Safety and T cell modulating effects of high dose vitamin D3 supplementation in multiple sclerosis

Background

A poor vitamin D status has been associated with a high disease activity of multiple sclerosis (MS). Recently, we described associations between vitamin D status and peripheral T cell characteristics in relapsing remitting MS (RRMS) patients. In the present study, we studied the effects of high dose vitamin D₃ supplementation on safety and T cell related outcome measures.

Methodology/Principal Findings

Fifteen RRMS patients were supplemented with 20 000 IU/d vitamin D₃ for 12 weeks. Vitamin D and calcium metabolism were carefully monitored, and T cell characteristics were studied by flowcytometry. All patients finished the protocol without side-effects, hypercalcaemia, or hypercalciuria. The median vitamin D status increased from 50 nmol/L (31–175) at week 0 to 380 nmol/L (151–535) at week 12 (P<0.001). During the study, 1 patient experienced an exacerbation of MS and was censored from the T cell analysis. The proportions of (naïve and memory) CD4⁺ Tregs remained unaffected. Although Treg suppressive function improved in several subjects, this effect was not significant in the total cohort (P = 0.143). An increased proportion of IL-10⁺ CD4⁺ T cells was found after supplementation (P = 0.021). Additionally, a decrease of the ratio between IFN-γ⁺ and IL-4⁺ CD4⁺ T cells was observed (P = 0.035).

Conclusion/Significance

Twelve week supplementation of high dose vitamin D₃ in RRMS patients was well tolerated and did not induce decompensation of calcium metabolism. The skewing towards an anti-inflammatory cytokine profile supports the evidence on vitamin D as an immune-modulator, and may be used as outcome measure for upcoming randomized placebo-controlled trials.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00940719","term_id":"NCT00940719"}}NCT00940719     

Interaction of ouabain with the Na(+) pump in intact epithelial cells

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.     

Interleukin-1 Receptor–Associated Kinase M–Deficient Mice Demonstrate an Improved Host Defense during Gram-negative Pneumonia

Pneumonia is a common cause of morbidity and mortality and the most frequent source of sepsis. Bacteria that try to invade normally sterile body sites are recognized by innate immune cells through pattern recognition receptors, among which toll-like receptors (TLRs) feature prominently. Interleukin-1 receptor (IL-1R)–associated kinase (IRAK)-M is a proximal inhibitor of TLR signaling expressed by epithelial cells and macrophages in the lung. To determine the role of IRAK-M in host defense against bacterial pneumonia, IRAK-M-deficient (IRAK-M^−/−) and normal wild-type (WT) mice were infected intranasally with Klebsiella pneumoniae. IRAK-M mRNA was upregulated in lungs of WT mice with Klebsiella pneumonia, and the absence of IRAK-M resulted in a strongly improved host defense as reflected by reduced bacterial growth in the lungs, diminished dissemination to distant body sites, less peripheral tissue injury and better survival rates. Although IRAK-M^−/− alveolar macrophages displayed enhanced responsiveness toward intact K. pneumoniae and Klebsiella lipopolysaccharide (LPS) in vitro, IRAK-M^−/− mice did not show increased cytokine or chemokine levels in their lungs after infection in vivo. The extent of lung inflammation was increased in IRAK-M^−/− mice shortly after K. pneumoniae infection, as determined by semiquantitative scoring of specific components of the inflammatory response in lung tissue slides. These data indicate that IRAK-M impairs host defense during pneumonia caused by a common gram-negative respiratory pathogen.     

Citrullination, a possible functional link between susceptibility genes and rheumatoid arthritis

Antibodies directed to citrullinated proteins (anti-cyclic citrullinated peptide) are highly specific for rheumatoid arthritis (RA). Recent data suggest that the antibodies may be involved in the disease process of RA and that several RA-associated genetic factors might be functionally linked to RA via modulation of the production of anti-cyclic citrullinated peptide antibodies or citrullinated antigens.     

