Laser Scribing Fabrication of Graphitic Carbon Biosensors for Label-Free Detection of Interleukin-6.

Interleukin-6 (IL-6) is an important immuno-modulating cytokine playing a pivotal role in inflammatory processes in disease induction and progression. As IL-6 serves as an important indicator of disease state, it is of paramount importance to develop low cost, fast and sensitive improved methods of detection. Here we present an electrochemical immunosensor platform based on the use of highly porous graphitic carbon electrodes fabricated by direct laser writing of commercial polyimide tapes and chemically modified with capture IL-6 antibodies. The unique porous and 3D morphology, as well as the high density of edge planes of the graphitic carbon electrodes, resulted in a fast heterogeneous electron transfer (HET) rate, k0 = 0.13 cm/s. The resulting immunosensor showed a linear response to log of concentration in the working range of 10 to 500 pg/mL, and low limit of detection (LOD) of 5.1 pg/mL IL-6 in phosphate buffer saline. The total test time was approximately 90 min, faster than the time required for ELISA testing. Moreover, the assay did not require additional sample pre-concentration or labelling steps. The immunosensor shelf-life was long, with stable results obtained after 6 weeks of storage at 4 °C, and the selectivity was high, as no response was obtained in the presence of another inflammatory cytokine, Interlukin-4. These results show that laser-fabricated graphitic carbon electrodes can be used as selective and sensitive electrochemical immunosensors and offer a viable option for rapid and low-cost biomarker detection for point-of-care analysis.

Comparison of edible brown algae extracts for the inhibition of intestinal carbohydrate digestive enzymes involved in glucose release from the diet.

Type II diabetes is considered the most common metabolic disorder in the developed world and currently affects about one in ten globally. A therapeutic target for the management of type II diabetes is the inhibition of α- glucosidase, an essential enzyme located at the brush border of the small intestinal epithelium. The inhibition of α-glucosidase results in reduced digestion of carbohydrates and a decrease in postprandial blood glucose. Although pharmaceutical synthetic inhibitors are available, these are usually associated with significant gastrointestinal side effects. In the present study, the impact of inhibitors derived from edible brown algae is being investigated and compared for their effect on glycaemic control. Carbohydrate- and polyphenolic-enriched extracts derived from Ascophyllum nodosum, Fucus vesiculosus and Undaria pinnatifida were characterised and screened for their inhibitory effects on maltase and sucrase enzymes. Furthermore, enzyme kinetics and the mechanism of inhibition of maltase and sucrase were determined using linear and nonlinear regression methods. All tested extracts showed a dose-dependent inhibitory effect of α-glucosidase with IC50 values ranging from 0⋅26 to 0⋅47 mg/ml for maltase; however, the only extract that was able to inhibit sucrase activity was A. nodosum, with an IC50 value of 0⋅83 mg/ml. The present study demonstrates the mechanisms in which different brown seaweed extracts with varying composition and molecular weight distribution differentially inhibit α-glucosidase activities. The data highlight that all brown seaweed extracts are not equal in the inhibition of carbohydrate digestive enzymes involved in postprandial glycaemia.

Identification and treatment of obstructive sleep apnea by a primary care team with a subset focus on chronic pain management.

BACKGROUND: Patients diagnosed with obstructive sleep apnea (OSA), who also consume prescription opioids, have a greater likelihood of morbidity and mortality. This study evaluated whether a primary care team, focused on chronic pain care management, could use a validated questionnaire (STOP-Bang) and motivational follow-up, to increase identification and treatment of OSA. METHODS: This study was a retrospective, dual arm, pre/post controlled study. Participants of this study included the complete chronic pain management sub group treated by this primary care team. Participants were ≥ 18 years old and prescribed daily opioids for treatment of chronic pain. All participants had a multifaceted, individualized, educational meeting that included completing a STOP-Bang questionnaire. Participants who received a score ≥ three were advised to follow up with their primary care physician. Participants were seen quarterly throughout the study. RESULTS: The primary outcome of this study was that 65% of participants with likely OSA were using CPAP for a minimum of 12 months (range of 12-25 months, 18-month average) post-intervention vs. 37% CPAP-use in the control group (12 months of observation), both groups were chronic opioid users with OSA. This was a 28% relative improvement (p = 0.0034). A secondary outcome was that 8.9% of non-prior CPAP users obtained CPAP post- intervention; a 56.7% pre-post improvement (p = 0.0064, x2 = 10.08 with 1 degree of freedom). Also, participants who were likely to have OSA (STOP-Bang score ≥ 3 or had a positive polysomnography (AHI >5 with comorbidities)) compared to those unlikely to have OSA (STOP-Bang score <3 or had a negative polysomnography (AHI <5)) in this study were more likely to be male, have a higher BMI, have hypertension, have cardiovascular disease and/or have diabetes (all types). CONCLUSION: Team based care management for participants taking prescription opioids, where STOP-Bang questionnaires were completed, were associated with an increase in the identification and treatment of OSA.

Protection from EAE by IL-4Ralpha(-/-) macrophages depends upon T regulatory cell involvement.

