Induktion einer TH1-Reaktion nach Stimulation von dendritischen Zellen mit MALP-2 und IFN-[gamma] in einem in vitro Allergiemodell

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The Pyrazolo[3,4-d]pyrimidine Derivative, SCO-201, Reverses Multidrug Resistance Mediated by ABCG2/BCRP

ATP-binding cassette (ABC) transporters, such as breast cancer resistance protein (BCRP), are key players in resistance to multiple anti-cancer drugs, leading to cancer treatment failure and cancer-related death. Currently, there are no clinically approved drugs for reversal of cancer drug resistance caused by ABC transporters. This study investigated if a novel drug candidate, SCO-201, could inhibit BCRP and reverse BCRP-mediated drug resistance. We applied in vitro cell viability assays in SN-38 (7-Ethyl-10-hydroxycamptothecin)-resistant colon cancer cells and in non-cancer cells with ectopic expression of BCRP. SCO-201 reversed resistance to SN-38 (active metabolite of irinotecan) in both model systems. Dye efflux assays, bidirectional transport assays, and ATPase assays demonstrated that SCO-201 inhibits BCRP. In silico interaction analyses supported the ATPase assay data and suggest that SCO-201 competes with SN-38 for the BCRP drug-binding site. To analyze for inhibition of other transporters or cytochrome P450 (CYP) enzymes, we performed enzyme and transporter assays by in vitro drug metabolism and pharmacokinetics studies, which demonstrated that SCO-201 selectively inhibited BCRP and neither inhibited nor induced CYPs. We conclude that SCO-201 is a specific, potent, and potentially non-toxic drug candidate for the reversal of BCRP-mediated resistance in cancer cells

Induktion einer TH1-Reaktion nach Stimulation von dendritischen Zellen mit MALP-2 und IFN-γ in einem in vitro Allergiemodell

In der vorliegenden Arbeit wurde der Einfluss von dendritischen Zellen (DC) auf die allergische TH2−Reaktion untersucht. Dazu wurde zunächst die Generierung von humanen DC aus Monozyten etabliert und optimiert. Monozyten wurden aus dem Vollblut von freiwilligen gesunden Blutspendern mittels Dichtegradientenzentrifugation und anschließender magnetischer Zellsortierung(MACS) in hoher Reinheit (97%) isoliert. In einer 7−tägigen Zellkulturphase entstanden aus den Monozyten durch die Zugabe von GM−CSF und IL−4 unreife DC. Diese waren durch eine hohe Endozytosefähigkeit und eine schwache Expression von CD 80 und CD 86 sowie ein Fehlen von CD 83 gekennzeichnet. Dieses Protokoll wurde angewendet, um DC von Spendern, die gegen das Majorallergen der Hausstaubmilbe Dermatophagoides pteronyssinus, DerP1,allergisch sind, bzw. von gesunden Kontrollspendern zu generieren. Zur funktionellen Charakterisierung wurden diese DC anschließend mit autologen Lymphozytenkokultiviert. In diesem in vitro Allergiemodell stellte sich nach DerP1 Vorstimulation in der Allergiker−Gruppe eine TH2 Reaktion ein. Diese war durch die gesteigerte Produktion des TH2−Zytokins IL−4 und die reduzierte Produktion des TH1−ZytokinsIFN−γ durch die Lymphozyten gekennzeichnet. In der gesunden Kontrollgruppe hatte die Allergenstimulation keinen Effekt. Mit diesem Modell kann die Beeinflussung deslymphozyten−modulatorischen Potenzials von DC durch pharmakologische Kandidatensubstanzen untersucht werden. Als Kandidatensubstanz wurden DC von Allergikern mit 100 pg/ml MALP−2, einemsynthetischen Lipopeptid abgeleitet aus Mycoplasma fermentans, und mit 500 U/mlIFN−γ sowie dem Allergen stimuliert. Diese Stimulation induzierte die Reifung von DC: Die Expression von CD 80, CD 83 und CD 86 sowie CD 40 und HLA−DR wurde hochreguliert, der Monozyten−Marker CD 14 herunterreguliert. Des Weiteren induzierte die Gabe beider Substanzen die Ausschüttung der Zytokine IL−12 undTNF−α. In der Kokultur von DC mit autologen Lymphozyten wurde durch diese Behandlung die allergenspezifische TH2−Reaktion in eine TH1−Reaktion überführt: Die IFN−γ Ausschüttung wurde massiv gesteigert, was mit der Reduktion der IL−4Konzentration einher ging. Des Weiteren induzierten mit MALP−2 und IFN−γ sowie Allergen vorbehandelte DC die lymphozytäre Proliferation. Diese Effekte waren von der Produktion von IL−12 abhängig, da die Blockierung dieses Zytokins in der Kokultur durch Antikörper die lymphozytäre IFN−γ Produktion und die Proliferation reduzierte. Die hier beschriebene Modulation der allergischen TH2−Reaktion nach TH1 durchMALP−2 und IFN−γ stimulierte DC stellt eine Rationale für die Applikation von DC in der Allergietherapie dar

Evaluation of the environmental risk assessment procedure according to Directive 2001/18/EC for Gene Modified Organism used as medicinal products