Insertion of leader peptidase into the thylakoid membrane during synthesis in a chloroplast translation system

The mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins are poorly understood. In this study, we have used a translation system isolated from chloroplasts to begin to investigate these mechanisms. The bacterial membrane protein leader peptidase (Lep) was used as a model protein because its targeting and insertion mechanisms are well understood for Escherichia coli and for the endoplasmic reticulum. Lep could thus provide insight into the functional homologies between the different membrane systems. Lep was efficiently expressed in the chloroplast translation system, and the protein could be inserted into thylakoid membranes with the same topology as in E. coli cytoplasmic membranes, following the positive-inside rule. Insertion of Lep into the thylakoid membrane was stimulated by the trans-thylakoid proton gradient and was strongly inhibited by azide, suggesting a requirement for SecA activity. Insertion most likely occurred in a cotranslational manner, because insertion could only be observed if thylakoid membranes were present during translation reactions but not when thylakoid membranes were added after translation reactions were terminated. To halt the elongation process at different stages, we translated truncated Lep mRNAs without a stop codon, resulting in the formation of stable ribosome nascent chain complexes. These complexes showed a strong, salt-resistant affinity for the thylakoid membrane, implying a functional interaction of the ribosome with the membrane and supporting a cotranslational insertion mechanism for Lep. Our study supports a functional homology for the insertion of Lep into the thylakoid membrane and the E. coli cytoplasmic membrane.     

Abdominal fat mass is associated with adaptive immune activation: the CODAM Study

Abdominal fat-related activation of the innate immune system and insulin resistance (IR) are implicated in the pathogenesis of cardiovascular diseases. Recent data support an important role of the adaptive immune system as well. In this study, we investigate the association between waist circumference and markers of systemic adaptive immune activation, and the potential mediating role of innate immune activation and/or IR herein. The study population consisted of 477 (304 men) individuals (mean age: 59.4 ± 7.0 years) in whom waist circumference, HOMA2-IR (IR derived from homeostasis model assessment), and markers of innate (C-reactive protein (CRP), interleukin (IL)-6, serum amyloid A (SAA)) and adaptive (neopterin, soluble CD25 (sCD25)) immune activation were measured. These markers were compiled into an adaptive and innate immune activation score by averaging the respective z-scores. After adjustments for age, sex, glucose metabolism, smoking status, prior cardiovascular disease, and other risk factors, waist circumference was associated with the adaptive (standardized regression coefficient β = 0.12 (95% confidence intervals: 0.04-0.20)) and the innate immune activation scores (β = 0.24 (0.17-0.31)), and with HOMA2-IR (β = 0.49 (0.42-0.56)). The innate immune activation score and HOMA2-IR were also positively associated with the adaptive immune activation score (β = 0.31 (0.21-0.40) and β = 0.11 (0.02-0.21), respectively). The association between waist circumference and the adaptive immune activation score was completely abolished when further adjusted for innate immune activation and HOMA2-IR (to β = -0.01 (-0.10-0.08)), and the specific mediation "effects" attributable to each of these variables were 58% and 42%, respectively. We conclude that abdominal obesity is associated with systemic adaptive immune activation and that innate immune activation and IR constitute independent and equally important pathways explaining this association.     

Staphylococcus aureus and Wegener's granulomatosis

Wegener's granulomatosis (WG) is a form of systemic vasculitis. It is characterized by granulomatous inflammation in the upper and lower airways, vasculitis and necrotizing glomerulonephritis, and is strongly associated with antineutrophil cytoplasmic antibodies against proteinase 3. Since the etiology of the disease is not clear, treatment, consisting of corticosteroids and immunosuppressives, is nonspecific and associated with severe side effects. Pinpointing the trigger(s) of the disease would highly improve treatment. Clinical evidence shows that an infectious agent, the bacterium Staphylococcus aureus, is a risk factor for disease relapse, suggesting its involvement in the pathogenesis of WG. Here we review both clinical and experimental data that either indicate or support a role for S. aureus in WG.