Mesenchymal expression of the <Emphasis Type="Italic">FRAS1/FREM2</Emphasis> gene unit is decreased in the developing fetal diaphragm of nitrofen-induced congenital diaphragmatic hernia

Purpose

Developmental mutations that inhibit normal formation of extracellular matrix (ECM) in fetal diaphragms have been identified in congenital diaphragmatic hernia (CDH). FRAS1 and FRAS1-related extracellular matrix 2 (FREM2), which encode important ECM proteins, are secreted by mesenchymal cells during diaphragmatic development. The FRAS1/FREM2 gene unit has been shown to form a ternary complex with FREM1, which plays a crucial role during formation of human and rodent diaphragms. Furthermore, it has been demonstrated that the diaphragmatic expression of FREM1 is decreased in the nitrofen-induced CDH model. We hypothesized that FRAS1 and FREM2 expression is decreased in the developing diaphragms of fetal rats with nitrofen-induced CDH.

Methods

Pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on D13, D15 and D18. Microdissected diaphragms were divided into nitrofen-exposed/CDH and control samples (n = 12 per time-point and experimental group, respectively). Diaphragmatic gene expression levels of FRAS1 and FREM2 were analyzed by qRT-PCR. Immunofluorescence double staining for FRAS1 and FREM2 was combined with the mesenchymal marker GATA4 in order to evaluate protein expression and localization in pleuroperitoneal folds (PPFs) and fetal diaphragmatic tissue.

Results

Relative mRNA expression of FRAS1 and FREM2 were significantly reduced in PPFs of nitrofen-exposed fetuses on D13 (1.76 ± 0.86 vs. 3.09 ± 1.15; p < 0.05 and 0.47 ± 0.26 vs. 0.82 ± 0.36; p < 0.05), developing diaphragms of nitrofen-exposed fetuses on D15 (1.45 ± 0.80 vs. 2.63 ± 0.84; p < 0.05 and 0.41 ± 0.16 vs. 1.02 ± 0.49; p < 0.05) and fully muscularized diaphragms of CDH fetuses on D18 (1.35 ± 0.75 vs. 2.32 ± 0.92; p < 0.05 and 0.37 ± 0.24 vs. 0.70 ± 0.32; p < 0.05) compared to controls. Confocal laser scanning microscopy revealed markedly diminished FRAS1 and FREM2 immunofluorescence in diaphragmatic mesenchyme, which was associated with reduced proliferation of mesenchymal cells in nitrofen-exposed PPFs and fetal CDH diaphragms on D13, D15 and D18 compared to controls.

Conclusion

Decreased mesenchymal expression of FRAS1 and FREM2 in the nitrofen-induced CDH model may cause failure of the FRAS1/FREM2 gene unit to activate FREM1 signaling, disturbing the formation of diaphragmatic ECM and thus contributing to the development of diaphragmatic defects in CDH.

     

Using transprofessional care in the emergency department to reduce patient admissions: A retrospective audit of medical histories

The demand for emergency department (ED) services has increased significantly, due to our increasingly ageing population and limited access to primary care. This article reports outcomes from a transprofessional model of care in an ED in Victoria, Australia. Nurses, physiotherapists, social workers, and occupational therapists undertook additional education to increase the range of services they could provide and thereby expedite patient flow through the ED. One hundred patients who received this service were matched against 50 patients who did not. The most common reasons for patient admission were limb injury/limb pain (n = 47, 23.5%) and falls (n = 46, 23.0%). Transprofessional interventions included applying supportive bandages, slings, zimmer splints and controlled ankle motion (CAM) boots, and referral to new services such as case management and mental health teams. The rate of hospital admissions was significantly lower in the transprofessional group (n = 27, 18.0%) than in the reference group (n = 19, 38%, p = 0.005). This group also had a slightly lower re-presentation rate (n = 4, 2.7%) than patients in the reference group (n = 2, 4.0%). There are many benefits that support this model of care that in turn reduces ED overcrowding and work stress. A transprofessional model may offer a creative solution to meeting the varied needs of patients presenting for emergency care.     

