Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

Background

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis.

Methodology/Principal Findings

Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner.

Conclusion/Significance

Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases.     

In Vivo Dynamic Deformation of Articular Cartilage in Intact Joints Loaded by Controlled Muscular Contractions

When synovial joints are loaded, the articular cartilage and the cells residing in it deform. Cartilage deformation has been related to structural tissue damage, and cell deformation has been associated with cell signalling and corresponding anabolic and catabolic responses. Despite the acknowledged importance of cartilage and cell deformation, there are no dynamic data on these measures from joints of live animals using muscular load application. Research in this area has typically been done using confined and unconfined loading configurations and indentation testing. These loading conditions can be well controlled and allow for accurate measurements of cartilage and cell deformations, but they have little to do with the contact mechanics occurring in a joint where non-congruent cartilage surfaces with different material and functional properties are pressed against each other by muscular forces. The aim of this study was to measure in vivo, real time articular cartilage deformations for precisely controlled static and dynamic muscular loading conditions in the knees of mice. Fifty and 80% of the maximal knee extensor muscular force (equivalent to approximately 0.4N and 0.6N) produced average peak articular cartilage strains of 10.5±1.0% and 18.3±1.3% (Mean ± SD), respectively, during 8s contractions. A sequence of 15 repeat, isometric muscular contractions (0.5s on, 3.5s off) of 50% and 80% of maximal muscular force produced cartilage strains of 3.0±1.1% and 9.6±1.5% (Mean ± SD) on the femoral condyles of the mouse knee. Cartilage thickness recovery following mechanical compression was highly viscoelastic and took almost 50s following force removal in the static tests.     

No evidence for DUP25 in patients with panic disorder using a quantitative real-time PCR approach

A duplication of chromosome 15q24-q26 (DUP25) has been reported to be associated with anxiety disorders. We tested for the presence of DUP25 in a sample of 50 patients with panic disorder and 50 controls using a quantitative real-time PCR approach. Contrary to the original finding, our results were compatible with the absence of DUP25, and no significant difference could be detected between patients and controls (P=1.0). Thus, our study does not support the hypothesis of an involvement of DUP25 in panic disorder.     

Tuberous sclerosis genes regulate cellular 14-3-3 protein levels

The genes TSC1, encoding hamartin, and TSC2, encoding tuberin are responsible for tuberous sclerosis. This autosomal dominant tumor suppressor gene syndrome affects about 1 in 6000 individuals. A variety of tumors characteristically occur in different organs of tuberous sclerosis patients and are believed to result from defects in cell cycle/cell size control. We performed a proteomics approach of two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after ectopic overexpression of human TSC1 or TSC2. We found the cellular levels of four isoforms of the 14-3-3 protein family, 14-3-3 gamma, 14-3-3, 14-3-3 sigma, and 14-3-3 zeta, to be regulated by the two tuberous sclerosis gene products. In the same experiments the protein levels of keratin 7, capZ alpha-1 subunit, ezrin, and nedasin were not affected by ectopic TSC1 or TSC2. Western blot analyses confirmed the deregulation of 14-3-3 proteins upon ectopic overexpression of TSC1 and TSC2. A TSC1 mutant not encoding the transmembrane domain and the tuberin-binding domain but harbouring most of the coiled-coil region and the ERM protein interaction domain of hamartin did not affect 14-3-3 protein levels. The here presented findings suggest that deregulation of 14-3-3 protein amounts might contribute to the development of tumors in tuberous sclerosis patients. These data provide important new insights into the molecular development of this disease especially since both, the TSC genes and the 14-3-3 proteins, are known to be involved in mammalian cell cycle control.     

Body adiposity index and other indexes of body composition in the SAPHIR study: Association with cardiovascular risk factors

Objective:

The accuracy of anthropometric surrogate markers such as the body adiposity index (BAI) and other common indexes like the body mass index (BMI), waist‐to‐hip ratio (WHR) and waist‐to‐height ratio (WHtR) to predict metabolic sequelae is essential for its use in clinical practice.

Design and Methods:

Thus, we evaluated the strength of BAI and other indexes to relate with anthropometric parameters, adipocytokines, blood lipids, parameters of glucose‐homeostasis and blood pressure in 1,770 patients from the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study in a crosssectional design. Measurements were BAI, BMI, WHR, WHtR, abdominal subcutaneous and visceral adipose tissue (aSAT and VAT), total body adipose tissue mass, body weight, waist‐ and hip circumference (WC and HC), leptin, adiponectin, high‐density lipoprotein‐cholesterol (HDL‐C), low‐density lipoprotein‐cholesterol (LDL‐C), triglycerides (TG), fasting plasma glucose, fasting plasma insulin, the homeostasis model assessment of insulin resistance (HOMAIR), systolic and diastolic blood pressure.

