017 ABSTRACT The anti-apoptotic cellular FLICE-like inhibitory protein cFLIP plays a pivotal role in normal tissues homoeostasis and the development of many tumors, but its role in normal thymus (NT), thymomas and thymic carcinomas (TC) is largely unknown. Expression, regulation and function of cFLIP were analyzed in biopsies of NT, thymomas, thymic squamous cell carcinomas (TSCC), thymic epithelial cells (TECs) derived thereof and in the TC line 1889c by qRT-PCR, western blot, shRNA techniques, and functional assays addressing survival, senescence and autophagy. More than 90% of thymomas and TSCCs showed increased cFLIP expression compared to NT. cFLIP expression declined with age in NTs but not in thymomas. During short term culture cFLIP expression levels declined significantly slower in neoplastic than non-neoplastic primary TECs. Down-regulation of cFLIP by shRNA or NF-κB inhibition accelerated senescence and induced autophagy and cell death in neoplastic TECs. The results suggest a role of cFLIP in the involution of normal thymus and the development of thymomas and TSCC. Since increased expression of cFLIP is a known tumor escape mechanism, it may serve as tissue-based biomarker in future clinical trials, including immune checkpoint inhibitor trials in the commonly PD-L1 high thymomas and TCs.

BACKGROUND The diagnostic significance of the atopy patch test for the management of dermatitis possibly triggered by aeroallergens is still controversial. However, sufficiently large studies with routinely tested standardized aeroallergen patch test preparations in dermatitis patients are lacking. OBJECTIVE To evaluate the reaction frequency and the reaction profiles of 10 until mid-2015 commercially available, standardized aeroallergen patch test preparations of the 'Stallerpatch' test series (Stallergenes, Antony Cedex, France) in a large multicentre patient cohort. METHODS A retrospective data analysis of patients with suspected aeroallergen-dependent eczematous skin lesions was performed, who were patch tested in 15 Information Network of Departments of Dermatology-associated clinics between 2000 and 2015. Patients were stratified according to their atopic dermatitis (AD) status. RESULTS The study group included 3676 patients (median age 41 years, 34.8% males, 54.5% AD). The most common aeroallergens causing positive patch test reactions were Dermatophagoides pteronyssinus (19.6%), Dermatophagoides farinae (16.9%), birch (6.2%), timothy grass (6.0%), cat dander (5.4%), mugwort (4.9%) and dog dander (4.6%). Reactions to other pollen allergen preparations, that is 5 grasses (3.2%), cocksfoot (2.1%) and plantain (1.6%), were less common. Positive patch test reactions to aeroallergens were consistently more frequent in patients with AD. These patients showed proportionally less dubious, follicular, irritant and weak positive reactions. Independent of AD status, a patient history of past or present allergic rhinitis was associated with an increased chance of a positive aeroallergen patch test reaction to pollen allergens. CONCLUSION The aeroallergen patch test is a useful add-on tool in clinical routine, especially in patients with AD and/or respiratory allergy. A patch test series comprising Dermatophagoides pteronyssinus, Dermatophagoides farinae, birch, timothy grass, cat dander and mugwort seems to be suitable. Controlled studies with specific provocation and elimination procedures are required to further evaluate the diagnostic significance of the proposed screening series.

The mechanisms of lung cancer evading the immune system are still not fully understood. As regulatory T cells (Tregs) exhibit a pro-tumoral function they are in focus of immunotherapies. Peripheral Treg differentiation is driven by TCR and its co-stimulation. A further crucial signal displays the binding of IL-2 to the high affinity receptor CD25 with downstream involvement of Signal Transducer and Activator of Transcription 5 (STAT5). STAT5 activation as well as TGF-beta stimulation in the periphery leads to expression of the Treg key factor FoxP3. In this study, we wanted to investigate the role of STAT5 and its activated status via phosphorylation (pSTAT5) within lung cancer. We thus isolated peripheral blood mononuclear cells (PBMC) from NSCLC patients and healthy donors. PBMCs were cultured under different stimulation conditions for 4 days and analyzed by FACS. Here we found CD4+ T cells with high induction of pSTAT5A and CD25 upon aCD3 and aCD28 antibody treatment. Moreover, CD4+CD25+FoxP3+ Tregs were detected under T cell activating conditions, and were found to be increased by TGFβ in smokers and NSCLC patients. The Treg population was further inducible by adding low doses of IL-2 while CD4+CD25+FoxP3− effector T cells decreased in the presence of TGFβ. Our findings point to an immunosuppressive microenvironment induced by the tumor with increased TGFβ and reduced IL-2 forcing effector T cells into a Treg state. These preliminary data suggest that current immunotherapy by aCD3/aCD28 antibody treatment could be improved by an additional blockade of TGFβ and induction of IL-2 levels. This treatment would result in an increase of CD4+CD25+pSTAT5+FoxP3− effector cells and a reduction of CD4+CD25+FoxP3+STAT5+ Tregs.

