Effect of confounding factors on a phospho-flow assay of ribosomal S6 protein for therapeutic drug monitoring of the mTOR-inhibitor everolimus in heart transplanted patients.

CONTEXT: Several assays of monitoring immune cell function have been developed to enhance therapeutic drug monitoring. OBJECTIVE: An in vitro-validated whole-blood assay of phosphorylated ribosomal protein S6 (pS6RP) was evaluated for confounders to monitor the mTOR-inhibitor everolimus (ERL). MATERIALS AND METHODS: Whole blood samples from 87 heart transplant recipients were analyzed for pS6RP-expression in CD3-positive T-cells by phospho-flow analysis. RESULTS: ERL blood concentration, laboratory parameters, co-medications, demographic and clinical data were reviewed. CONCLUSION: Evaluating the pS6RP-assay revealed that pS6RP is influenced by cyclosporine A (CsA) blood concentration, duration of ERL treatment, co-medication with thiazide diuretics and different metabolic parameters.

Impact of family-oriented rehabilitation and prevention: an inpatient program for mothers with breast cancer and their children.

OBJECTIVE: This pilot intervention study assessed the impact of a 3-week inpatient program for mothers with primary breast cancer and their children. The program combined rehabilitative treatment and preventive, psychosocial, child-centered interventions. METHODS: A total of 116 mothers (age 29-57 years, mean = 41.1) with primary, non-metastasized breast cancer completed standardized questionnaires before (Pre1), at the beginning of (Pre2), and at the end of (Post) a 3-week inpatient intervention. The standardized scales included mothers' and children's quality of life and children's psychological symptoms. Mothers' emotional functioning and their 116 children's (age 3-14 years, mean = 7.4; 47% female) psychological health and emotional symptoms were defined as primary outcome measures. A within-subject-control-group design was used to compare changes before the intervention (measurements Pre1-Pre2) to changes during the intervention (measurements Pre2-Post) via dependent one-sided t-tests. Additional exploratory analyses for further outcome variables were performed. RESULTS: Changes during the intervention period were significantly greater than changes during the waiting period for all primary outcome measures (mothers' emotional functioning: p < 0.0001; children's psychological health: p = 0.0035; and children's emotional symptoms: p = 0.0005). CONCLUSIONS: Data suggest that the family-oriented intervention 'getting well together' seems to be beneficial to mothers' and children's quality of life and psychological well-being. Combining oncological rehabilitation and preventive child-centered interventions might be a feasible approach to supporting breast cancer patients and their children and improving their emotional state. Further research is warranted.

[Mothers with breast cancer and their children: initial results regarding the effectiveness of the family oriented oncological rehabilitation program "gemeinsam gesund werden"]. / Brustkrebskranke Mütter und ihre Kinder: Erste Ergebnisse zur Effektivität der familienorientierten onkologischen Rehabilitationsmassnahme, gemeinsam gesund werden".

When a parent has a serious somatic illness, children suffer grave distress, their risk to develop a mental disorder increases. Sick mothers, in comparison to patients without children, experience additional strain, e. g. because they worry about their children. Although many studies focus on the quality of life of breast cancer patients, little is known about the special situation of young mothers with breast cancer. Currently, a prospective study with four assessment points (pre, post, 3- and 12-months-follow-up) is conducted on a large sample of women taking part in the family-oriented inpatient rehabilitation program "gemeinsam gesund werden'. In this paper a longitudinal sample of 173 women and 153 children, their quality of life and psychological impairment prior to, after and in the course of one year after the rehabilitation is presented. Compared to the general population, the children as well as the mothers are considerably impaired, especially in regard to Psychological Health/Emotional Functioning. These impairments improve markedly within the survey period. One year after the rehabilitation, the children show no more impairment than the reference groups. The mothers improve significantly as well, however, after one year their quality of life scores are still lower than those of the general population. In conclusion, a considerable impairment of the analyzed group of mothers with breast cancer and their children, the necessity of special supportive measures as well as the effectiveness of the model rehabilitation program can be shown.

