MUG Mel3 Cell Lines Reflect Heterogeneity in Melanoma and Represent a Robust Model for Melanoma in Pregnancy

Melanomas are aggressive tumors with a high metastatic potential and an increasing incidence rate. They are known for their heterogeneity and propensity to easily develop therapy-resistance. Nowadays they are one of the most common cancers diagnosed during pregnancy. Due to the difficulty in balancing maternal needs and foetal safety, melanoma is challenging to treat. The aim of this study was to provide a potential model system for the study of melanoma in pregnancy and to illustrate melanoma heterogeneity. For this purpose, a pigmented and a non-pigmented section of a lymph node metastasis from a pregnant patient were cultured under different conditions and characterized in detail. All four culture conditions exhibited different phenotypic, genotypic as well as tumorigenic properties, and resulted in four newly established melanoma cell lines. To address treatment issues, especially in pregnant patients, the effect of synthetic human lactoferricin-derived peptides was tested successfully. These new BRAF-mutated MUG Mel3 cell lines represent a valuable model in melanoma heterogeneity and melanoma pregnancy research. Furthermore, treatment with anti-tumor peptides offers an alternative to conventionally used therapeutic options—especially during pregnancy

The ARCA Registry: A Collaborative Global Platform for Advancing Trial Readiness in Autosomal Recessive Cerebellar Ataxias.

Autosomal recessive cerebellar ataxias (ARCAs) form an ultrarare yet expanding group of neurodegenerative multisystemic diseases affecting the cerebellum and other neurological or non-neurological systems. With the advent of targeted therapies for ARCAs, disease registries have become a precious source of real-world quantitative and qualitative data complementing knowledge from preclinical studies and clinical trials. Here, we review the ARCA Registry, a global collaborative multicenter platform (>15 countries, >30 sites) with the overarching goal to advance trial readiness in ARCAs. It presents a good clinical practice (GCP)- and general data protection regulation (GDPR)-compliant professional-reported registry for multicenter web-based capture of cross-center standardized longitudinal data. Modular electronic case report forms (eCRFs) with core, extended, and optional datasets allow data capture tailored to the participating site's variable interests and resources. The eCRFs cover all key data elements required by regulatory authorities [European Medicines Agency (EMA)] and the European Rare Disease (ERD) platform. They capture genotype, phenotype, and progression and include demographic data, biomarkers, comorbidity, medication, magnetic resonance imaging (MRI), and longitudinal clinician- or patient-reported ratings of ataxia severity, non-ataxia features, disease stage, activities of daily living, and (mental) health status. Moreover, they are aligned to major autosomal-dominant spinocerebellar ataxia (SCA) and sporadic ataxia (SPORTAX) registries in the field, thus allowing for joint and comparative analyses not only across ARCAs but also with SCAs and sporadic ataxias. The registry is at the core of a systematic multi-component ARCA database cluster with a linked biobank and an evolving study database for digital outcome measures. Currently, the registry contains more than 800 patients with almost 1,500 visits representing all ages and disease stages; 65% of patients with established genetic diagnoses capture all the main ARCA genes, and 35% with unsolved diagnoses are targets for advanced next-generation sequencing. The ARCA Registry serves as the backbone of many major European and transatlantic consortia, such as PREPARE, PROSPAX, and the Ataxia Global Initiative, with additional data input from SPORTAX. It has thus become the largest global trial-readiness registry in the ARCA field

Heat-Induced Structural Changes Affect OVA-Antigen Processing and Reduce Allergic Response in Mouse Model of Food Allergy

BACKGROUND AND AIMS: The egg protein ovalbumin (OVA) belongs to six most frequent food allergens. We investigated how thermal processing influences its ability to induce allergic symptoms and immune responses in mouse model of food allergy. METHODOLOGY/PRINCIPAL FINDINGS: Effect of increased temperature (70°C and 95°C) on OVA secondary structure was characterized by circular dichroism and by the kinetics of pepsin digestion with subsequent HPLC. BALB/c mice were sensitized intraperitoneally and challenged with repeated gavages of OVA or OVA heated to 70°C (h-OVA). Levels of allergen-specific serum antibodies were determined by ELISA (IgA and IgGs) or by β-hexosaminidase release test (IgE). Specific activities of digestive enzymes were determined in brush border membrane vesicles of jejunal enterocytes. Cytokine production and changes in regulatory T cells in mesenteric lymph nodes and spleen were assessed by ELISA and FACS. Heating of OVA to 70°C caused mild irreversible changes in secondary structure compared to boiling to 95°C (b-OVA), but both OVA treatments led to markedly different digestion kinetics and Tregs induction ability in vitro, compared to native OVA. Heating of OVA significantly decreased clinical symptoms (allergic diarrhea) and immune allergic response on the level of IgE, IL-4, IL-5, IL-13. Furthermore, h-OVA induced lower activities of serum mast cell protease-1 and enterocyte brush border membrane alkaline phosphatase as compared to native OVA. On the other hand h-OVA stimulated higher IgG2a in sera and IFN-γ secretion by splenocytes. CONCLUSIONS: Minor irreversible changes in OVA secondary structure caused by thermal processing changes both its digestion and antigenic epitopes formation, which leads to activation of different T cell subpopulations, induces shift towards Th1 response and ultimately reduces its allergenicity

Conditional Stat1 Ablation Reveals the Importance of Interferon Signaling for Immunity to Listeria monocytogenes Infection

