Identifikation von ABCC1 als potenziellen Angriffspunkt in Actinomycin D-resistenten, pädiatrischen Hochrisiko-Rhabdomyosarkomen: ein CRISPR-Aktivationsscreen

Rhabdomyosarcomas (RMS) are the most common childhood soft tissue tumors. Despite multimodal treatment regimen, around 20% of pediatric sarcoma patients still suffer from local relapses, indicating underlying resistance to common therapy. Translocation of PAX3 and FOXO1, and the expression of the respective fusion protein, is known to predict adverse prognosis, but no significant improvement in patient outcome of these high-risk patients has been achieved in the last three decades. Here, we performed a genome-wide CRISPR activation screen in the fusion-positive cell line Rh4 to search for genes associated with resistance to actinomycin D (ActD), a drug commonly used in the treatment of RMS and other pediatric cancers such as nephroblastoma and Ewing’s sarcoma. The top-ranking hit was the gene ABCC1, encoding for a drug efflux-pump. The expression of ATP-binding cassette (ABC) transporters like ABCC1 has long been suspected to play a role in multidrug resistance, however small molecule inhibitors targeting them are still not approved for clinical use. In this study, we were able to observe a synergistic relationship between ActD and the ABCC1 inhibitor tetrandrine, implying a possible benefit of the inhibition of ABCC1 in ActD-resistant RMS and highlighting the necessity of further preclinical and clinical investigations into the role of ABCC1 in RMS in response to treatment.Rhabdomyosarkome (RMS) sind die häufigsten pädiatrischen Weichteilsarkome. Trotz eines multimodalen Behandlungsansatzes leiden 20% aller pädiatrischen Sarkompatient*innen unter lokalen Rezidiven, was auf zugrundeliegende Resistenzmechanismen schließen lässt. Die Translokation von PAX3 und FOXO1 und die Expression des entsprechenden Fusionsproteins sind bekannte negative Prädiktoren für die Prognose von RMS. In den letzten 30 Jahren konnte jedoch keine Verbesserung des Therapieerfolges von Hochrisikopatienten erzielt werden. In dieser Studie nutzten wir einen genomweiten CRISPR-Aktivationsscreen in der fusionspositiven RMS-Zelllinie Rh4, um nach Genen zu suchen, die mit einer Resistenz gegenüber Actinomycin D (ActD) assoziiert sind. Dieses Chemotherapeutikum findet nicht nur bei RMS-Patient*innen, sondern auch in der Behandlung anderer pädiatrischer Tumoren wie dem Nephroblastom und dem Ewing-Sarkom Verwendung. Das Gen mit dem höchsten Rang war die Effluxpumpe ABCC1. Obwohl die Expression von ATP-binding cassette (ABC)-Transportern wie ABCC1 schon lange unter dem Verdacht steht, zu Multiresistenzen von Tumoren beizutragen, sind gezielte small molecule-Therapien noch nicht für den klinischen Gebrauch zugelassen. In dieser Studie konnten wir eine synergistische Beziehung zwischen ActD und dem ABCC1-Inhibitor Tetrandrine nachweisen, welches auf einen möglichen Vorteil einer Kombinationstherapie in ActD-resistenten RMS hinweist. Zudem unterstreicht unsere Studie die Notwendigkeit, die Rolle von ABCC1 in RMS in präklinischen und klinischen Studien weiter zu erforschen

Langerhans cells are negative regulators of the anti-Leishmania response

Langerhans cells suppress the immune response to low-dose Leishmania major infection in part by inducing regulatory T cells

Die Integration von Frauen in die Wissenschaft - eine Echternacher Springprozession

"Vor gut zwanzig Jahren entwickelte feministische Gewißheiten über Frauen sind aufgrund vielfältiger Erfahrungen neuen Suchbewegungen gewichen, denn das Bild von der Frau als besserem Menschen und hohe Glückserwartungen an Frauenzusammenhänge haben sich als problematisch erwiesen. Die positive Besetzung des eigenen Geschlechts ist zwar weiterhin die conditio sine qua non für Fraueninitiativen, ein überhöhtes Frauenbild stellt aber nicht selten auch einen Hemmschuh für strukturelle und psychosoziale Entwicklungen dar, wie sie besonders in den Projekten der Neuen Frauenbewegung erforderlich sind. Das Ideal des 'Anderen' ist sowohl Antriebskraft als auch Behinderung, indem es die Frauenbewegung maßgeblich vorangetrieben hat, im Rahmen einer beruflich organisierten Praxis aber zwiespältige Wirkungen zeitigt. Ohne die Privatsphäre zum Politikbereich zu erheben, wäre es nicht möglich gewesen, kollektiv an die Interessen und Problemlagen von Frauen anzuknüpfen, statt sie auszugrenzen. Gleichzeitig enthält diese Ausweitung die Gefahr, private und berufliche Umgangsformen, politische Interessen und psychische Bedürfnisse unreflektiert miteinander zu vermischen. Die Differenzierungen der Neuen Frauenbewegung und zunehmende Institutionalisierungen weisen auf die notwendige Entwicklung neuer Organisations-, Verhaltens- und Kommunikationsprinzipien hin, die weder als Abweichung noch als Entwertung von früheren Formen feministischen Denkens und Handelns angesehen werden und ihrerseits gleichberechtigte Teilhabe und Selbstbestimmung nicht aus dem Auge verlieren." (Autorenreferat