Individualised stepwise adaptive treatment for 3-6-year-old preschool children impaired by attention-deficit/hyperactivity disorder (ESCApreschool): study protocol of an adaptive intervention study including two randomised controlled trials within the consortium ESCAlife.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is a psychosocially impairing and cost-intensive mental disorder, with first symptoms occurring in early childhood. It can usually be diagnosed reliably at preschool age. Early detection of children with ADHD symptoms and an early, age-appropriate treatment are needed in order to reduce symptoms, prevent secondary problems and enable a better school start. Despite existing ADHD treatment research and guideline recommendations for the treatment of ADHD in preschool children, there is still a need to optimise individualised treatment strategies in order to improve outcomes. Therefore, the ESCApreschool study (Evidence-Based, Stepped Care of ADHD in Preschool Children aged 3 years and 0 months to 6 years and 11 months of age (3;0 to 6;11 years) addresses the treatment of 3-6-year-old preschool children with elevated ADHD symptoms within a large multicentre trial. The study aims to investigate the efficacy of an individualised stepwise-intensifying treatment programme. METHODS: The target sample size of ESCApreschool is 200 children (boys and girls) aged 3;0 to 6;11 years with an ADHD diagnosis according to Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) or a diagnosis of oppositional defiant disorder (ODD) plus additional substantial ADHD symptoms. The first step of the adaptive, stepped care design used in ESCApreschool consists of a telephone-assisted self-help (TASH) intervention for parents. Participants are randomised to either the TASH group or a waiting control group. The treatment in step 2 depends on the outcome of step 1: TASH responders without significant residual ADHD/ODD symptoms receive booster sessions of TASH. Partial or non-responders of step 1 are randomised again to either parent management and preschool teacher training or treatment as usual. DISCUSSION: The ESCApreschool trial aims to improve knowledge about individualised treatment strategies for preschool children with ADHD following an adaptive stepped care approach, and to provide a scientific basis for individualised medicine for preschool children with ADHD in routine clinical care. TRIAL REGISTRATION: The trial was registered at the German Clinical Trials Register (DRKS) as a Current Controlled Trial under DRKS00008971 on 1 October 2015. This manuscript is based on protocol version 3 (14 October 2016).

Antisense gapmers selectively suppress individual oncogenic p73 splice isoforms and inhibit tumor growth in vivo.

BACKGROUND: Differential mRNA splicing and alternative promoter usage of the TP73 gene results in the expression of multiple NH2-truncated isoforms that act as oncogenes. Abundant levels of these p73 variants in a variety of human cancers correlated with adverse clinical prognosis and response failure to conventional therapies, underscoring their relevance as marker for disease severity and target for cancer intervention. With respect to an equally important role for amino-truncated p73 splice forms (DeltaTAp73) and DeltaNp73 (summarized as DNp73) in the tumorigenic process, we designed locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers against individual species that were complementary to DeltaEx2 and DeltaEx2/3 splice junctions and a region in exon 3B unique for DeltaN' and DeltaN. RESULTS: Treatment of cancer cells with these ASOs resulted in a strong and specific reduction of tumorigenic p73 transcripts and proteins, importantly, without abolishing the wild-type p73 tumor suppressor form as observed with p73-shRNA. The specific antisense oligonucleotides rescued cells from apoptosis inhibition due to overexpression of their corresponding amino-truncated p73 isoform and decreased tumor cell proliferation. Furthermore, ASO-116 against DeltaEx2/3 coupled to magnetic nanobead polyethyleneimine (MNB/PEI) carriers significantly inhibited malignant melanoma growth, which correlated with a shift in the balance between endogenous TAp73 and DeltaEx2/3 towards apoptotic full-length p73. CONCLUSION: Our study demonstrates the successful development of LNA-ASOs that selectively differentiate between the closely related p73 oncoproteins, and provide new tools to further delineate their biological properties in different human malignancies and for therapeutic cancer targeting.

Autonomous growth and hepatocarcinogenesis in transgenic mice expressing the p53 family inhibitor DNp73.

p53 family proteins carry on a wide spectrum of biological functions from differentiation, cell cycle arrest, apoptosis and chemosensitivity of tumors. Conversely, N-terminally truncated p73 (DNp73) functions as a potent inhibitor of all these tumor suppressor properties, implicating its participation in malignant transformation and oncogenesis. Several reports indicated considerable up-regulation of DNp73 in hepatocellular carcinoma (HCC) that correlates with reduced survival of patients, but little is known about the functional significance of DNp73 to tumorigenesis in vivo due to the lack of an appropriate model. To address this, we generated transgenic mice in which DNp73 expression is directed to the liver by the albumin promoter. Gene expression was tested by mRNA and protein analyses. Transgenic mice exhibited prominent hepatic histological abnormalities including increased hepatocyte proliferation resulting in preneoplastic lesions (liver cell adenomas) at 3-4 months. Among 12- to 20-month-old mice, 83% of animals developed hepatic carcinoma. HCC displayed a significant increase of hyperphosphorylated inactive retinoblastoma, whereas p53-regulated inhibitors of cell cycle progression were down-regulated in the tumors. Our data firmly establish the unique oncogenic capability of DNp73 to drive hepatocarcinogenesis in vivo, supporting its significance as a marker for disease severity in patients and as target for cancer prevention. This model offers new opportunities to further delineate DNp73-mediated liver oncogenesis but may also enable the development of more effective cancer therapies.

Dendritic Cells and Their Role in Cardiovascular Diseases: A View on Human Studies.