The transcription factor PU box binding-1 (PU.1), which is encoded by the SPI1 gene, is required for the development of most myeloid cell lineages as well as B- and T cells. Consequently, an alteration of the SPI1 gene and thus PU.1 can have severe impact on the immune system. However, the role of PU.1 in cancer is not yet fully understood. In this study, we were looking for a hint on the influence of PU.1 expression on various cell types during non-small cell lung cancer (NSCLC). To start the investigation, we isolated peripheral blood mononuclear cells (PBMCs) from patients with NSCLC as well as from healthy controls. The PBMCs were cultured with different cytokines for 4 days and subsequently analyzed by flow cytometry. Furthermore, RNA was isolated from the PBMCs and qPCR was performed. The qPCR results showed induced PU.1 mRNA levels in NSCLC patients in relation to GAPDH, compared to control patients. By flow cytometry, we could detect higher PU.1 expression in CD56+ Natural killer (NK) cells from lung cancer patients compared to the healthy donors. Moreover, NK-cells from lung cancer patients, in contrast to healthy controls, expressed the immunosuppressive protein Programmed Death 1 (PD-1). Furthermore, PBMCs from NSCLC patients that were cultured with aCD3/aCD28 antibodies, showed suppression of PU.1 as well as PD-1 in NK-cells, which was even more prominent after addition of IL-2. These preliminary data suggest, that conditions inducing T cell activation like IL-2 and antiCD3/antiCD28 stimulation, can downregulate both the transcription factor PU.1 and PD-1 in NK-cells in tumor patients. Thus, improvement of current immunotherapy could use additional IL-2, which targets PU-1 as well as PD-1 in NK cells in lung cancer.

Immunotherapy improves the life expectancy of patients with lung cancer by targeting the immune inhibitory T cell surface markers resulting in the activation of anti-tumor T cell survival and suppression of tumor cell growth. The transcription factor repressor Positive Regulatory Domain Containing Protein 1 (encoded in humans by the Prdm1 gene) also known as B lymphocytes-induced maturation protein-1 (Blimp-1) affects the homeostasis and function of CD4+ and CD8+ T cells as well as T regulatory cells (T regs). We recently reported that Blimp-1 is induced in the lung tumoral region of patients with lung ADC. To further investigate whether Blimp-1 could be a candidate gene involved in the regulation of the anti-tumor immune-response in lung cancer, we started to investigate peripheral blood mononuclear cells (PBMCs) isolated from healthy control subjects and NSCLC patients for Blimp-1 expression in immunosuppressive CD4+CD25+Foxp3+ T regs. The PBMCs were cultured with and without antiCD3/CD28 antibodies, without and with TGF-beta, to mimic the tumor microenvironment, for 4 days and subsequently analyzed by flow cytometry using antibodies against Blimp-1, Foxp3, CD4 and CD25(IL-2R alpha chain). Here we found, by trend, induced CD4+Blimp1+CD25+Foxp3+ Tregs in unstimulated PBMCs from patients with lung cancer. Moreover, TGF-beta further induced these Blimp1+CD4+CD25+Foxp3+ Tregs in PBMCs obtained both from healthy controls and from NSCLC patients. Taken together these data indicate that the tumor microenvironment induces Blimp-1 in immunosuppressive CD4+CD25+Foxp3+ Tregs via TGF-beta. Thus, current immunotherapy should be combined to TGF-beta inhibitors to target CD4+CD25+Blimp1+Foxp-3+ T regulatory cells.

