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TRPCs (transient receptor potential classical or cation channels) play a crucial role in tumor biology, especially in the Ca2+ homeostasis in cancer cells. TRPC4 is a pH-sensitive member of this family of proteins. As solid tumors exhibit an inversed pH-gradient with lowered extracellular and increased intracellular pH, both contributing to tumor progression, TRPC4 might be a signaling molecule in the altered tumor microenvironment. This is the first study to investigate the expression profiles of TRPC4 in common skin cancers such as basal cell carcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma (MM) and nevus cell nevi (NCN). We found that all SCCs, NCNs, and MMs show positive TRPC4-expression, while BCCs do only in about half of the analyzed samples. These data render TRPC4 an immunohistochemical marker to distinguish SCC and BCC, and this also gives rise to future studies investigating the role of TRPC4 in tumor progression, and especially metastasis as BCCs very rarely spread and are mostly negative for TRPC4.

Hintergrund Die Ultraschalldiagnostik gilt für den Radiologen, Hals-Nasen-Ohren-Arzt (HNO) oder Mund-Kiefer-Gesichts-Chirurgen als Standard in der Abklärung zahlreicher Pathologien. Es besteht ein Konsens, dass die digitale Dokumentation heute dringend notwendig ist, um die Qualität der sonographischen Dokumentationen zu verbessern und zu standardisieren. Es häufen sich Publikationen zur Implementierung standardisierter Befunddokumentation einschließlich der Kopf- und Halssonographie. Ziel der Arbeit Die vorliegende Arbeit zielt darauf ab, die Qualität von routinemäßig angefertigten Kopf- und Halssonographiebefunden nach Kriterien der Kassenärztlichen Vereinigung (KV) Bayern an einer Auswahl deutscher HNO-Universitätskliniken stichprobenartig zu ermitteln. Material und Methoden Insgesamt wurden retrospektiv 70 zufällig ausgewählte, anonymisierte schriftliche Befunde einschließlich Bildmaterial von insgesamt 7 HNO-Universitätskliniken stichprobenartig nach KV-Kriterien durch einen erfahrenen Prüfer der KV Bayern ausgewertet und deskriptiv analysiert. Ergebnisse Von 70 Befunden konnten 69 ausgewertet werden. Die Dokumentationsvollständigkeit lag im Mittel bei 80,6 %. Neun Befunde waren vollständig korrekt dokumentiert (13 %). Die Dokumentationsvollständigkeit der einzelnen Kliniken lag zwischen 68,1 % und 93 %. Mit 88,5 % vs. 75 % erbrachte eine strukturierte Befundung eine höhere Befundvollständigkeit. In 75 % der Fälle verfügten die Kliniken mit strukturiertem Befund auch über digitale Dokumentationslösungen. Schlussfolgerung Die Vollständigkeit und Qualität von routinemäßig angefertigten Kopf- und Halssonographiebefunden an einer Auswahl von HNO-Universitätskliniken ist insgesamt optimierbar. Die Implementierung strukturierter Befundmasken und die Umstellung der analogen Dokumentation auf digitale Lösungen sowie Vernetzung mit dem Klinikinformationssystem (KIS) und Bildarchivierungs- und Kommunikationssystem (PACS) sollte weiter vorangetrieben werden. Darüber hinaus sind leitende Ärzte dazu angehalten, die Befundqualität unerfahrener Kollegen regelmäßig zu prüfen und im Rahmen der Facharztausbildung auf die Erfüllung entsprechender Standards wie der KV-Ultraschallvereinbarung hinzuarbeiten. Background Ultrasound diagnostics are widely used and are standard for radiologists, otolaryngologists, and oral and maxillofacial surgeons in the diagnostic work-up of various pathologies. There is agreement that digital documentation is urgently needed at present to improve and standardize the quality of sonographic documentation. There are more and more publications on the implementation of standardized documentation of findings in imaging diagnostics, including head and neck sonography. Objective The present work aims to determine the quality of routine head and neck sonography findings on a random basis, according to the criteria of the Bavarian Association of Statutory Health Insurance Physicians (KVB) at a selection of German university otolaryngology departments (ENT). Materials and methods A total of 70 randomly selected anonymized written findings including image documentation from seven ENT departments were retrospectively analyzed by an experienced KVB examiner concerning fulfilment of KVB criteria. The data were evaluated descriptively. Results Of the 70 reports, 69 were eligible for evaluation. The average documentation completeness was 80.6%. A total of 9 findings were correctly documented in full (13%). The documentation completeness of the individual departments was sorted in ascending order from 68.1% to 93%. With 88.5% vs. 75%, the hospitals with a structured report showed a higher level of completeness. In 75% of the cases the hospitals with structured reports also had digital solutions for reporting and image archiving. Conclusion In general, there is potential for optimization regarding the completeness and quality of routinely prepared head and neck sonography findings at the selected university ENT departments. The implementation of structured reporting masks and the conversion of analogue documentation into digital solutions as well as digital networking with the hospital information systems, picture archiving and communication systems should be promoted. Supervision by senior doctors is required to ensure the quality of findings of inexperienced colleagues and to help to achieve standards in reporting.