The deliberate release of genetically modified organisms (GMOs) including GMOs used as medicinal products, e.g. gene therapies, into the environment is regulated by directive 2001/18/EC of the European Parliament and of the Council of 12 March2001. An integral part of the directive regulates the provision of information on the GMO and, based on this, the risk management with regard to the environmental effects of such releases. As regulated by this directive, a publicly accessible database is the "GMO Register" of the JOINT RESEARCH CENTER of the EC (http://gmoinfo.jrc.ec.europa.eu/Default.aspx), which contains information about all releases under the guideline. As of 07.11.2016, there were 238 entries of medicinal GMOs” in the "Summary Notification Information Format (SNIF).SNIFs are prepared as a summary document of the confidential environmental risk assessments (ERA) by the respective Sponsors of clinical trials in the EU and evaluated during the clinical trial application by the national competent authorities. They comprise, inter alia, information regarding the GMOs and the parental organism’s nature, release, environmental interactions, monitoring, waste treatment and emergency response plans. We strived to assess information concerning the environmental risk, derived measures and the overall standard of SNIFs concerning compliance with the regulatory requirements. To do so, we picked a homogeneous group of GMOs, namely gene modified Adenovirus, the most frequently used vector in gene therapy trials worldwide. Relevant information were entered into a database and categorized, applying unified vocabulary. Different challenges regarding the information available within the SNIFs were identified by analyzing the database: in several cases mandatory information was not available, e.g. monitoring plans, and in other cases the SNIF documents were misinterpreted, e.g. the connection between replication, dissemination and survivability was interpreted heterogeneously. Although this analysis has been performed using only Adenovirus data, information gaps and inconsistencies are transferable to other species as well. Consequently, it is proposed to specify some parts of the SNIFs in order to make more reliable information transparently available

Human effector memory T helper cells engage with mouse macrophages and cause Graft-versus-Host-like pathology in skin of humanized mice used in a nonclinical immunization study

Humanized mice engrafted with human hematopoietic stem cells and developing functional human T-cell adaptive responses are in critical demand to test human-specific therapeutics. We previously showed that humanized mice immunized with long-lived induced-dendritic cells loaded with the pp65 viral antigen (iDCpp65) exhibited a faster development and maturation of T cells. Herein, we evaluated these effects in a long-term (36 weeks) nonclinical model using two stem cell donors to assess efficacy and safety. Relative to baseline, iDCpp65 immunization boosted the output of effector memory CD4+ T cells in peripheral blood and lymph nodes. No weight loss, human malignancies, or systemic graft-versus-host (GVH) disease were observed. However, for one reconstitution cohort, some mice immunized with iDCpp65 showed GVH-like signs on the skin. Histopathology analyses of the inflamed skin revealed intrafollicular and perifollicular human CD4+ cells near F4/80+ mouse macrophages around hair follicles. In spleen, CD4+ cells formed large clusters surrounded by mouse macrophages. In plasma, high levels of human T helper 2-type inflammatory cytokines were detectable, which activated in vitro the STAT5 pathway of murine macrophages. Despite this inflammatory pattern, human CD8+ T cells from mice with GVH reacted against the pp65 antigen in vitro. These results uncover a dynamic cross-species interaction between human memory T cells and mouse macrophages in the skin and lymphatic tissues of humanized mice

In Vivo Metabolic Imaging of [1-13C]Pyruvate-d3 Hyperpolarized By Reversible Exchange With Parahydrogen

Metabolic magnetic resonance imaging (MRI) using hyperpolarized (HP) pyruvate has shown promise as a non-invasive technique for diagnosing, staging, and monitoring response to treatment in cancer and other diseases. The clinically established method for producing HP pyruvate is dynamic nuclear polarization; however, it is rather expensive and slow. Here, we demonstrate fast (6 min), low-cost production of HP [1-13C]pyruvate-d3 in aqueous solution using Signal Amplification By Reversible Exchange (SABRE), and in vivo metabolic MRI. The injected solution was sterile, non-toxic, pH neutral and contained ≈30 mM [1-13C]pyruvate-d3 polarized to ≈11% (residual 250 mM methanol and 20 µM catalyst). It was obtained by rapid solvent evaporation and metal filtering. The procedure was well tolerated by all four mice studied here. This achievement is a significant step of making HP MRI available to a wider community. Fast, low-cost, and high-throughput parahydrogen-hyperpolarization has become a viable alternative for metabolic MRI of living organisms

Preoperative hemoglobin thresholds for survival equity in women and men

Anemia affects humans throughout life, and is linked to higher morbidity and mortality. Unclear is whether hemoglobin values are equivalent between women and men. This study evaluates the association of preoperative hemoglobin levels with in-hospital mortality and estimates thresholds for survival equity between men and women. All adult patients undergoing surgery between 2010 and 2019 from 14 German hospitals were included in the study. Thresholds for survival equity were determined with generalized additive models. In total, 842,130 patients with a median in-hospital follow-up time of 7 days were analyzed. During follow-up 20,370 deaths occurred. Preoperative hemoglobin stratified in-hospital mortality (log-rank test p < 0.001) and was associated with mortality independently of demographic risk, surgical risk and health status. For each 1 g/dL reduction in preoperative hemoglobin, the odds of mortality increased by a factor of 1.22 (95% CI 1.21–1.23, p < 0.001). A preoperative hemoglobin threshold of 10.5 g/dL reflected equivalent risk for both male and female patients. Hemoglobin levels below 10.5 g/dL had higher risk of mortality for women than for men. The findings from this study aid evidence-based thresholds, inform anemia management and promote equitable care, thus enhancing patient outcomes