Lung cancer is one of the most common cancer types in the western world, with a high lethality and increasing incidence. One major factor, supporting tumor growth is a high sugar diet. Thus, reducing the sugar intake by different diets might be a way to improve current cancer treatment. These metabolic changes are accompanied by regulation of systemic levels of Insulin and insulin like growth factors. As oppose to normal cells, tumor cells metabolism mainly depends on glycolysis. Thus, reducing diet carbohydrate intake might be a way to improve current immunotherapy by inducing immunological changes caused by the extracellular glucose concentration in tumor microenvironment. Therefore, we cultured the human lung adenocarcinoma (ADC) cell line A549 with different extracellular glucose concentrations to analyze effects by flow cytometry and qPCR. Moreover, lung tissue samples from patients with non small cell lung cancer (NSCLC), were analyzed by qPCR. In a murine model of lung ADC, tumor cells were injected intravenously and mice were fed with different diets. Lung cells were isolated and analyzed by flow cytometry. Here we show that, IGF1R expression is upregulated in A549 cells line, depending on increasing extracellular glucose concentration. Moreover, in studies on human cohorts of patients with NSCLC, we found a direct correlation between BMI and IGF1R expression and its downstream signaling. Finally, in a murine model of disease, diet glucose restriction decreased significantly tumor load associated with increased lung infiltrating CD8+ T-cells and tissue-resident memory T-cells. Thus, this study supports the need to introduce glucose restrictions in the diet of patients with NSCLC in association with current immunotherapy.

Thymomas and thymic squamous cell carcinomas (TSQCCs) are rare thymic epithelial tumours. Data on angiogenesis and vascular phenotype in these tumours are limited, and no study has taken histological World Health Organization (WHO) subtypes into account. The aim of this study was to compare vascularization, pericytes coverage and expression of angiogenic growth factors in different WHO-defined subtypes of thymoma METHODS AND RESULTS: Vascular density, diameter and architecture and expression of α-smooth muscle actin (SMA), platelet-derived growth factor (PDGF) receptor-β (PDGFRβ), vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) were investigated in WHO type A, AB, B1, B2 and B3 thymomas and TSQCCs, by the use of immunostaining, quantitative morphometry, and tumour vessel isolation by trypsin digestion. Expression levels of angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2), VEGF-A, PDGF-B and Hif-1α were examined by quantitative reverse transcription polymerase chain reaction. A and AB thymomas were characterized by a dense network of capillary-like vessels with tight pericyte coverage, whereas B thymomas showed a loose vascular network with increasing vascular diameters and increasing expression of SMA and PDGFRβ from B1 to B3 thymomas and TSQCCs. VEGFR1 and VEGFR2 were expressed in vessels of all analysed tumour entities, and at higher levels in epithelial cells of A and B3 thymomas and TSQCCs. mRNA of Ang-2, but not of Ang-1, was significantly up-regulated in all thymoma subtypes, with the highest levels being found in A thymomas. In TSQCCs, Ang-1 and VEGF were the predominantly up-regulated growth factors. Hif-1α was only up-regulated in B3 thymomas and TSQCCs.Thymomas and TSQCCs differ significantly in their vascular architecture and expression of key angiogenic growth factors. The findings could help to improve the differential diagnosis of difficult-to-classify thymic epithelial tumours, and indicate different mechanisms of tumour angiogenesis and functional differences of tumour vessels of major thymoma subtypes and TSQCCs.

Mitochondrial homeostasis has recently emerged as a focal point in the pathophysiology of idiopathic pulmonary fibrosis (IPF), but conflicting data have been reported regarding its regulation. We speculated that phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein at the intersection of multiple cell death and mitochondrial turnover pathways, might be involved in the pathogenesis of IPF. PGAM5-deficient mice and human pulmonary epithelial cells were analyzed comparatively with PGAM5-proficient controls in a bleomycin-based model of pulmonary fibrogenesis. Mitochondria were visualized by confocal and transmission electron microscopy. Mitochondrial homeostasis was assessed using JC1 (ΔΨ) and flow cytometry. PGAM5 plays an important role in pulmonary fibrogenesis. Pgam5−/− mice displayed significantly attenuated lung fibrosis compared to controls. Complementary, in vitro studies demonstrated that PGAM5 impaired mitochondrial integrity on a functional and structural level independently of mtROS-production. On a molecular level, reduced mitophagy caused by PGAM5 deficiency improved mitochondrial homeostasis. Our study identifies PGAM5 as an important regulator of mitochondrial homeostasis in pulmonary fibrosis. Our data further indicate PGAM5-mediated mitophagy itself as a pivotal gateway event in the mediation of self-sustaining mitochondrial damage and membrane depolarization. Our work hereby highlights the importance of mitochondrial dynamics and identifies a potential therapeutic target that warrants further studies. Toxic agents lead to mitochondrial damage resulting in depolarization of the mitochondrial membrane potential (ΔΨ) which is a gateway event for the initiation of PGAM5-mediated mitophagy. PGAM5-mediated mitophagy in turn leads to a self-perpetuating escalation of ΔΨ depolarization. Loss of the mitophagy-based damage-enhancing loop under PGAM5-deficient conditions breaks this vicious cycle, leading to improved mitochondrial homeostasis.