Background Ultrasound diagnostics are widely used and are standard for radiologists, otolaryngologists, and oral and maxillofacial surgeons in the diagnostic work-up of various pathologies. There is agreement that digital documentation is urgently needed at present to improve and standardize the quality of sonographic documentation. There are more and more publications on the implementation of standardized documentation of findings in imaging diagnostics, including head and neck sonography. Objective The present work aims to determine the quality of routine head and neck sonography findings on a random basis, according to the criteria of the Bavarian Association of Statutory Health Insurance Physicians (KVB) at a selection of German university otolaryngology departments (ENT). Materials and methods A total of 70 randomly selected anonymized written findings including image documentation from seven ENT departments were retrospectively analyzed by an experienced KVB examiner concerning fulfilment of KVB criteria. The data were evaluated descriptively. Results Of the 70 reports, 69 were eligible for evaluation. The average documentation completeness was 80.6%. A total of 9 findings were correctly documented in full (13%). The documentation completeness of the individual departments was sorted in ascending order from 68.1% to 93%. With 88.5% vs. 75%, the hospitals with a structured report showed a higher level of completeness. In 75% of the cases the hospitals with structured reports also had digital solutions for reporting and image archiving. Conclusion In general, there is potential for optimization regarding the completeness and quality of routinely prepared head and neck sonography findings at the selected university ENT departments. The implementation of structured reporting masks and the conversion of analogue documentation into digital solutions as well as digital networking with the hospital information systems, picture archiving and communication systems should be promoted. Supervision by senior doctors is required to ensure the quality of findings of inexperienced colleagues and to help to achieve standards in reporting.

Background: L-kynurenine is a tryptophan-derived immunosuppressive metabolite and precursor to neurotoxic anthranilate and quinolinate. We evaluated the stereoisomer D-kynurenine as an immunosuppressive therapeutic which is hypothesized to produce less neurotoxic metabolites than L-kynurenine. Methods: L-/D-kynurenine effects on human and murine T cell function were examined in vitro and in vivo (homeostatic proliferation, colitis, cardiac transplant). Kynurenine effects on T cell metabolism were interrogated using [13C] glucose, glutamine and palmitate tracing. Kynurenine was measured in tissues from human and murine tumours and kynurenine-fed mice. Findings: We observed that 1 mM D-kynurenine inhibits T cell proliferation through apoptosis similar to L-kynurenine. Mechanistically, [13C]-tracing revealed that co-stimulated CD4+ T cells exposed to L-/D-kynurenine undergo increased β-oxidation depleting fatty acids. Replenishing oleate/palmitate restored effector T cell viability. We administered dietary D-kynurenine reaching tissue kynurenine concentrations of 19 μM, which is close to human kidney (6 μM) and head and neck cancer (14 μM) but well below the 1 mM required for apoptosis. D-kynurenine protected Rag1–/– mice from autoimmune colitis in an aryl-hydrocarbon receptor dependent manner but did not attenuate more stringent immunological challenges such as antigen mismatched cardiac allograft rejection. Interpretation: Our dietary kynurenine model achieved tissue concentrations at or above human cancer kynurenine and exhibited only limited immunosuppression. Sub-suppressive kynurenine concentrations in human cancers may limit the responsiveness to indoleamine 2,3-dioxygenase inhibition evaluated in clinical trials. Funding: The study was supported by the NIH, the Else Kröner-Fresenius-Foundation, Laffey McHugh foundation, and American Society of Nephrology.

