Fostering school reintegration after psychiatric inpatient treatment: description and study protocol of an evaluation study about a rehabilitation program for children and adolescents with chronic school refusal (SchuTIng-stAR)

BackgroundSchool refusal among children and adolescents with mental health issues carries long-term risks for their educational trajectories, future employment, mental health, and social participation. Despite the availability of multiple treatment approaches, a significant number of adolescents continue to experience difficulties with school attendance following inpatient therapy or partial hospitalization. To enhance reintegration into school, a rehabilitation project called “educational participation and integration for children and adolescents with mental illness through a seamless stepwise rehabilitation program” (“SchuTIng-stAR”) was developed specifically for children and adolescents with severe and persistent school refusal associated with psychiatric disorders who are at risk of continued school attendance problems after psychiatric inpatient treatment or partial hospitalization.Methods/study designAfter describing the therapeutic rationale, the development, and the content of the program, the study protocol for its evaluation using both quantitative and qualitative methods is presented. The primary objectives of the evaluation are firstly to assess the effects of the treatment on psychological symptoms and school attendance, and secondly to identify factors that influence the participation and engagement of patients, parents, and other stakeholders involved (teachers, youth welfare services). The operationalization of outcomes, measurement methods and hypotheses regarding effectiveness are described. Measurements will be taken at three points in time: at the beginning of the rehabilitation intervention (T1), at the end of treatment (T2) for the main outcome and after a six-month follow-up period (T3) for follow-up assessment. Therefore, it is a one-group pretest–posttest design with follow-up period. Additionally, it is explained how interviews with families will be analyzed using qualitative content analysis.DiscussionThe formative and summative evaluation of innovative treatment programs for children and adolescents, including the perspectives of relevant stakeholders, is essential to ensure their sustainability and their integration into already existing services provided by health and social care systems. As chronic school avoidance is a multifactorial and complex condition and its course is often characterized by relapses, it is important to develop sustainable treatment approaches and to closely examine treatment commitment using qualitative methods. The discussion focuses on the extent to which the rehabilitation intervention and the study produce the expected results, and what factors might contribute to divergent outcomes

Einfluss von IFN-beta auf die Lebensspanne und apoptose tumorinfiltrierender neutrophiler Granulozyten