Background The immunosuppressive role of the cytokine IL-35 in patients with non-small cell lung cancer (NSCLC) is poorly understood. In this study, we analysed the localisation and regulation of IL-35 in the lung of patients with non-small cell lung cancer (NSCLC) to further elucidate the immune-escape of cancer cells in perioperative course of disease. Methods Interleukin 35 (IL-35) was measured by ELISA in postoperative serum from 7 patients with NSCLC as well as 8 samples from healthy controls. Immunohistochemistry, FACS analysis, real-time PCR, as well as western blot from samples of the control (CTR), peri-tumoural (PT) and the tumoural (TU) region of the lung derived from patients with NSCLC and 10 controls were performed. Results Here we found elevated levels of IL-35 in the TU region as well as postoperative serum from patients with lung adenocarcinoma. Consistently, we found an increased expression of IL-35+Foxp-3+ cells, which associated with ARG1 mRNA expression and decreased TNFA in the TU region of the lung of patients with NSCLC as compared to their CTR region. Furthermore, in the CTR region of the lung of patients with NSCLC, CD68+ macrophages were induced and correlated with IL-35+ cells. Finally, IL-35 positively correlated with TTF-1+PD-L1+ cells in the TU region of NSCLC patients. Conclusions Induced IL-35+Foxp3+ cell numbers in the TU region of the lung of patients with NSCLC associated with ARG1 mRNA expression and with TTF-1+PD-L1+ cells. In the tumour-free CTR area, IL-35 correlated with CD68+ macrophages. Thus inhibitors to IL-35 would probably succeed in combination with antibodies against immune checkpoints like PD-L1 and PD-1 currently used against NSCLC because they would inhibit immunosuppressive macrophages and T regulatory cells while promoting T cell-mediated anti-tumoural immune responses in the microenvironment as well as the TU region of NSCLC patients.

Summary Autoimmunologic phenomena triggered by CD8-positive cytotoxic T cells may be involved in the pathogenesis of achalasia, but the pathogenic mechanisms generally remain unknown, and the concomitant histopathologic changes in the esophageal epithelium in achalasia are incompletely described. This study was designed to characterize the inflammation within the esophageal epithelium of these patients. We collected 72 esophageal specimens from 38 consecutive patients with achalasia (16 men, 22 women) representing different disease stages. The inflammatory cells were characterized immunohistochemically. Seven end-stage disease esophagectomy specimens were also included. Our data show a T-cell–rich inflammatory response predominantly composed of CD4-positive T cells with an overall CD4/CD8 ratio of 1.82 and only few CD4-CD25-FoxP3–positive regulatory T cells or CD20-positive B cells. Signs of inflammation were most pronounced in the middle esophagus, followed by upper and lower esophagus. Between different stages of disease, there were statistically significant differences among CD25-positive lymphocytes in the upper esophagus and CD4-, CD8-, and CD25-positive lymphocytes in the middle esophagus. The esophageal epithelial inflammation in achalasia seems to be of reactive and unspecific nature and does not reflect the composition of the CD8-dominant inflammatory infiltrate within the muscular esophageal wall in active disease. The statistically significant differences in unspecific inflammation between different stages of disease are more likely due to effects of the dysmotility and cannot be assumed pathognomic for the disease. In conclusion, we assume that it is not possible to diagnose or confirm achalasia by means of esophageal biopsy alone.