Health care providers’ support of patients’ autonomy,phosphate medication adherence,race and gender in end stage renal disease

This study was designed to assess dialysis subjects’ perceived autonomy support association with phosphate binder medication adherence, race and gender. A multi-site cross-sectional study was conducted among 377 dialysis subjects. The Health Care Climate (HCC) Questionnaire assessed subjects’ perception of their providers’ autonomy support for phosphate binder use, and adherence was assessed by the self-reported Morisky Medication Adherence Scale. Serum phosphorus was obtained from the medical record. Regression models were used to examine independent factors of medication adherence, serum phosphorus, and differences by race and gender. Non-white HCC scores were consistently lower compared with white subjects’ scores. No differences were observed by gender. Reported phosphate binder adherence was associated with HCC score, and also with phosphorus control. No significant association was found between HCC score and serum phosphorus. Autonomy support, especially in non-white end stage renal disease subjects, may be an appropriate target for culturally informed strategies to optimize mineral bone health.     

Identification of coeliac disease triggering glutenin peptides in adults

Objective: Coeliac disease affects approximately 1% of Northern American and European populations. It is caused by an inappropriate immune response to dietary gluten. Gluten comprises of two major protein fractions: gliadins and glutenins. Glutenins have recently been found to be toxic to coeliac individuals. Proliferation assays suggest in some but not all paediatric coeliac individuals there may be immunological stimulation with high molecular weight (HMW) glutenins. Less evidence pertains to low molecular weight (LMW) glutenins. The aim is to assess adaptive, T-cell driven, and innate immune response in adult coeliac individuals towards HMW glutenin peptide, glut04, and LMW glutenin peptide, glt156.

Materials and methods: Coeliac patients were recruited attending endoscopy for routine monitoring. Adaptive immune response towards glut04 and glt156 was measured by proliferation assays and measurement of interferon-γ secretion in 28 T-cell lines. The innate immune response was assessed by measurement of enterocyte cell height (ECH) in coeliac small intestinal biopsies following overnight incubation in organ culture chambers in a further nine individuals.

Results: There were 3/28 and 2/28 positive proliferation results using gluten-sensitive T-cells with glut04 and glt156, respectively. All coeliac biopsies tested in organ culture chambers demonstrated clear reduction in ECH with peptic-tryptic digest of whole industrial gluten, glut04 and glt156 when compared to negative control ovalbumin (p?Conclusions: This study demonstrates glutenin epitopes glut04 and glt156, while minor T-cell epitopes, are important in their ability to trigger the innate immune response.     

Effects of Cycling Versus Running Training on Sprint and Endurance Capacity in Inline Speed Skating

The purpose of this study was to compare the effects of running versus cycling training on sprint and endurance capacity in inline speed skating. Sixteen elite athletes (8 male, 8 female, 24 ± 8 yrs) were randomly assigned into 2 training groups performing either 2 session per week of treadmill running or ergometer cycling in addition to 3 skating specific sessions (technique, plyometrics, parkour) for 8 weeks. Training intensity was determined within non-specific (cycling or running) and effects on specific endurance capacity within a specific incremental exercise test. Before and after the intervention all athletes performed a specific (300m) and one non-specific (30s cycling or 200m running) all-out sprint test according to the group affiliation. To determine the accumulation of blood lactate (BLa) and glucose (BGL) 20 μl arterialized blood was drawn at rest, as well as in 1 min intervals for 10 min after the sprint test. The sport-specific peak oxygen uptake (VO₂ peak) was significantly increased (+17%; p = 0.01) in both groups and highly correlated with the sprint performance (r = -0.71). BLa values decreased significantly (-18%, p = 0.02) after the specific sprint test from pre to post-testing without any group effect. However, BGL values only showed a significant decrease (-2%, p = 0.04) in the running group. The close relationship between aerobic capacity and sprint performance in inline speed skating highlights the positive effects of endurance training. Although both training programs were equally effective in improving endurance and sprint capacities, the metabolic results indicate a faster recovery after high intensity efforts for all athletes, as well as a higher reliance on the fat metabolism for athletes who trained in the running group.