Efficient immunosurveillance is a major factor in protecting the individual against cancer. Components of the innate immune system like neutrophilic granulocytes and type I IFNs were demonstrated to significantly influence this complex process by altering tumor angiogenesis and growth. Consistently, many neutrophil activities seem to be controlled by endogenous IFN-beta. Nevertheless, little is known about its detailed impact on life span and polarization of such cells. Moreover, whether and how type I IFN is induced in the tumor context and from which cellular sources it is derived is not clear. These issues were addressed in the present study. Using different transplantable tumors in an IFN- beta reporter mouse model, it could be demonstrated for the first time that IFN-beta is induced in solid tumors. This induction is mediated in a STING-IRF3/5 dependent manner and tumor infiltrating myeloid dendritic cell were identified as a main cellular source of this cytokine. Furthermore, the present results provide evidence that endogenous IFN-beta is regulating apoptosis, maturation and turnover of pro-angiogenic tumor infiltrating neutrophils by influencing both, the extrinsic as well as the intrinsic apoptosis pathways. Accordingly, the life span of tumor associated neutrophils (TANs) was remarkably prolonged in tumor bearing Ifnb1ko mice, compared to wild type (WT) controls. Lower expression of Fas, reactive oxygen species, active caspase 3 and 9 as well as a change in expression ratio of pro- and anti-apoptotic members of the Bcl-2 family were observed under such conditions. In addition, the major apoptosome constituent Apaf-1 was downregulated. In line with inhibition of apoptosis and increased neutrophil longevity in the absence of endogenous IFN-beta, a strong enhancement of G-CSF expression and PI3 kinase phosphorylation was detected. During the present study substantial evidence was accumulated supporting the role of endogenous IFN-beta as factor responsible for the induction of an N1 anti-tumor neutrophil polarization. Hence, a significant down regulation of N1 associated neutrophil features such as ICAM1 expression, TNF-alpha production and tumor killing capacity were observed in Ifnb1ko mice, compared to WT controls. Of note, exogenous IFN- beta therapy of tumor bearing WT mice significantly influenced neutrophil polarization at the primary tumor site and in the pre metastatic lung, inducing a N1 anti-tumor neutrophil phenotype. Taken together, the present work provides important insight into the molecular mechanisms underlying type I IFN-mediated cancer immune surveillance via the polarization of tumor associated neutrophilic granulocytes.Ein intaktes Immunsystem ist ein essentieller Faktor zum Schutz des Menschen vor bösartigen Tumorerkrankungen. Besonders Komponenten des angeborenen Immunsystems, wie neutrophile Granulozyten und Typ I Interferone, beeinflussen in signifikantem Umfang die Entwicklung von Tumoren indem sie deren Wachstum und Angiogenese kontrollieren. Im Konsens damit konnte gezeigt werden, dass viele funktionelle Eigenschaften von neutrophilen Granulozyten durch IFN-beta reguliert werden. Trotz allem ist der genaue Einfluss dieses Zytokins auf die Lebensspanne und Polarisation der Granulozyten bislang unbekannt. Es ist darüberhinaus unklar, ob, in welchem Umfang und von welchen Zellen IFN- beta im Tumorgewebe produziert wird. Diese Fragen waren Gegenstand der vorliegenden Dissertation. Mit Hilfe von transplantabelen Tumoren konnte in einem IFN- beta -Reporter-Maus-Model zum ersten Mal gezeigt werden, dass es während des Wachstums solider Tumore zur Induktion von IFN- beta kommt. Diese Induktion ist abhängig von den Adaptormolekülen STING, IRF3 und IRF5. Tumor-assoziierte myeloide dendritische Zellen konnten als Hauptquelle des IFN- beta identifiziert werden. In der vorliegenden Arbeit konnte zudem gezeigt werden, dass endogenes IFN- beta sowohl die Reifung als auch den Zelltod pro-angiogenetischer, neutrophiler Granulozyten reguliert indem es sowohl Komponenten des intrinsischen als auch des extrinsischen Apoptosepathways beeinflusst. In Ifnb1-/- Mäusen war die Lebensspanne der Granulozyten im Vergleich zum Wildtyp drastisch reduziert. Dies wurde bedingt durch eine abgeschwächte Expression von Fas, eine geringere Produktion reaktiver Sauerstoffspezies, reduzierte Aktivität von Caspasen (3,9), sowie ein verändertes Genexpressionsmuster pro- und anti-apoptotischer Mitglieder der Bcl-2 Proteinfamilie. Zusätzlich wurde in Abwesenheit von IFN- beta eine signifikant geringere Expression von Apaf-1, einem Hauptbestandteil des Apoptosoms detektiert. Mechanistisch ist dies evtl. auf eine gesteigerte Produktion des Zytokins G-CSF zurückzuführen. In der vorliegenden Dissertation wurde außerdem ein deutlicher Einfluss von Typ I IFN auf die Polarisation von Granulozyten hin zu einem N1 anti-tumor Phänotyp deutlich. Viele wichtige Marker dieses Granulozytenphänotyps, wie ICAM1 oder TNF-alpha waren in Abwesenheit von IFN- beta drastisch in ihrer Expression reduziert. Eine niedrig dosierte Typ I IFN Therapie war im Gegensatz dazu in der Lage die Polarisation der Neutrophilen in tumortragenden WT Mäusen gegenläufig zu beeinflussen und einen N1 Phänotyp zu induzieren. Zusammenfassend gehen aus der vorliegenden Dissertation wichtige Erkenntnisse hervor die zum besseren Verständnis der Polarisation und Repolarisation tumor-unterstützender Granulozyten beitragen

Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration.

The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase "cut-and-paste" engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared

Type I IFNs induce anti-tumor polarization of tumor associated neutrophils in mice and human.

The importance of tumor associated neutrophils (TANs) in cancer development is in the meantime well established. Numerous of clinical data document the adverse prognostic effects of neutrophil infiltration in solid tumors. However, certain tumor therapies need functional neutrophils to be effective, suggesting altered neutrophil polarization associated with different outcomes for cancer patients. Therefore, modulation of neutrophilic phenotypes represents a potent therapeutic option, but factors mediating neutrophil polarization are still poorly defined. In this manuscript we provide evidence that type I IFNs alter neutrophilic phenotype into anti-tumor, both in mice and human. In the absence of IFN-β, pro-tumor properties, such as reduced tumor cytotoxicity with low neutrophil extracellular traps (NETs) expression, low ICAM1 and TNF-α expression, dominated neutrophil phenotypes in primary lesion and premetastatic lung. Interestingly, such neutrophils have significantly prolonged life-span. Notably, interferon therapy in mice altered TAN polarization towards anti-tumor N1. Similar changes in neutrophil activation could be observed in melanoma patients undergoing type I IFN therapy. Altogether, these data highlight the therapeutic potential of interferons, suggesting optimization of its clinical use as potent anti-tumor agent

The Mechanism of Type I Interferon-Mediated Polarization of Tumor-Associated Neutrophils in Mice and Human

Abstract Neutrophils are the most abundant cells of all white blood cells and play a key role in host inflammatory responses. Importantly, inflammation has been associated with increased susceptibility for cancer and neutrophils, as a crucial component of this process, play essential role in inflammation-driven tumorigenesis. Neutrophils also represent an independent prognostic marker in a broad variety of neoplasias. The tumor microenvironment represents a special niche that is extremely influencing infiltrating immune cells. The concept of immune cell polarization was first described for macrophages (anti-tumor M1/pro-tumor M2). Recently neutrophil polarization has been postulated. Neutrophils appear to have diverse phenotypes in the tumor microenvironment i.e. tumor promoting (N2) or inhibiting (N1). Previously, we could show that significantly elevated numbers of neutrophils accumulate in tumors of mice that lack endogenous type I IFNs (Ifnb1-/-). Such tumor associated neutrophils (TANs) do not only efficiently support tumor angiogenesis and growth by up-regulating pro-angiogenic molecules (VEGF and MMP9), but also secrete higher amounts of neutrophil-attracting chemokines and display prolonged survival, compared to their WT counterparts thus representing pro-tumor N2 phenotype. Moreover, we could show that these N2 neutrophils efficiently support metastatic processes, due to up-regulation of pro-metastatic proteins, like Bv8, MMP9, S100A8 and S100A9, and due to impaired tumor cell killing. Treatment of such cells with rmIFNb reversed such an effect leading to anti-tumor N1 polarization. Here, we add further evidence emphasizing the importance of type I IFNs for neutrophil polarization in tumor microenvironment and reveal possible mechanism responsible for this phenomenon. In Ifnb1-/- mice, we observe a significant down-regulation of anti-tumor neutrophil markers, like ICAM1 and TNF-α. Moreover, neutrophils show reduced formation of NETs, accompanied by lower tumor killing capacity. Under these conditions, massively enhanced neutrophil turnover in combination with accumulation of immature neutrophils is observed. Importantly, therapeutic intervention in both Ifnb1-/- and WT mice using low dose IFN-β, induced anti-tumor activation of neutrophils. Correspondingly, in human melanoma patients undergoing type I IFN therapy, neutrophil anti-tumor characteristics were augmented, compared to untreated patients, suggesting effective outcome of this therapy. Further, we evaluated the mechanism of prolonged neutrophil survival in the absence of endogenous IFN-β. Importantly, we found that G-CSF mRNA expression levels in Ifnb1-/- neutrophils from different anatomical compartments as well as G-CSF blood serum levels were markedly up-regulated in such mice. G-CSF-expression levels were strongly reduced when the Ifnb1-/- neutrophils were incubated with rmIFN-β suggesting involvement of type I interferons in G-CSF down-regulation. Notably, we could recently show that G-CSF induces synthesis of enzyme Nicotinamide phosphoribosyltransferase (NAMPT), which is a rate-limiting enzyme converting nicotinamide (NA) into NAD+ that in turn activates NAD+ -dependent protein deacetylases sirtuins (SIRTs). NAMPT serves as an inhibitor of neutrophil apoptosis and as neutrophil chemoattractant by upregulation of CXCL8. It is a potent pro-inflammatory factor (upregulation of ROS release) and pro-angiogenic factor (smooth muscle maturation). At the same time, NAMPT was found to be strongly overexpressed in tumors and serum of leukemia patients. Analysis of NAMPT and SIRTs levels in tumor bearing Ifnb1-/- mice revealed highly upregulated levels of NAMPT and SIRT1 in blood neutrophils of these animals, in comparison to WT mice, which was in line with elevated levels of G-CSF. It also correlates with enhanced tumor angiogenesis, growth and metastasis. Based on these observations, we identified a new mechanism of interferon-mediated activation of pro-tumor neutrophils. Since tumor associated neutrophils represent a highly potent therapeutic target, these data highlight the therapeutic potential of interferons and NAMPT inhibitors, suggesting optimization of their clinical use as potent anti-tumor agent. Disclosures No relevant conflicts of interest to declare. </jats:sec