Results and Conclusions:

BAI was significantly associated with leptin and HC. We conclude that BAI was the best calculator for leptin. BAI was inferior to BMI to predict anthropometric parameters other than HC, adiponectin, blood lipids, parameters of glucose homeostasis, and blood pressure in this cross‐sectional study.     

Combination of high-throughput microfluidics and FACS technologies to leverage the numbers game in natural product discovery

High-throughput platforms facilitating screening campaigns of environmental samples are needed to discover new products of natural origin counteracting the spreading of antimicrobial resistances constantly threatening human and agricultural health. We applied a combination of droplet microfluidics and fluorescence-activated cell sorting (FACS)-based technologies to access and assess a microbial environmental sample. The cultivation performance of our microfluidics workflow was evaluated in respect to the utilized cultivation media by Illumina amplicon sequencing of a pool of millions of droplets, respectively. This enabled the rational selection of a growth medium supporting the isolation of microbial diversity from soil (five phyla affiliated to 57 genera) including a member of the acidobacterial subgroup 1 (genus Edaphobacter). In a second phase, the entire diversity covered by 1071 cultures was used for an arrayed bioprospecting campaign, resulting in > 6000 extracts tested against human pathogens and agricultural pests. After redundancy curation by using a combinatorial chemical and genomic fingerprinting approach, we assigned the causative agents present in the extracts. Utilizing UHPLC-QTOF-MS/MS-guided fractionation and microplate-based screening assays in combination with molecular networking the production of bioactive ionophorous macrotetrolides, phospholipids, the cyclic lipopetides massetolides E, F, H and serratamolide A and many derivatives thereof was shown.     

Development of a new real-time TaqMan PCR assay for quantitative analyses of Candida albicans resistance genes expression

Candida albicans is an important opportunistic pathogen that can cause serious fungal diseases in immunocompromised patients including cancer patients, transplant patients, and patients receiving immunosuppressive therapy in general, those with human immunodeficiency virus infections and undergoing major surgery. Its emergence spectrum varies from mucosal to systemic infections and the first line treatment is still based on fluconazole, a triazole derivate with a potent antifungal activity against most of C. albicans strains. Nevertheless the emergence of fluconazole-resistant C. albicans strains can lead to treatment failures and thus become a clinical problem in the management of such infections. For that reason we consider it important to study mechanisms inducing azole resistance and the possibilities to influence this process. In this work we give a short report on a real-time PCR (TaqMan) assay, which can be used for quantitative analyses of gene expression levels of MDR1, CDR1 and ERG11, genes supposed to contribute to development of the resistance mechanisms. We show some results achieved with that assay in fluconazole susceptible and resistant strains that confirm results seen earlier in experiments using Northern blot hybridisation and prove that the comparative DeltaCt method is valid for our system.     

Regulation of differential pro- and anti-apoptotic signaling by glucocorticoids

More than a quarter of a century ago, the phenomenon of glucocorticoid-induced apoptosis in the majority of hematological cells was first recognized. More recently, glucocorticoid-induced antiapoptotic signaling associated with apoptosis resistance has been identified in cells of epithelial origin, most of malignant solid tumors and some other tissues. Despite these huge amount of data demonstrating differential pro- and anti-apoptotic effects of glucocortioids, the underlying mechanisms of cell type specific glucocorticoid signaling are just beginning to be described. This review summarizes our present understanding of cell type-specific pro- and anti-apoptotic signaling induced by glucocorticoids. In the first section we give a summary and update of known glucocorticoid-induced pathways mediating apoptosis in hematological cells. We shortly introduce mechanisms of glucocorticoid resistance of hematological cells. We highlight and discuss the emerging molecular evidence of a general induction of survival signaling in epithelial cells and carcinoma cells by glucocorticoids. We provide a model for glucocorticoid-induced resistance in cells growing in a tissue formation. Thus, attachment to the extracellular matrix and cell-cell contacts typical for e.g. epithelial and tumor cells may be crucially involved in switching the balance of several interacting pathways to survival upon treatment with glucocorticoids.