The administration of Th2 cytokines or immune deviation to a Th2 phenotypic response has been shown to protect against the autoimmune pathology of experimental autoimmune encephalomyelitis (EAE). To better understand the function of Th2 cytokines in the induction stage of EAE in the absence of an overt Th2 response, we immunized IL-4 receptor alpha-deficient (IL-4Ralpha(-/-)) mice, which are unable to respond to either IL-4 or IL-13. Contrary to expectations, mice lacking IL-4Ralpha had a lower incidence of EAE and a delayed onset compared to WT BALB/c mice; however, this delay did not correlate to an alteration in the Th1/Th17 cytokine balance. Instead, IL-4Ralpha-responsive macrophages were essential promoters of disease as macrophage-specific IL-4Ralpha-deficient (LysM(cre)IL-4Ralpha(-/lox)) mice were protected from EAE. The protection afforded by IL-4Ralpha-deficiency was not due to IL-10-, IFN-gamma-, NO- or IDO-mediated suppression of T-cell responses but was dependent upon the presence of regulatory T cells (Tregs). This investigation highlights the importance of macrophages and Tregs in regulating central nervous system inflammation and demonstrates that macrophages activated in the absence of Th2 cytokines can promote disease suppression by Tregs.

Type II-activated macrophages suppress the development of experimental autoimmune encephalomyelitis.

Treatment with immune complexes, which ligate Fcgamma receptors (FcgammaRs), suppresses the development of experimental autoimmune encephalomyelitis (EAE). To determine the mechanism of action, we investigated how these immune complexes affected type II activation of macrophages (that is, exposure to immune complexes in a proinflammatory environment). Our results show that lower doses of interferon-gamma (IFN-gamma) were more effective at priming bone marrow-derived macrophages (BMMphi) to produce more interleukin 10 (IL-10) and less IL-12p40 in response to lipopolysaccharide (LPS) and immune complexes compared with LPS alone. Moreover, at the lowest level of IFN-gamma (20 U ml(-1)), a significant downregulation in the surface expression of CD40, CD80 and PD-L1 was observed in LPS and immune complex-stimulated macrophages (that is, type II activated) than macrophages stimulated with LPS alone (that is, classically activated). Finally, treatment of mice with type II-activated macrophages protected them from developing EAE, suggesting that administration of immune complexes is protective against EAE by inducing type II-activated macrophages.

Fcgamma receptor-ligating complexes improve the course of experimental autoimmune encephalomyelitis by enhancing basal Th2 responses.

IL-12p40 and macrophages are essential for the induction of disease in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. In this paper, we show that treatment of mice with opsonized erythrocytes, which have been shown to ligate Fcgamma receptors on macrophages and alter their cytokine profile, significantly delayed the onset of experimental autoimmune encephalomyelitis. This protection correlated to the induction of Th2 responses by autoreactive T cells, enhanced basal systemic responses and a significant downregulation of IL-12p40 and nitric oxide synthase-2, but not IFN-gamma expression. IL-4 was essential for the protection by opsonized erythrocytes as the effects of treatment were eliminated in IL-4-deficient mice. Together these studies suggest that the ligation of Fcgamma receptors can modify the development of autoimmune disease by altering macrophage activation and enhancing Th2 responses.

The synthetic form of a novel chicken beta-defensin identified in silico is predominantly active against intestinal pathogens.

Antimicrobial peptides are essential components of innate immunity and are generally thought to act by disrupting the membrane integrity of microbes. Here we report the discovery of two novel chicken beta-defensins, gallinacin (Gal)-11 and Gal-12, found by hidden Markov model profile searching of the chicken genome. We have sequenced the genes and elucidated the 3'UTR of Gal-11. Differential mRNA expression of these novel genes has been shown across a panel of chicken tissues. Gal-11 mRNA was highly expressed in the small intestine, the liver, the gall bladder and the spleen and also showed moderate expression in several other areas of the chicken anatomy, whilst Gal-12 mRNA was found only in the liver and the gall bladder. Antimicrobial activity of synthetic Gal-11 has been demonstrated against a range of bacteria and is predominantly active against the intestinal pathogens Salmonella typhimurium and Listeria monocytogenes.

Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken.

Antimicrobial peptides (AMPs) are essential components of innate immunity in a range of species from Drosophila to humans and are generally thought to act by disrupting the membrane integrity of microbes. In order to discover novel AMPs in the chicken, we have implemented a bioinformatic approach that involves the clustering of more than 420,000 chicken expressed sequence tags (ESTs). Similarity searching of proteins-predicted to be encoded by these EST clusters-for homology to known AMPs has resulted in the in silico identification of full-length sequences for seven novel gallinacins (Gal-4 to Gal-10), a novel cathelicidin and a novel liver-expressed antimicrobial peptide 2 (LEAP-2) in the chicken. Differential gene expression of these novel genes has been demonstrated across a panel of chicken tissues. An evolutionary analysis of the gallinacin family has detected sites-primarily in the mature AMP-that are under positive selection in these molecules. The functional implications of these results are discussed.