Signal transducer and activator of transcription 1 (Stat1) is a key player in responses to interferons (IFN). Mutations of Stat1 cause severe immune deficiencies in humans and mice. Here we investigate the importance of Stat1 signaling for the innate and secondary immune response to the intracellular bacterial pathogen Listeria monocytogenes (Lm). Cell type-restricted ablation of the Stat1 gene in naïve animals revealed unique roles in three cell types: macrophage Stat1 signaling protected against lethal Lm infection, whereas Stat1 ablation in dendritic cells (DC) did not affect survival. T lymphocyte Stat1 reduced survival. Type I IFN (IFN-I) signaling in T lymphocytes reportedly weakens innate resistance to Lm. Surprisingly, the effect of Stat1 signaling was much more pronounced, indicating a contribution of Stat1 to pathways other than the IFN-I pathway. In stark contrast, Stat1 activity in both DC and T cells contributed positively to secondary immune responses against Lm in immunized animals, while macrophage Stat1 was dispensable. Our findings provide the first genetic evidence that Stat1 signaling in different cell types produces antagonistic effects on innate protection against Lm that are obscured in mice with complete Stat1 deficiency. They further demonstrate a drastic change in the cell type-dependent Stat1 requirement for memory responses to Lm infection

Dynamic models of drug users and susceptibles : optimal mix of use reduction and harm reduction in Australia and the U.S.A.

Zsfassung in dt. SpracheDiese Dissertation analysiert Zwei-Zustands-Modelle zur optimalen Kontrolle von illegalem Drogenkonsum. Im Unterschied zu vielen bestehenden Modellen werden Personen, die für Drogenkonsum empfänglich sind, in einem separaten Zustand betrachtet. Die zweite Zustandsvariable gibt die Anzahl der Konsumenten an.Zunächst wird das Grundmodell, ein nichtlineares, autonomes Modell mit unendlichem Planungshorizont, beschrieben. Der Einstieg in den Drogenkonsum ist eine Funktion der beiden Zustände, wobei die Wahrscheinlichkeit, dass es zu einem Neueinstieg kommt, nicht-linear von der momentanen Anzahl an Konsumenten abhängt. Die Kontrolle ``harm reduction'' wirkt sich einerseits positiv auf die im Zielfunktional akkumulierten sozialen Kosten aus, gleichzeitig bewirkt sie aber einen erhöhten Drogeneinstieg. Das Modell wird für die Kokainepidemie in den Vereinigten Staaten von Amerika und für ``injecting drug users'' in Australien betrachtet.Für die Kontrollvariable gibt es Kontrollbeschränkungen.Gleichgewichtslösungen mit Randkontrolle sind in den optimalen Lösungen von signifikanter Relevanz. ``Harm reduction'' soll in Australien nahezu immer umgesetzt werden, hingegen ist der maximal mögliche Einsatz von ``harm reduction'' in den U.S.A. nur in Phasen der Epidemie mit entweder sehr wenigen oder sehr vielen Drogenkonsumenten optimal. In den Bereichen dazwischen ist ``pure use reduction'' optimal.Der zweite Teil der Dissertation präsentiert die optimalen Lösungen für einige Abwandlungen des Basismodelles. Es wird angenommen, dass die Kokainepidemie in den USA weniger ansteckend ist. Danach wird die Funktion für den Drogeneinsteig um sogenannte Innovatoren ergänzt. Die dritte Abwandlung berücksichtigt die für die Kontrolle anfallenden Kosten.Abschließend wird die funktionale Form für den Drogeneinstieg verändert.Die Lösung der Modelle erfolgt mit dem Pontryagin'schen Maximumprinzip.Hierbei treten in fast allen Fällen Gleichgewichtspunkte mit Randkontrolle auf. In den Einzugsgebieten der Gleichgewichte existieren sogenannte Indifferenz-Kurven oder DNSS-Kurven (benannt nach Dechert, Nishimura, Sethi und Skiba). Entlang solcher Kurven ist ein Entscheidungsträger indifferent zwischen zwei Pfaden, die auf verschiedene Weise zu einem optimalen Gleichgewicht führen, beziehungsweise zwischen zwei Pfaden, die zu unterschiedlichen optimalen Gleichgewichten führen.17

When in a drug epidemic should the policy objective switch from use reduction to harm reduction?

A heated debate in drug policy concerns the relative merits of "harm reduction" (e.g., reducing drug-related HIV/AIDS transmission) vs. "use reduction" (controlling drug use per se). This paper models whether shifting emphasis between these goals over the course of a drug epidemic might reduce social costs relative to pursuing one or the other exclusively. Results suggest different answers for different drugs and/or countries. In particular, harm reduction may have always been effective for Australia's injection drug use problem, but for US cocaine it may not have been in the past even if it could be so today. In certain circumstances harm reduction may "tip" an epidemic toward a high- rather than low-use equilibrium. The location in state space of regions where this occurs can be sensitive to parameter changes, suggesting caution may be in order when advocating harm reduction, unless there is confidence the epidemic has been modeled and parameterized accurately.OR in societal problem analysis Control Health Drug policy Epidemics

Energy metabolic state in hypothermically stored boar spermatozoa using a revised protocol for efficient ATP extraction

Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are determined in many sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiency in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to twenty-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane intact spermatozoa supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa

CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

The transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance