Vitamin D(3) down-regulates proinflammatory cytokine response to Mycobacterium tuberculosis through pattern recognition receptors while inducing protective cathelicidin production

A well-known association between vitamin D(3) and infection with Mycobacterium tuberculosis has previously been reported, but little is known regarding the underlying mechanisms. We have investigated how 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] affects the proinflammatory cytokine production induced by M. tuberculosis. Furthermore, we explored whether 1,25(OH)(2)D(3) influence the production of the protective antimycobacterial peptide cathelicidin. Upon in vitro stimulation with M. tuberculosis, 1,25(OH)(2)D(3) induced a dose-dependent down-regulation of IL-6, TNFα and IFNγ, while increasing the production of IL-10 in culture supernatant as well as cathelicidin mRNA expression. This effect on cytokine response was not due to modulation of T-helper cell differentiation, as T-bet, GATA3, Foxp3 and ROR-γt mRNA expression remained unaffected. Similarly, 1,25(OH)(2)D(3) did not affect suppressor of cytokine signaling (SOCS)1 and SOCS3 mRNA expression. The mechanism whereby 1,25(OH)(2)D(3) inhibited the proinflammatory cytokine response was through reduced expression of the pattern recognition receptors (PRR) - TLR2, TLR4, Dectin-1 and mannose receptor, whose mRNA and protein expression were both reduced. The suppression of PRRs could be restored by a VDR antagonist. Upon M. tuberculosis stimulation, 1,25(OH)(2)D(3) modulates the balance in cytokine production towards an anti-inflammatory profile by repression of TLR2, TLR4, Dectin-1 and mannose receptor expression, while increasing cathelicidin production. These two effects may have beneficial consequences, by reducing the collateral tissue damage induced by proinflammatory cytokines, while the antibacterial effects of cathelicidin are enhanced.     

Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat

Background  

Relaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors.     

Lifestyle modifications to prevent and control hypertension. 1. Methods and an overview of the Canadian recommendations. Canadian Hypertension Society,Canadian Coalition for High Blood Pressure Prevention and Control,Laboratory Centre for Disease Control at Health Canada,Heart and Stroke Foundation of Canada

OBJECTIVE: To provide updated, evidence-based recommendations for health care professionals on lifestyle changes to prevent and control hypertension in otherwise healthy adults (except pregnant women). OPTIONS: For people at risk for hypertension, there are a number of lifestyle options that may avert the condition--maintaining a healthy body weight, moderating consumption of alcohol, exercising, reducing sodium intake, altering intake of calcium, magnesium and potassium, and reducing stress. Following these options will maintain or reduce the risk of hypertension. For people who already have hypertension, the options for controlling the condition are lifestyle modification, antihypertensive medications or a combination of these options; with no treatment, these people remain at risk for the complications of hypertension. OUTCOMES: The health outcomes considered were changes in blood pressure and in morbidity and mortality rates. Because of insufficient evidence, no economic outcomes were considered. EVIDENCE: A MEDLINE search was conducted for the period January 1996 to September 1996 for each of the interventions studied. Reference lists were scanned, experts were polled, and the personal files of the authors were used to identify other studies. All relevant articles were reviewed, classified according to study design and graded according to level of evidence. VALUES: A high value was placed on the avoidance of cardiovascular morbidity and premature death caused by untreated hypertension. BENEFITS, HARMS AND COSTS: Lifestyle modification by means of weight loss (or maintenance of healthy body weight), regular exercise and low alcohol consumption will reduce the blood pressure of appropriately selected normotensive and hypertensive people. Sodium restriction and stress management will reduce the blood pressure of appropriately selected hypertensive patients. The side effects of these therapies are few, and the indirect benefits are well known. There are certainly costs associated with lifestyle modification, but they were not measured in the studies reviewed. Supplementing the diet with potassium, calcium and magnesium has not been associated with a clinically important reduction in blood pressure in people consuming a healthy diet. RECOMMENDATIONS: (1) It is recommended that health care professionals determine the body mass index (weight in kilograms/[height in metres]2) and alcohol consumption of all adult patients and assess sodium consumption and stress levels in all hypertensive patients. (2) To reduce blood pressure in the population at large, it is recommended that Canadians attain and maintain a healthy body mass index. For those who choose to drink alcohol intake should be limited to 2 or fewer standard drinks per day (maximum of 14/week for men and 9/week for women). Adults should exercise regularly. (3) To reduce blood pressure in hypertensive patients, individualized therapy is recommended. This therapy should emphasize weight loss for overweight patients, abstinence from or moderation in alcohol intake, regular exercise, restriction of sodium intake and, in appropriate circumstances, individualized cognitive behaviour modification to reduce the negative effects of stress. VALIDATION: The recommendations were reviewed by all of the sponsoring organizations and by participants in a satellite symposium of the fourth international Conference on Preventive Cardiology. They are similar to those of the World Hypertension League and the Joint National committee, with the exception of the recommendations on stress management, which are based on new information. They have not been clinically tested. SPONSORS: The Canadian Hypertension Society, the Canadian Coalition for High Blood Pressure Prevention and Control, the Laboratory Centre for Disease Control at health Canada, and the Heart and Stroke Foundation of Canada.     

