IL-22RA2 associates with multiple sclerosis and macrophage effector mechanisms in experimental neuroinflammation

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the CNS. Recent advances in whole-genome screening tools have enabled discovery of several MS risk genes, the majority of which have known immune-related functions. However, disease heterogeneity and low tissue accessibility hinder functional studies of established MS risk genes. For this reason, the MS model experimental autoimmune encephalomyelitis (EAE) is often used to study neuroinflammatory disease mechanisms. In this study, we performed high-resolution linkage analysis in a rat advanced intercross line to identify an EAE-regulating quantitative trait locus, Eae29, on rat chromosome 1. Eae29 alleles from the resistant strain both conferred milder EAE and lower production of proinflammatory molecules in macrophages, as demonstrated by the congenic line, DA.PVG-Eae29 (Dc1P). The soluble IL-22R α2 gene (Il-22ra2) lies within the Eae29 locus, and its expression was reduced in Dc1P, both in activated macrophages and splenocytes from immunized rats. Moreover, a single nucleotide polymorphism located at the end of IL-22RA2 associated with MS risk in a combined Swedish and Norwegian cohort comprising 5019 subjects, displaying an odds ratio of 1.26 (p = 8.0 × 10(-4)). IL-22 and its receptors have been implicated in chronic inflammation, suggesting that IL-22RA2 regulates a central immune pathway. Through a combined approach including genetic and immunological investigation in an animal model and large-scale association studies of MS patients, we establish IL-22RA2 as an MS risk gene.     

The corticosteroid hormone induced factor: a new modulator of KCNQ1 channels?

The corticosteroid hormone induced factor (CHIF) is a member of the one-transmembrane segment protein family named FXYD, which also counts phospholemman and the Na,K-pump gamma-subunit. Originally it was suggested that CHIF could induce the expression of the I(Ks) current when expressed in Xenopus laevis oocytes, but recently CHIF has attracted attention as a modulatory subunit of the Na,K-pump. In renal and intestinal epithelia, the expression of CHIF is dramatically up-regulated in response to aldosterone stimulation, and regulation of epithelial ion channels by CHIF is an attractive hypothesis. To study a potential regulatory effect of the CHIF subunit on KCNQ1 channels, co-expression experiments were performed in Xenopus laevis oocytes and mammalian CHO-K1 cells. Electrophysiological characterization was obtained by two-electrode voltage-clamp and patch-clamp, respectively. In both expression systems, we find that CHIF drastically modulates the KCNQ1 current; in the presence of CHIF, the KCNQ1 channels open at all membrane potentials. Thereby, CHIF is the first accessory subunit shown to be capable of modulating both the Na,K-pump and an ion channel. To find a possible physiological function of the constitutively open KCNQ1/CHIF complex, the precise localization of KCNQ1 and CHIF in distal colon and kidney from control and salt-depleted rats was determined by confocal microscopy. However, in these tissues, we did not detect an obvious overlap in expression between KCNQ1 and CHIF. In conclusion, the hormone-regulated subunit CHIF modulates the voltage sensitivity of the KCNQ channels, but so far evidence for an actual co-localization of CHIF and KCNQ1 channels in native tissue is lacking.     

Calcium influx determines the muscular response to electrotransfer

Cell membrane permeabilization by electric pulses (electropermeabilization), results in free exchange of ions across the cell membrane. The role of electrotransfer-mediated Ca(2+)-influx on muscle signaling pathways involved in degeneration (β-actin and MurF), inflammation (IL-6 and TNF-α), and regeneration (MyoD1, myogenin, and Myf5) was investigated, using pulse parameters of both electrochemotherapy (8 HV) and DNA delivery (HVLV). Three pulsing conditions were used: 8 high-voltage pulses (8 HV), resulting in large permeabilization and ion flux, and a combination of one high-voltage pulse and one low-voltage pulse (HVLV), either alone or in combination with injection of DNA. Mice and rats were anesthetized before pulsing. At the times given, animals were killed, and intact tibialis cranialis muscles were excised for analysis. Uptake of Ca(2+) was assessed using (45)Ca as a tracer. Using gene expression analyses and histology, we showed a clear association between Ca(2+) influx and muscular response. Moderate Ca(2+) influx induced by HVLV pulses results in activation of pathways involved in immediate repair and hypertrophy. This response could be attenuated by intramuscular injection of EGTA reducing Ca(2+) influx. Larger Ca(2+) influx as induced by 8-HV pulses leads to muscle damage and muscle fiber regeneration through recruitment of satellite cells. The extent of Ca(2+) influx determines the muscular response to electrotransfer and, thus, the success of a given application. In the case of electrochemotherapy, in which the objective is cell death, a large influx of Ca(2+) may be beneficial, whereas for DNA electrotransfer, muscle recovery should occur without myofiber loss to ensure preservation of plasmid DNA.     