Mutations in isocitrate dehydrogenase (IDH) or a reduced expression of L-2-hydroxyglutarate (HG)-dehydrogenase result in accumulation of D-2-HG or L-2-HG, respectively, in tumor tissues. D-2-HG and L-2-HG have been shown to affect T-cell differentiation and activation, however, effects on human myeloid cells have not been investigated so far. In this study we analyzed the impact of D-2-HG and L-2-HG on activation and maturation of human monocyte-derived dendritic cells (DCs). 2-HG was taken up by DCs and had no impact on cell viability but diminished CD83 expression after Lipopolysaccharides (LPS) stimulation. Furthermore, D-2-HG and L-2-HG significantly reduced IL-12 secretion but had no impact on other cytokines such as IL-6, IL-10 or TNF. Gene expression analyses of the IL-12 subunits p35/IL-12A and p40/IL-12B in DCs revealed decreased expression of both subunits. Signaling pathways involved in LPS-induced cytokine expression (NFkB, Akt, p38) were not altered by D-2-HG. However, 2-HG reprogrammed LPS-induced metabolic changes in DCs and increased oxygen consumption. Addition of the ATP synthase inhibitor oligomycin to DC cultures increased IL-12 secretion and was able to partially revert the effect of 2-HG. Our data show that both enantiomers of 2-HG can limit activation of DCs in the tumor environment.

The accelerated metabolism of tumor cells, inevitable for maintaining high proliferation rates, is an emerging target for tumor therapy. Increased glucose and lipid metabolism as well as mitochondrial activity have been shown in solid tumors but also in leukemic cells. As tumor cells are able to escape the blockade of one metabolic pathway by a compensatory increase in other pathways, treatment strategies simultaneously targeting metabolism at different sites are currently developed. However, the number of clinically applicable anti-metabolic drugs is still limited. Here, we analyzed the impact of the anti-diabetic drug metformin alone or in combination with two non-steroidal anti-inflammatory drugs (NSAIDs) diclofenac and diflunisal on acute myeloid leukemia (AML) cell lines and primary patient blasts. Diclofenac but not diflunisal reduced lactate secretion in different AML cell lines (THP-1, U937, and KG-1) and both drugs increased respiration at low concentrations. Despite these metabolic effects, both NSAIDs showed a limited effect on tumor cell proliferation and viability up to a concentration of 0.2 mM. In higher concentrations of 0.4–0.8 mM diflunisal alone exerted a clear effect on proliferation of AML cell lines and blocked respiration. Single treatment with the anti-diabetic drug metformin blocked mitochondrial respiration, but proliferation and viability were not affected. However, combining all three drugs exerted a strong cytostatic and cytotoxic effect on THP-1 cells. Comparable to the results obtained with THP-1 cells, the combination of all three drugs significantly reduced proliferation of primary leukemic blasts and induced apoptosis. Furthermore, NSAIDs supported the effect of low dose chemotherapy with cytarabine and reduced proliferation of primary AML blasts. Taken together we show that low concentrations of metformin and the two NSAIDs diclofenac and diflunisal exert a synergistic inhibitory effect on AML proliferation and induce apoptosis most likely by blocking tumor cell metabolism. Our results underline the feasibility of applying anti-metabolic drugs for AML therapy.

Somatische Mutationen der Isozitratdehydrogenase (IDH) 1 und 2 wurden in verschiedenen Tumorentitäten nachgewiesen. Das mutierte Enzym IDH1/2 erlangte die Fähigkeit, α-Ketoglutarat in den Onkometaboliten 2-Hydroxyglutarat (2-HG) zu konvertieren. Bei der akuten myeloischen Leukämie (AML) ist bekannt, dass die Mutation der IDH1/2 positiv mit der Tumorprogression und einem schlechteren Outcome korreliert. Im Gegensatz dazu ist für Glioma WHO II und III und sekundäre Glioblastoma beschrieben, dass die Mutation mit einer guten Prognose korreliert. Auch beim Mamma Karzinom konnten erhöhte Level von 2-HG nachgewiesen werden, auch wenn keine Mutation der IDH vorlag und hier beeinflusste die Höhe des 2-HG-Levels die Prognose ebenfalls negativ. Bisher wird angenommen, dass 2-HG vor allem zu epigenetischen Veränderungen führt und somit die Prognose der Patienten beeinflusst. Aufgrund der wichtigen Rolle von Immunzellen für die Tumorentstehung und -entwicklung, sollte im Rahmen der Arbeit der Effekt von 2-HG auf Immunzellen untersucht werden. Hierbei