The chick embryo test agrees with the mouse bio-assay for assessment of the pathogenicity of Listeria species

The pathogenicity of 20 strains of Listeria was determined with the mouse intravenous bio-assay and the 10-day chick embryo chorioallontoic membrane test. In the former, survival in the spleen was measured and in the latter, the LD₁₀₀ and LD₅₀. There was good correlation between the results of the two tests. Listeria monocytogenes strains that grow in the mouse spleen had an LD₁₀₀ of < 125 organisms, while strains in which the numbers of organisms in the mouse spleen remain constant had an LD₁₀₀ > 125 organisms. Listeria seeligeri, L. innocua, L. welshimeri, L. grayi and L. murrayi did not persist in the spleen and the numbers of organisms used did not kill chick embryos.     

Parallel recordings of photosynthetic electron transport and K+-channel activity in single guard cells

Stomata open in response to red and blue light. Red light-induced stomatal movement is mediated by guard cell chloroplasts and related to K+-uptake into these motor cells. We have combined a new type of microchlorophyll fluorometer with the patch-clamp technique for parallel studies of the photosynthetic electron transport and activity of plasma membrane K+ channels in single guard cell protoplast. In the whole-cell configuration and presence of ATP in the patch-pipette, the activity of the K+-uptake channels remained constant throughout the course of an experiment (up to 30 min) while photosynthetic activity declined to about 50%. In the absence of ATP inward K+ currents declined in a time-dependent manner. Under these ATP-free conditions, photosynthetic electron transport was completely blocked within 8 min. ADP together with orthophosphate was able to prevent inhibition of photosynthetic electron transport and run-down of K+-channel activity. The results demonstrate that the combination of these two techniques is suited to directly study cytosolic factors as common regulators of photosynthesis and plasma membrane transport within a single-cell.     

Improved and validated method for the determination of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC in serum,and in human liver microsomal preparations using gas chromatography-mass spectrometry

A validated method for the quantification of Delta(9)-tetrahydrocannabinol (THC) and its main metabolites 11-hydroxy-tetrahydrocannabinol (OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) in serum is presented. The substances were isolated by solid-phase extraction, derivatised by methylation, and analysed by means of GC-MS in the selected ion monitoring mode. Quantitation was achieved by the addition of deuterated analogues as internal standards. The method was linear up to 10 ng/ml for THC and OH-THC, and up to 50 ng/ml for THC-COOH. The limits of quantification were 0.62 ng/ml for THC, 0.68 ng/ml for OH-THC and 3.35 ng/ml for THC-COOH. The limits of detection for the least intensive ions were 0.52 ng/ml for THC, 0.49 ng/ml for OH-THC and 0.65 ng/ml for THC-COOH. The method was validated according to the requirements of the Journal of Chromatography B. The method has been routinely used on samples from drivers suspected of "driving under the influence". In addition to the forensic application, a cross-validation was carried out by applying the method developed for serum to human liver microsomal preparation samples.     

Molecular basis of inverse agonism in a G protein-coupled receptor

G protein-coupled receptors (GPCRs) recognize a wide variety of extracellular ligands to control diverse physiological processes. Compounds that bind to such receptors can either stimulate, fully or partially (full or partial agonists), or reduce (inverse agonists) the receptors' basal activity and receptor-mediated signaling. Various studies have shown that the activation of receptors through binding of agonists proceeds by conformational changes as the receptor switches from a resting to an active state leading to G protein signaling. Yet the molecular basis for differences between agonists and inverse agonists is unclear. These different classes of compounds are assumed to switch the receptors' conformation in distinct ways. It is not known, however, whether such switching occurs along a linear 'on-off' scale or whether agonists and inverse agonists induce different switch mechanisms. Using a fluorescence-based approach to study the alpha2A-adrenergic receptor (alpha(2A)AR), we show that inverse agonists are differentiated from agonists in that they trigger a very distinct mode of a receptor's switch. This switch couples inverse agonist binding to the suppression of activity in the receptor.     

High-throughput single-cell manipulation in brain tissue

The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution.