NF-κB Inhibition through Proteasome Inhibition or IKKβ Blockade Increases the Susceptibility of Melanoma Cells to Cytostatic Treatment through Distinct Pathways

Metastasized melanoma is almost universally resistant to chemotherapy. Given that constitutive or drug-induced upregulation of NF-κB activity is associated with this chemoresistance, NF-κB inhibition may increase the susceptibility to antitumoral therapy. On the cellular level, two principles of NF-κB inhibition, proteasome inhibition by bortezomib and IκB kinase-β (IKKβ) inhibition by the kinase inhibitor of NF-κB-1 (KINK-1), significantly increased the antitumoral efficacy of camptothecin. When combined with camptothecin, either of the two NF-κB-inhibiting principles synergistically influenced progression-related in vitro functions, including cell growth, apoptosis, and invasion through an artificial basement membrane. In addition, when C57BL/6 mice were intravenously injected with B16F10 melanoma cells, the combination of cytostatic treatment with either of the NF-κB-inhibiting compounds revealed significantly reduced pulmonary metastasis compared to either treatment alone. However, on the molecular level, nuclear translocation of p65, cell cycle analysis, and expression of NF-κB-dependent gene products disclosed distinctly different molecular mechanisms, resulting in the same functional effect. That proteasome inhibition and IKKβ inhibition affect distinct molecular pathways downstream of NF-κB, both leading to increased chemosensitivity, is previously unreported. Thus, it is conceivable that switching the two principles of NF-κB inhibition, once resistance to one of the agents occurs, will improve future treatment regimens

Oxaliplatin-Induced Leukocytoclastic Vasculitis under Adjuvant Chemotherapy for Colorectal Cancer: Two Cases of a Rare Adverse Event

Leukocytoclastic vasculitis is a multicausal systemic inflammatory disease of the small vessels, histologically characterized by inflammation and deposition of both nuclear debris and fibrin in dermal postcapillary venules. The clinical picture typically involves palpable purpura of the lower legs and may be associated with general symptoms such as fatigue, arthralgia and fever. Involvement of the internal organs, most notably the kidneys, the central nervous system or the eyes, is possible and determines the prognosis. Oxaliplatin-induced leukocytoclastic vasculitis is a very rare event that limits treatment options in affected patients. We report 2 patients who developed the condition under chemotherapy for advanced rectal and metastatic colon carcinoma, respectively; a termination of the therapy was therefore necessary. While current therapies for colorectal cancer include the combination of multimodal treatment with new and targeted agents, rare and unusual side effects elicited by established agents also need to be taken into account for the clinical management

Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5–10%), heat-inactivated (hiFCS, 0.1–10%) or human serum albumin (HSA, 0.05–2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research

Plättchen-Oberflächenrezeptoren und Melanom-Metastasierung

Ziel dieser Arbeit war es, die Rollen der Plättchen-Rezeptoren GPVI und GPIbα bei der hämatogenen Melanom-Metastasierung zu untersuchen. Die letzten Jahrzehnte haben zunehmend Indizien für eine Beteiligung von Plättchen und Plättchen-Rezeptoren bei der hämatogenen Tumor-Metastasierung geliefert. Man weiß heute, dass funktionsfähige Plättchen eine Voraussetzung für die hämatogene Verbreitung der Tumorzellen darstellen. Die Blockade verschiedener Plättchen-Rezeptoren oder anderer Faktoren der primären Hämostase kann die Metastasierung in vivo deutlich abschwächen. Aus diesem Grund wurden die Rollen der beiden bei der Hämostase essentiellen Plättchen-Rezeptoren GPVI und GPIbα in einem Modell der experimentellen pulmonalen Metastasierung untersucht. Sowohl GPVI als auch GPIbα wurden in einem syngenen, murinen Modell der experimentellen pulmonalen Metastasierung durch monoklonale, monovalente Antikörper (p0p/B Fab-Fragmente) gehemmt. Für die Experimente wurden B16F10 Melanomzellen und C57BL/6 Mäuse eingesetzt. Die Antikörper-Behandlung führte zu keiner Thrombozytopenie, welche die Metastasierungs-Ergebnisse hätte verfälschen können. Während die Blockade von GPVI in dem von uns verwendeten System keinen Einfluss auf die in vivo Metastasierung hatte, führte die Blockade von GPIbα zu einem unerwarteten und signifikanten Anstieg der Anzahl der metastatischen Foci. Dieser Effekt konnte nur beobachtet werden, wenn GPIbα vor oder zusammen mit der Tumorzellinjektion blockiert wurde. Dies deutet auf eine Rolle von GPIbα in der frühen Phase der metastatischen Kaskade hin. Diese Beobachtung konnte durch die Untersuchung des Verhaltens von mit GFP (green fluorescent protein) markierten B16F10 Melanomzellen im Lungengewebe unmittelbar nach der Injektion bestätigt werden. GFP-markierte Tumorzellen reicherten sich unter GPIbα-Blockade vermehrt im Lungengewebe an. Dagegen hatte die Blockade von GPIbα in P-Selektin defizienten Mäusen keinen Einfluss auf die pulmonale Melanom-Metastasierung, was eine Rolle für GPIbα in den frühen, P-Selektin abhängigen Schritten der Metastasierung nahe legt. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass GPIbα bei der Kontrolle der frühen Schritte der metastatischen Kaskade eine Rolle spielt, da seine Blockade zu einem Anstieg der Melanom-Metastasierung führt. Es konnte erstmalig demonstriert werden, dass die Blockade eines Plättchenrezeptors nicht in jedem Fall die Reduktion der Metastasierung zum Ergebnis hat, sondern auch pro-metastastisch wirken kann.The aim of this thesis was to investigate the roles of the platelet surface receptors GPVI and GPIbα in the process of hematogenous melanoma metastasis. In the last decades, there has been accumulating evidence for a contribution of platelets and platelet receptors to hematogenous tumor metastasis. It is known today that functional platelets are a prerequisite to hematogenous dissemination of tumor cells and that the blockade of different platelet surface receptors or other factors of primary hemostasis can diminish metastasis in vivo. Therefore, the two important platelet receptors GPVI and GPIbα, which both play a central role in hemostasis, were studied in the context of experimental pulmonary metastasis. GPVI as well as GPIbα were inhibited by monoclonal, monovalent antibodies (p0p/B Fab fragments) in a syngeneic murine model of experimental pulmonary metastasis using C57BL/6 mice and B16F10 melanoma cells. Antibody treatment of the mice did not induce thrombocytopenia, excluding an influence of platelet numbers on experimental metastasis. While blockade of GPVI showed no effect on in vivo melanoma metastasis in our experimental system, GPIbα-blockade led to an unexpected and significant increase in the number of metastatic foci. This effect could only be seen when GPIbα was blocked either before or together with tumor cell injection, pointing to a role of GPIbα in the early steps of the metastatic cascade. This observation was confirmed by the assessment of early fate of melanoma cells transfected with green fluorescent protein (GFP) under GPIbα blockade. GFP-transfected cells showed improved survival and pulmonary arrest of tumor cells shortly after GPIbα inhibition. In contrast, GPIbα blockade in P-selectin-deficient mice had no enhancing effect on metastasis, suggesting the involvement of GPIbα in the initial, P-selectin-dependent steps of metastasis. In conclusion, these findings show that GPIbα contributes to the control of early steps in tumor metastasis, in addition to its role in hemostasis. It could be demonstrated for the first time, that blockade of a platelet surface receptor does not always lead to a reduction of metastasis but can also have a pro-metastatic effect