sollten insbesondere dendritische Zellen (DC) untersucht werden, da diese Antigen präsentierenden Zellen zum einen die Verbindung zwischen angeborener und adaptiver Immunität bilden und zum anderen essentiell sind für die Initiierung einer effektiven Anti-Tumor-Immunantwort. Vordaten der Arbeitsgruppe zeigten, dass 2-HG die Interleukin (IL)-12p70 Sekretion von DC signifikant hemmt. In der Arbeit sollte diese verminderte Sekretion bestätigt und der Mechanismus der Hemmung anhand verschiedener Parameter näher untersucht werden. Zudem sollte die Expression der Interleukin Untereinheiten p35 und p40 auf mRNA Ebene, sowie die Biosynthese der Untereinheiten des Interleukins unter Einfluss von 2-HG analysiert werden. Da eine effektive Differenzierung der DC die Basis der DC Aktivität darstellt, sollten weitere mögliche Effekte auf die Differenzierung und Aktivierung der DC untersucht werden. In der Arbeit konnte gezeigt werden, dass das Dinatriumsalz von 2-HG spezifisch die IL-12p70 Produktion von LPS-aktivierten DC beeinflusst, jedoch keine Veränderung der Produktion anderer Zytokine bewirkt. Es zeigte sich zwar eine leichte Erhöhung der IL-10 Konzentration sowie eine leichte Reduktion der TNF Konzentration im Überstand der Zellen, diese Veränderungen waren jedoch nicht signifikant. Auch die Genexpression der IL-12p70 Untereinheiten p35 und p40 zeigte sich durch Na2-2-HG reduziert, jedoch ohne Signifikanz. Auf Proteinebene, mittels Durchflusszytometrie untersucht, führte Na2-2-HG zu einer Reduktion des IL-12p70-Dimers sowie zur Verminderung der p40 Untereinheit. Zur Verifizierung dieser Beobachtung sollten noch weitere Untersuchungen folgen. In der MLR schien sich ein leichter Effekt vor allem durch das L-Enantiomer von Na2-2-HG im Vergleich zur LPS-Kontrolle vorzuliegen, v.a. bei einer DC-Zellzahl von 5000 pro Well, da im Rahmen meiner Arbeit jedoch nur ein Versuch durchgeführt wurde, müssen weitere Versuche folgen. Bei der Untersuchung des Stoffwechsels der DC, wurden das Enzym Indolamin 2,3-Dioxigenase (IDO), die Atmung und die Laktatproduktion der Zellen unter Einfluss von Na2-2-HG analysiert. Die Untersuchung von IDO sowie der Laktatproduktion zeigten keinen Effekt von Na2-2-HG auf diese Stoffwechselwege. Die Untersuchung der Atmung der Zellen mittels PreSens ergab eine leichte Verstärkung der Atmung nach ca. drei Stunden, dieser Effekt war aber nur vorübergehend und sollte in weiteren Versuchen genauer untersucht werden. Um den Mechanismus der Beeinflussung des Zellmetabolismus durch Na2-2-HG genauer zu untersuchen, wurden erste Analysen von LPS-Signaltranduktionswegen mittels WesternBlot durchgeführt. Weder die Degradation von IκBα, noch dessen Phosphorylierung wurden hierbei durch Na2-2-HG beeinflusst. Auch bei der Untersuchung der MAPK Akt und p38 und deren Phosphorylierung konnte kein Effekt von Na2-2-HG festgestellt werden. Da in dieser Arbeit der spezifische Effekt von Na2-2-HG auf die IL-12p70 Produktion in DC bestätigt werden konnte, bei den weiteren Untersuchungen sich jedoch nur bei der Zellatmung ein signifikanter Effekt durch den Metaboliten ergab, sollte in weiteren Experimenten der mögliche Zusammenhang zwischen beiden Effekten analysiert werden., As you know, tumor metabolism is modified in many ways, for example the changes in the glucose metabolism, described with the warburg effect. It is known that a lot of lactate is produced by tumor cells. This is due to for example the upregulation of oncogenes and the loss of tumor suppressor genes. Furthermore, there are mutations in the succinate Dehydrogenase, fumarate hydrogenase and isocitrate dehydrogenase (IDH), which are enzymes of the TCA cycle. One of these three enzymes, the IDH, is mutated in several cancers and associated with different overall survival. IDH mutations lead to production of L- and D-2Hydroxyglutarat (D-2HG). In tumor cells, IDH is often mutated. This is a gain of function mutation and the mutated IDH converts αKG to D-2HG. As there is no backreaction possible, D-2HG accumulates. Until now, there are some publications about effects of 2HG. But this is still under debate. The question now is: how does the 2HG affect on immune cells, especially dendritic cells (DC)? Last year the researching group startet to analyze effects of 2HG on immune cells. Cytokines secreation was measured by ELISA after 24h. we coud see that L- and D-HG lead to a reduction of IL-12 production in DC. The aim of the project is to analyze this effect of 2HG on DC more in detail. As a control we used metabolites involved in similar pathways: α-Ketoglutarate (aKG), Glutamic acid, Glutamic acid Natrium and Lactate. For activated DCs (mDC), it is known that the production of IL-12, IL-10, TNF, MCO-1 and IL-6 increases. Furthermore, the Antigenpresentation and Lactate production increase, but the respiration decreases in mDC. So, we investigated the effect of 2HG on LPS mDC and on the control metabolites. With LPS the production of IL-12 in DC increases a lot. With LPS and 2HG, doesnt matter which enantiomer, we see a significantly decreased production. We found also an significant effect of aKG, but there is a significant difference between 2HG and aKG. We also investigated the influence of 2HG on the production of other cytokines, but there was no significant effect of 2HG and also no effect of the controls on IL10, TNF, IL-6, MCP-1. Only Lactate lead to lower concentrations of the cytokines in the supernatant. Next we focused on the expression of Indolamin 2,3-Dioxygenase (IDO) in activated DC. IDO is upregulated in DCs after LPS. There was no effect of 2HG on IDO expression. As we know that DC are antigen presenting cells, we had a look at the Antigenpresentation in mDC treated with 2HG in a mixed lymphocyte reaction (MLR). Therefore we cultered mDC with naive Lymphozyte and the metabolites. As compared to mDC, the antigenpresentation of mDC with L-HG decreased, but there is no effect with D-HG. Therefore, more experiments are nessessairy to investigate this effect. Then we studied the effect of 2-HG on Lactate production and Respiration in mDC. It is known that the activation of DC with LPS induces lactate production. But there was no effect of 2-HG or of one of the controls on lactate production. As we found an effect of 2HG on IL-12 production in DC, we also checked, if there is an effect of metabolic modulators on IL-12 production. The first drug we focused on was Diclofenac. We measured the concentration of lactate in the supernatant. As expected before, with Diclo we found less lactate in the supernatant at different concentrations. In addition Diclofenac leads to a reduction of IL-12 but also TNF production as well as IL10 and MCP-1. This is different from 2HG which only reduces the IL-12 production. The next metabolic modulator we investigated, was Rotenon, a blocker of complex 1 in the respiration chain. we had a look at respiration in the preSense, which measures Oxygen use of the cells over 24h. Oxygen consumption of mDC is reduced in comparison to immature DC. Rotenon, as expectet before, lead to a strong inhibition of oxygen use. Then weh ad a look at 2HG. D-2HG had no additional effect on oxygen use as compared to LPS control. So The effect of 2HG on the IL12 production may not be related to an effect on respiration. In summary, we had a very specific effect of 2HG only on IL-12, no effect on IDO, and a slight effect on antigenpresentation in the MLR. Concerning the effect of 2HG on metabolism, we had no effect on lactate production and no effect on respiration. This is why we concentrated now on IL-12 and its subunits p35 and p40. Their encoding genes are Il-12A and IL-12B. Now we were interested in the regulation of these subunits on mRNA level and protein level which we analyzed by intracellular FACS staining. On mRNA level, there was a decreased expression of IL-12A by D- and L-2HG. We also had an effect with the control metabolites. The same is true for IL-12B. This is different from IL-12 production in the ELISA where we had a significantly stronger effect of 2HG as compared to aKG. Then we started to analyze the subunits of IL12 on protein level and the effect of 2HG on protein subunits. We treated the cells with monensin, a substance which blocks the transport of proteins in the golgi and therefore blocks the transport out of the cell. We found, that p35 is constitutive produced. After maturation, there were after incubation with LPS and Monensin, 17% double positive cells (p35 and p40). When we added D-2-HG, we saw that the amount of double positive cells was reduced compared to the mDC. Therefore, we suggest that there is an effect of D-2HG on the production of p40 in activated DC. Summerizing all the experiments, we had a specific effect of 2HG on IL-12 production in mDC and a significant effect on the respiration of the DC, but there was no other very specific effect of 2HG on DC. Therefore, more experiments will follow, to analyze the effect of 2HG on immune cells.