The antigen-presenting dendritic cells (DCs) are key to the immunological response, with different functions ascribed ranging from cellular immune activation to induction of tolerance. Such immunological responses are involved in the pathophysiological mechanisms of cardiovascular diseases, with DCs shown to play a role in atherosclerosis, hypertension, and heart failure and most notably following heart transplantation. A better understanding of the interplay between the immune system and cardiovascular diseases will therefore be critical for developing novel therapeutic treatments as well as innovative monitoring tools for disease progression. As such, the present review will provide an overview of DCs involvement in the pathophysiology of cardiovascular diseases and how targeting these cells may have beneficial effects for the prognosis of patients.

A data set from flash X-ray imaging of carboxysomes.

Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere.

Assessment of Immunological Biomarkers in the First Year after Heart Transplantation.

BACKGROUND: Pharmacodynamic biomarkers that detect changes of immunological functions have been recognized as a helpful tool to increase the efficacy of immunosuppressive drug therapies. However, physiological changes of immunological biomarkers following transplantation are not investigated. Therefore, we assessed frequently used immunological biomarkers of the circulating blood in the first year following heart transplantation (HTx). METHODS: Activation markers CD25 and CD95, intracellular cytokines IL-2 and IFNγ, chemokines IP10 and MIG, and subsets of dendritic cells as well as antibodies against human leukocyte antigens (HLA) and major histocompatibility complex class I-related chain A (MICA) antigens were analyzed at different time points using flow cytometry and Luminex xMAP technology. RESULTS: Expression of IL-2, IFNγ, and plasmacytoid dendritic cells (pDCs) significantly increased (p < 0.01) during the first year. Anti-HLA antibodies decreased continuously, while anti-MICA antibodies showed minor increase within the first year. An association between percentage of pDCs and anti-MICA antibody positivity was proven. pDCs, IFNγ-producing T cells, and IP10 concentration were associated in a stronger way with age and gender of HTx recipients than with antibodies against HLA or MICA. CONCLUSIONS: We conclude that certain immunological biomarkers of the circulating blood change during the first year after HTx. These changes should be considered for interpretation of biomarkers after transplantation.

Role of dendritic cells in the context of acute cellular rejection: comparison between tacrolimus- or cyclosporine A-treated heart transplanted recipients.

BACKGROUND: In the last years many studies have been designed to predict risk of acute rejection and to adapt the immunosuppressive therapy. The importance of dendritic cells (DCs) in the immune response, especially their role in tolerance is known. Thus, we investigated the influence of tacrolimus (TAC)-based and of cyclosporine A (CsA)-based immunosuppressive therapies on dendritic cells and the incidence of rejection in heart transplant recipients. METHODS: Groups consisted of 14 CsA treated and 15 TAC treated patients. At different study time points (0, 3 and 6 months after study begin) peripheral blood from the patients was drawn to analyse (1) blood concentration of CsA or TAC (trough value) and (2) percentages of plasmacytoid and myeloid DC (p and mDC) subsets using flow cytometry. Histological rejection grading was performed of endomyocardial biopsies. RESULTS: TAC treated patients had significantly higher values of pDCs (CsA group 53.9%±13.0%; TAC group 67.5%±8.4%; p<0.05) and significantly lower values of mDCs than CsA treated patients (CsA group 58%±19.0%; TAC group 45.2%±10.7%; p<0.05). In general, HTx patients with rejection grade of ≥2 had significant lower values of pDCs (55.1%±16.2%) compared to patients without rejection (63.6%±10.5%; p<0.05). TAC-treated patients had significantly less rejections CsA-treated patients (CsA group 0.86±0.95; TAC group 0.2±0.4; p<0.05). CONCLUSIONS: Our results showed that HTx patients with high pDCs had a lower risk for rejection and that TAC-treated patients had higher pDCs values compared to CsA-treated patients. Future studies need to define individual pDC values to predict acute cellular rejection.

The effectiveness of child and adolescent psychiatric treatments in a naturalistic outpatient setting.

Data concerning the effectiveness of naturalistic treatments (treatment-as-usual) in child and adolescent psychiatric (CAP) services are scarce. The purpose of this prospective observational study was to examine the effectiveness of CAP treatments in a naturalistic outpatient setting. Three hundred six patients (attention-deficit/hyperactivity disorder, ADHD, n=94; conduct disorder, CD, n=57; anxiety disorder, AD, n=53; depressive disorder, DD, n=38; other diagnostic categories, n=64), from nine child and adolescent psychiatric practices in Germany, were evaluated. Treatment effects were compared between patients who received frequent treatment and patients who only participated in diagnostics and short interventions. Since randomization was not feasible, propensity score analysis methods were used. Regarding the total sample, no significant treatment effects were found. However, a subgroup analysis of the four most frequent disorders (ADHD, CD, AD, DD) showed small to moderate treatment effects in patients with ADHD and AD. In CD and DD subgroups, no significant treatment effects could be found. "Real-world" CAP outpatient treatment seems to produce significant effects for ADHD and AD, but not for CD and DD. Compared to efficacy studies, our results show that naturalistic treatment might be better than expected.