The Interleukin-23/Interleukin-17 Axis Links Adaptive and Innate Immunity in Psoriasis

Research into the pathophysiology of psoriasis has shed light onto many fascinating immunological interactions and underlying genetic constellations. Most prominent among these is the crosstalk between components of the innate and the adaptive immune system and the crucial role of interleukins (IL)-23 and -17 within this network. While it is clear that IL-23 drives and maintains the differentiation of Th17 lymphocytes, many aspects of the regulation of IL-23 and IL-17 are not quite as straightforward and have been unraveled only recently. For example, we know now that Th17 cells are not the only source of IL-17 but that cells of the innate immune system also produce considerable amounts of this central effector cytokine. In addition, there is IL-23-independent production of IL-17. Besides other innate immune cells, neutrophilic granulocytes prominently contribute to IL-17-related immune regulations in psoriasis, and it appears that they employ several mechanisms including the formation of neutrophil extracellular traps. Here, we strive to put the central role of the IL-23/IL-17 axis into perspective within the crosstalk between components of the innate and the adaptive immune system. Our aim is to better understand the complex immune regulation in psoriasis, a disorder that has become a model disease for chronic inflammation

The Impact of COVID-19 Pandemic on Medical Doctors’ Work-Family Balance at German University Clinics

The measures taken to cope with the COVID-19 pandemic by governments worldwide have vast consequences on all areas of life. To assess the impact of the COVID-19 pandemic on long-term career development, we evaluated the work-family balance of medical doctors at nine German university clinics. The results indicate a severely disturbed work-family balance, which was mostly due to insufficient childcare, based on restrictions in school operations and childcare. Despite the newly created emergency childcare options, aiming to ensure the functioning of the “systematically important” professional groups, medical doctors feel that they are not sufficiently supported by the measures taken by local governments. Women, in particular, see their professional development at risk. Our results underline that proper and flexible childcare is essential for the career advancement of female medical doctors and is particularly important in times of crises such as the current COVID-19 pandemic. At university medicine clinics, increased work time flexibility and optimized schooling and childcare are needed to promote the career development of female as well as male medical doctors in the early stage of their careers

Expansion microscopy of neutrophil nuclear structure and extracellular traps

Neutrophils are key players of the immune system and possess an arsenal of effector functions, including the ability to form and expel neutrophil extracellular traps (NETs) in a process termed NETosis. During NETosis, the nuclear DNA/chromatin expands until it fills the whole cell and is released into the extracellular space. NETs are composed of DNA decorated with histones, proteins, or peptides, and NETosis is implicated in many diseases. Resolving the structure of the nucleus in great detail is essential to understand the underlying processes, but so far, superresolution methods have not been applied. Here, we developed an expansion-microscopy-based method and determined the spatial distribution of chromatin/DNA, histone H1, and nucleophosmin with an over fourfold improved resolution (<40–50 nm) and increased information content. It allowed us to identify the punctate localization of nucleophosmin in the nucleus and histone-rich domains in NETotic cells with a size of 54–66 nm. The technique could also be applied to components of the nuclear envelope (lamins B1 and B2) and myeloperoxidase, providing a complete picture of nuclear composition and structure. In conclusion, expansion microscopy enables superresolved imaging of the highly dynamic structure of nuclei in immune cells