Key points

• In addition to a highly developed aerobic performance inline speed skaters also require a highly trained anaerobic capacity to be effective in the sprint sections such as the mass start, tactical attacks and finish line sprint.
• An 8-week low-intensity endurance training program of either cycling or running training combined with additional routine training improves classical aerobic characteristics (17% increase of VO₂ peak), as well as values for acceleration and speed.
• Athletes who trained in the running group demonstrated a higher reliance on the fat metabolism in the sport-specific post-testing.
• The significant reduction in anaerobic ATP turnover during repeated sprints appears to be partially compensated by an increase in VO₂ in subsequent sprint. The results revealed a close relationship between the aerobic capacity and sprint performance in inline speed skating.

Key words: Aerobic metabolism, blood glucose concentration, all-out sprint test     

Reversible HLA multimers (Streptamers) for the isolation of human cytotoxic T lymphocytes functionally active against tumor- and virus-derived antigens

The development of MHC/peptide multimers has facilitated the visualization and purification of antigen-specific T cells. However, the persistence of multimers leads to prolonged T cell receptor signaling and subsequently to altered T-cell function. We have recently developed a new type of MHC/peptide multimers, which can be dissociated from the T cell. Herein, we have generated and tested for the first time reversible HLA/peptide multimers, termed Streptamers, for the isolation of human T cells. The Streptamer technique demonstrates the specificity and sensitivity of conventional HLA/peptide tetramers with regards to the sorting of human T lymphocytes. This is shown for T cells directed against immunogenic peptides derived from viral and tumor-associated antigens. We show that antigen-specific cytotoxic T cells remain functionally active following Streptamer dissociation, whereas lytic function and proliferation of the T cells is impaired in the presence of conventional tetramers. These novel HLA/peptide Streptamer reagents allow the isolation of antigen-specific T cells with preserved function and, therefore, facilitate the development of adoptive T cell transfer regimens for the treatment of patients with cancer or infectious diseases.     

The rat mammary gland: morphologic changes as an indicator of systemic hormonal perturbations induced by xenobiotics

The development and morphology of the rat mammary gland are dependent upon several hormones including estrogens, androgens, progesterone, growth hormone and prolactin. In toxicology studies, treatment with xenobiotics may alter these hormones resulting in changes in the morphology of reproductive tissues such as the mammary gland. In the rat, male and female mammary glands exhibit striking morphologic differences that can be altered secondary to hormonal perturbations. Recognizing these morphologic changes can help the pathologist predict potential xenobiotic-induced perturbations in the systemic hormonal milieu. This review examines the development of the rat mammary gland and the influence of sex hormones on the morphology of the adult male and female rat mammary gland. Specific case examples from the literature and data from our laboratory highlight the dynamic nature of the rat mammary gland in response to hormonal changes.     

Three-dimensional mapping of ozone-induced injury in the nasal airways of monkeys using magnetic resonance imaging and morphometric techniques

Age-related changes in gross and microscopic structure of the nasal cavity may alter local tissue susceptibility as well as the dose of inhaled toxicant delivered to susceptible sites. This article describes a novel method for the use of magnetic resonance imaging, 3-dimensional airway modeling, and morphometric techniques to characterize the distribution and magnitude of ozone-induced nasal injury in infant monkeys. Using this method, we generated age-specific, 3-dimensional, epithelial maps of the nasal airways of infant Rhesus macaques. The principal nasal lesions observed in this primate model of ozone-induced nasal toxicology were neutrophilic rhinitis, along with necrosis and exfoliation of the epithelium lining the anterior maxilloturbinate. These lesions, induced by acute or cyclic (episodic) exposures, were examined by light microscopy, quantified by morphometric techniques, and mapped on 3-dimensional models of the nasal airways. Here, we describe the histopathologic, imaging, and computational biology methods developed to precisely characterize, localize, quantify, and map these nasal lesions. By combining these techniques, the location and severity of the nasal epithelial injury were correlated with epithelial type, nasal airway geometry, and local biochemical and molecular changes on an individual animal basis. These correlations are critical for accurate predictive modeling of exposure-dose-response relationships in the nasal airways, and subsequent extrapolation of nasal findings in animals to humans for determining risk.