Interleukin 1α Promotes Th1 Differentiation and Inhibits Disease Progression in Leishmania major–susceptible BALB/c Mice

Protective immunity against pathogens such as Leishmania major is mediated by interleukin (IL)-12–dependent Th1-immunity. We have shown previously that skin-dendritic cells (DCs) from both resistant C57BL/6 and susceptible BALB/c mice release IL-12 when infected with L. major, and infected BALB/c DCs effectively vaccinate against leishmaniasis. To determine if cytokines other than IL-12 might influence disease outcome, we surveyed DCs from both strains for production of a variety of cytokines. Skin-DCs produced significantly less IL-1α in response to lipopolysaccharide/interferon γ or L. major when expanded from BALB/c as compared with C57BL/6 mice. In addition, IL-1α mRNA accumulation in lymph nodes of L. major–infected BALB/c mice was ∼3-fold lower than that in C57BL/6 mice. Local injections of IL-1α during the first 3 d after infection led to dramatic, persistent reductions in lesion sizes. In L. major–infected BALB/c mice, IL-1α administration resulted in increased Th1- and strikingly decreased Th2-cytokine production. IL-1α and IL-12 treatments were similarly effective, and IL-1α efficacy was strictly IL-12 dependent. These data indicate that transient local administration of IL-1α acts in conjunction with IL-12 to influence Th-development in cutaneous leishmaniasis and prevents disease progression in susceptible BALB/c mice, perhaps by enhancing DC-induced Th1-education. Differential production of IL-1 by C57BL/6 and BALB/c mice may provide a partial explanation for the disparate outcomes of infection in these mouse strains

Treatment of melanoma cells with the synthetic retinoid CD437 induces apoptosis via activation of AP-1 in vitro, and causes growth inhibition in xenografts in vivo

Human malignant melanoma is notoriously resistant to pharmacological modulation. We describe here for the first time that the synthetic retinoid CD437 has a strong dose-dependent antiproliferative effect on human melanoma cells (IC50: 5 x 10(-6) M) via the induction of programmed cell death, as judged by analysis of cell morphology, electron microscopical features, and DNA fragmentation. Programmed cell death was preceded by a strong activation of the AP-1 complex in CD437-treated cells as demonstrated by gel retardation and chloramphenicol transferase (CAT) assays. Northern blot analysis showed a time-dependent increase in the expression of c-fos and c-jun encoding components of AP-1, whereas bcl-2 and p53 mRNA levels remained constant. CD437 also exhibited a strong growth inhibitory effect on MeWo melanoma cells in a xenograft model. In tissue sections of CD437-treated MeWo tumors from these animals, apoptotic melanoma cells and c-fos overexpressing cells were colocalized by TdT-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL) staining and in situ hybridization. Taken together, this report identifies CD437 as a retinoid that activates and upregulates the transcription factor AP-1, leading eventually to programmed cell death of exposed human melanoma cells in vitro and in vivo. Further studies are needed to evaluate whether synthetic retinoids such as CD437 represent a new class of retinoids, which may open up new ways to a more effective therapy of malignant melanoma

Uptake of Leishmania major by dendritic cells is mediated by Fcγ receptors and facilitates acquisition of protective immunity

Uptake of Leishmania major by dendritic cells (DCs) results in activation and interleukin (IL)-12 release. Infected DCs efficiently stimulate CD4− and CD8− T cells and vaccinate against leishmaniasis. In contrast, complement receptor 3–dependent phagocytosis of L. major by macrophages (MΦ) leads exclusively to MHC class II–restricted antigen presentation to primed, but not naive, T cells, and no IL-12 production. Herein, we demonstrate that uptake of L. major by DCs required parasite-reactive immunoglobulin (Ig)G and involved FcγRI and FcγRIII. In vivo, DC infiltration of L. major–infected skin lesions coincided with the appearance of antibodies in sera. Skin of infected B cell–deficient mice and Fcγ−/− mice contained fewer parasite-infected DCs in vivo. Infected B cell–deficient mice as well as Fcγ−/− mice (all on the C57BL/6 background) showed similarly increased disease susceptibility as assessed by lesion volumes and parasite burdens. The B cell–deficient mice displayed impaired T cell priming and dramatically reduced IFN-γ production, and these deficits were normalized by infection with IgG-opsonized parasites. These data demonstrate that DC and MΦ use different receptors to recognize and ingest L. major with different outcomes, and indicate that B cell–derived, parasite-reactive IgG and DC FcγRI and FcγRIII are essential for optimal development of protective immunity

An Alternative Pathway of Imiquimod-Induced Psoriasis-Like Skin Inflammation in the Absence of Interleukin-17 Receptor A Signaling