Modeling the Growth of Listeria monocytogenes in Soft Blue-White Cheese

The aim of this study was to develop a predictive model simulating growth over time of the pathogenic bacterium Listeria monocytogenes in a soft blue-white cheese. The physicochemical properties in a matrix such as cheese are essential controlling factors influencing the growth of L. monocytogenes. We developed a predictive tertiary model of the bacterial growth of L. monocytogenes as a function of temperature, pH, NaCl, and lactic acid. We measured the variations over time of the physicochemical properties in the cheese. Our predictive model was developed based on broth data produced in previous studies. New growth data sets were produced to independently calibrate and validate the developed model. A characteristic of this tertiary model is that it handles dynamic growth conditions described in time series of temperature, pH, NaCl, and lactic acid. Supplying the model with realistic production and retail conditions showed that the number of L. monocytogenes cells increases 3 to 3.5 log within the shelf life of the cheese.     

Planktonic aggregates of Staphylococcus aureus protect against common antibiotics

Bacterial cells are mostly studied during planktonic growth although in their natural habitats they are often found in communities such as biofilms with dramatically different physiological properties. We have examined another type of community namely cellular aggregates observed in strains of the human pathogen Staphylococcus aureus. By laser-diffraction particle-size analysis (LDA) we show, for strains forming visible aggregates, that the aggregation starts already in the early exponential growth phase and proceeds until post-exponential phase where more than 90% of the population is part of the aggregate community. Similar to some types of biofilm, the structural component of S. aureus aggregates is the polysaccharide intercellular adhesin (PIA). Importantly, PIA production correlates with the level of aggregation whether altered through mutations or exposure to sub-inhibitory concentrations of selected antibiotics. While some properties of aggregates resemble those of biofilms including increased mutation frequency and survival during antibiotic treatment, aggregated cells displayed higher metabolic activity than planktonic cells or cells in biofilm. Thus, our data indicate that the properties of cells in aggregates differ in some aspects from those in biofilms. It is generally accepted that the biofilm life style protects pathogens against antibiotics and the hostile environment of the host. We speculate that in aggregate communities S. aureus increases its tolerance to hazardous environments and that the combination of a biofilm-like environment with mobility has substantial practical and clinical importance.     

End-of-life practices in Danish ICUs: development and validation of a questionnaire

ABSTRACT: BACKGROUND: Practices for withholding or withdrawing therapy vary according to professional, cultural and religious differences. No Danish-validated questionnaire examining withholding and withdrawing practices exists, thus the aim of this study was to develop and validate a questionnaire for surveying the views of intensive care nurses, intensivists, and primary physicians regarding collaboration and other aspects of withholding and withdrawing therapy in the ICU. METHODS: A questionnaire was developed on the basis of literature, focus group interviews with intensive care nurses and intensivists, and individual interviews with primary physicians. The questionnaire was validated in the following 3 phases: a qualitative test with 17 participants; a quantitative pilot test with 60 participants; and a survey with 776 participants. The validation process included tests for face and content validity (by interviewing participants in the qualitative part of the pilot study), reliability (by assessing the distribution of responses within the individual response categories), agreement (by conducting a test-retest, evaluated by paired analyses), known groups' validity (as a surrogate test for responsiveness, by comparing two ICUs with a known difference in end-of-life practices), floor and ceiling effect, and missing data. RESULTS: Face and content validity were assessed as good by the participants in the qualitative pilot test; all considered the questions relevant and none of the participants found areas lacking. Almost all response categories were used by the participants, thus demonstrating the questionnaires ability to distinguish between different respondents, agreement was fair (the average test-retest agreement for the Likert scale responses was 0.54 (weighted kappa; range, 0.25-0.73), and known groups' validity was proved by finding significant differences in level of satisfaction with interdisciplinary collaboration and in experiences of withdrawal decisions being unnecessary postponed. Floor and ceiling effect was in accordance with other questionnaires, and missing data was limited to a range of 0-7% for all questions. CONCLUSIONS: The validation showed good and fair areas of validity of the questionnaire. The questionnaire is considered a useful tool to assess the perceptions of collaboration and other aspects of withholding and withdrawing therapy practices in Danish ICUs amongst nurses, intensivists, and primary physicians.     

Assessment of dietary exposure related to dietary GI and fibre intake in a nutritional metabolomic study of human urine

There is a need for a tool to assess dietary intake related to the habitual dietary glycaemic index (GI) and fibre in groups with large numbers of individuals. Novel metabolite-profiling techniques may be a useful approach when applied to human urine. In a long-term, controlled dietary intervention study, metabolomics were applied to assess dietary patterns. A targeted approach was used to evaluate the effects on urinary C-peptide excretion caused by the dietary treatments. Seventy-seven overweight subjects followed an 8-week low-calorie diet (LCD) and were then randomly assigned to a high-GI or low-GI diet for 6 month during which they completed 24-h urine collections at baseline (prior to the 8-week LCD) and after randomisation to the dietary intervention, at month 1, 3 and 6, respectively. Metabolite profiling in 24-h urine was performed by ¹H NMR and chemometrics. Partial least squares (PLS) analysis indicated that urinary formate could discriminate between high-GI and low-GI diets (correlation coefficient r = 0.82), and this finding was confirmed statistically (P = 0.01). PLS analysis also indicated that urinary hippurate could be associated with fibre intake, but this finding was not confirmed statistically. No associations between GI and urinary C-peptide were found. Our results emphasise that application of metabolomics is useful in the assessment of dietary exposure related to dietary GI and fibre seen at group level in a nutritional metabolomic study of human urine. As our design allowed for large variations in individually selected food items, biomarkers identified at group level may be interpreted as more general and robust markers, largely not confounded with markers from single dietary factors.