Topical application of imiquimod (IMQ) on the skin of mice induces inflammation with common features found in psoriatic skin. Recently, it was postulated that IL-17 has an important role both in psoriasis and in the IMQ model. To further investigate the impact of IL-17RA signaling in psoriasis, we generated IL-17 receptor A (IL-17RA)–deficient mice (IL-17RAdel) and challenged these mice with IMQ. Interestingly, the disease was only partially reduced and delayed but not abolished when compared with controls. In the absence of IL-17RA, we found persisting signs of inflammation such as neutrophil and macrophage infiltration within the skin. Surprisingly, already in the naive state, the skin of IL-17RAdel mice contained significantly elevated numbers of Th17- and IL-17-producing γδ T cells, assuming that IL-17RA signaling regulates the population size of Th17 and γδ T cells. Upon IMQ treatment of IL-17RAdel mice, these cells secreted elevated amounts of tumor necrosis factor-α, IL-6, and IL-22, accompanied by increased levels of the chemokine CXCL2, suggesting an alternative pathway of neutrophil and macrophage skin infiltration. Hence, our findings have major implications in the potential long-term treatment of psoriasis by IL-17-targeting drugs

Suppression of LPS-induced inflammatory responses in macrophages infected with Leishmania

<p>Abstract</p> <p>Background</p> <p>Chronic inflammation activated by macrophage innate pathogen recognition receptors such as TLR4 can lead to a range of inflammatory diseases, including atherosclerosis, Crohn's disease, arthritis and cancer. Unlike many microbes, the kinetoplastid protozoan pathogen <it>Leishmania </it>has been shown to avoid and even actively suppress host inflammatory cytokine responses, such as LPS-induced IL-12 production. The nature and scope of <it>Leishmania</it>-mediated inflammatory cytokine suppression, however, is not well characterized. Advancing our knowledge of such microbe-mediated cytokine suppression may provide new avenues for therapeutic intervention in inflammatory disease.</p> <p>Methods</p> <p>We explored the kinetics of a range of cytokine and chemokine responses in primary murine macrophages stimulated with LPS in the presence versus absence of two clinically distinct species of <it>Leishmania </it>using sensitive multiplex cytokine analyses. To confirm that these effects were parasite-specific, we compared the effects of <it>Leishmania </it>uptake on LPS-induced cytokine expression with uptake of inert latex beads.</p> <p>Results</p> <p>Whilst <it>Leishmania </it>uptake alone did not induce significant levels of any cytokine analysed in this study, <it>Leishmania </it>uptake in the presence of LPS caused parasite-specific suppression of certain LPS-induced pro-inflammatory cytokines, including IL-12, IL-17 and IL-6. Interestingly, <it>L. amazonensis </it>was generally more suppressive than <it>L. major</it>. We also found that other LPS-induced proinflammatory cytokines, such as IL-1α, TNF-α and the chemokines MIP-1α and MCP-1 and also the anti-inflammatory cytokine IL-10, were augmented during <it>Leishmania </it>uptake, in a parasite-specific manner.</p> <p>Conclusions</p> <p>During uptake by macrophages, <it>Leishmania </it>evades the activation of a broad range of cytokines and chemokines. Further, in the presence of a strong inflammatory stimulus, <it>Leishmania </it>suppresses certain proinflammatory cytokine responses in a parasite-specific manner, however it augments the production of other proinflammatory cytokines. Our findings highlight the complexity of inflammatory cytokine signalling regulation in the context of the macrophage and <it>Leishmania </it>interaction and confirm the utility of the <it>Leishmania</it>/macrophage infection model as an experimental system for further studies of inflammatory regulation. Such studies may advance the development of therapies against inflammatory disease.</p

Keratinocytes Determine Th1 Immunity during Early Experimental Leishmaniasis

Experimental leishmaniasis is an excellent model system for analyzing Th1/Th2 differentiation. Resistance to Leishmania (L.) major depends on the development of a L. major specific Th1 response, while Th2 differentiation results in susceptibility. There is growing evidence that the microenvironment of the early affected tissue delivers the initial triggers for Th-cell differentiation. To analyze this we studied differential gene expression in infected skin of resistant and susceptible mice 16h after parasite inoculation. Employing microarray technology, bioinformatics, laser-microdissection and in-situ-hybridization we found that the epidermis was the major source of immunomodulatory mediators. This epidermal gene induction was significantly stronger in resistant mice especially for several genes known to promote Th1 differentiation (IL-12, IL-1β, osteopontin, IL-4) and for IL-6. Expression of these cytokines was temporally restricted to the crucial time of Th1/2 differentiation. Moreover, we revealed a stronger epidermal up-regulation of IL-6 in the epidermis of resistant mice. Accordingly, early local neutralization of IL-4 in resistant mice resulted in a Th2 switch and mice with a selective IL-6 deficiency in non-hematopoietic cells showed a Th2 switch and dramatic deterioration of disease. Thus, our data indicate for the first time that epidermal cytokine expression is a decisive factor in the generation of protective Th1 immunity and contributes to the outcome of infection with this important human pathogen