Vitamin D supplementation for the prevention of type 2 diabetes in overweight adults: study protocol for a randomized controlled trial

Despite Australia's sunny climate, low vitamin D levels are increasingly prevalent. Sun exposure is limited by long working hours, an increase in time spent indoors, and sun protection practices, and there is limited dietary vitamin D fortification. While the importance of vitamin D for bone mineralization is well known, its role as a protective agent against chronic diseases, such as type 2 diabetes and cardiovascular disease, is less understood. Observational and limited intervention studies suggest that vitamin D might improve insulin sensitivity and secretion, mainly via its anti-inflammatory properties, thereby decreasing the risk of development and progression of type 2 diabetes. The primary aim of this trial is to investigate whether improved plasma concentrations of 25-hydroxyvitamin D (25(OH)D), obtained through vitamin D supplementation, will increase insulin sensitivity and insulin secretion. A secondary aim is to determine whether these relationships are mediated by a reduction in underlying subclinical inflammation associated with obesity.Fifty overweight but otherwise healthy nondiabetic adults between 18 and 60 years old, with low vitamin D levels (25(OH)D < 50 nmol/l), will be randomly assigned to intervention or placebo. At baseline, participants will undergo a medical review and anthropometric measurements, including dual X-ray absorptiometry, an intravenous glucose tolerance test, muscle and fat biopsies, a hyperinsulinemic euglycemic clamp, and questionnaires assessing diet, physical activity, sun exposure, back and knee pain, and depression. The intervention group will receive a first dose of 100,000 IU followed by 4,000 IU vitamin D (cholecalciferol) daily, while the placebo group will receive apparently identical capsules, both for a period of 16 weeks. All measurements will be repeated at follow-up, with the primary outcome measure expressed as a change from baseline in insulin sensitivity and secretion for the intervention group compared with the placebo group. Secondary outcome measures will compare changes in anthropometry, cardiovascular risk factors, and inflammatory markers.The trial will provide much needed clinical evidence on the impact of vitamin D supplementation on insulin resistance and secretion and its underlying mechanisms, which are relevant for the prevention and management of type 2 diabetes.Clinicaltrials.gov ID: NCT02112721 .Barbora de Courten, Aya Mousa, Negar Naderpoor, Helena Teede, Maximilian P J de Courten and Robert Scrag

Immature and Maturation-Resistant Human Dendritic Cells Generated from Bone Marrow Require Two Stimulations to Induce T Cell Anergy In Vitro

Immature dendritic cells (DC) represent potential clinical tools for tolerogenic cellular immunotherapy in both transplantation and autoimmunity. A major drawback in vivo is their potential to mature during infections or inflammation, which would convert their tolerogenicity into immunogenicity. The generation of immature DC from human bone marrow (BM) by low doses of GM-CSF (lowGM) in the absence of IL-4 under GMP conditions create DC resistant to maturation, detected by surface marker expression and primary stimulation by allogeneic T cells. This resistence could not be observed for BM-derived DC generated with high doses of GM-CSF plus IL-4 (highGM/4), although both DC types induced primary allogeneic T cell anergy in vitro. The estabishment of the anergic state requires two subsequent stimulations by immature DC. Anergy induction was more profound with lowGM-DC due to their maturation resistance. Together, we show the generation of immature, maturation-resistant lowGM-DC for potential clinical use in transplant rejection and propose a two-step-model of T cell anergy induction by immature DC

Patient-rated versus proxy-rated cognitive and functional measures in older adults

Molly Howland,1 Kevin C Allan,1 Caitlin E Carlton,1 Curtis Tatsuoka,2&ndash;4 Kathleen A Smyth,3 Martha Sajatovic1,2,4,5 1Case Western Reserve University School of Medicine, 2Neurological and Behavioral Outcomes Center, University Hospitals Case Medical Center, 3Department of Epidemiology and Biostatistics, 4Department of Neurology, 5Department of Psychiatry, Case Western Reserve University School of Medicine, Cleveland, OH,&nbsp;USA Objectives: Patients with cognitive impairment may have difficulty reporting their functional and cognitive abilities, which are important clinical outcomes. Health care proxies may be able to corroborate patient self-reports. Several studies reported discrepancy between patient and proxy ratings, though the literature is sparse on changes over time of these ratings. Our goals in this 12-month study were to compare patient and proxy reports on functioning, cognition, and everyday executive function, and to further elucidate correlates of patient&ndash;proxy discrepancy. Methods: This was a prospective cohort study of individuals older than 70 years who ranged from having no cognitive impairment to having moderate dementia who had a proxy available to complete instruments at baseline (N=76). Measurements included Alzheimer&rsquo;s Disease Cooperative Study&ndash;Activities of Daily Living Inventory (ADCS-ADLI), Neuro-QOL Executive Function, PROMIS Applied Cognition (PROMIS-Cog), Mini-Mental State Examination (MMSE), and Geriatric Depression Scale. Results: Patient- and proxy-rated ADCS-ADLI were correlated at baseline and at 1-year follow-up. Patient and proxy ratings were discrepant on Neuro-QOL Executive Function and PROMIS-Cog. Greater patient&ndash;proxy discrepancy on PROMIS-Cog was associated with younger age and less depression, and greater patient&ndash;proxy discrepancy on Neuro-QOL Executive Function was associated with less depression and worse cognitive impairment. Patient&ndash;proxy discrepancy increased over time for everyday executive function. Changes in proxy-rated but not patient-rated ADCS-ADLI correlated with MMSE changes. Conclusion: Patients and proxies generally agree in reporting on activities of daily living. Patient and proxy reports differ in their respective evaluation of cognitive functioning and everyday executive function. Ratings from both sources may be preferred for these two domains, though studies using gold standard measures are necessary. It is important that clinicians are aware of the differences between patient and proxy perspective to create an accurate clinical picture and guide treatment. Keywords: agreement, activities of daily living, dementia, executive function, caregive

Diagnosis of Mycobacterium tuberculosis infection using ESAT-6 and intracellular cytokine cytometry

Diagnosis of infection with Mycobacterium tuberculosis (MTB) using tuberculin skin testing (TST) is often hampered by prior Bacille Calmette–Guérin (BCG) vaccination. ESAT-6 is a protein that is expressed by MTB but absent in BCG. It has been postulated that it might be useful in distinguishing MTB-specific immune responses. This study measured CD4 T cell responder frequencies specific for ESAT-6 and the TST reagent purified protein derivative (PPD) in patients with tuberculosis (n = 16), controls with non-tuberculous pneumonia (n = 8) and normal subjects (n = 7). Responses were identified using the intracellular cytokine staining technique and flow cytometry on whole blood samples, and performed blinded to the patient condition. Antigen-specific CD4 cells were defined by CD69 positivity and one or more cytokine [interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-γ] and/or CD40L positivity. With ESAT-6 stimulation it was found that TB patients had significantly higher frequencies of IFN-γ and CD40L-positive CD4 T cells compared to the normal group, while no significant differences were measured with PPD stimulation. A responder frequency of 0·01% or higher for at least one of the measured cytokines/CD40L was defined as a positive response. Using this criterion to compare the two patient groups, PPD had 100% sensitivity but 0% specificity while ESAT-6 had 100% sensitivity and 88% specificity. Use of MTB-specific proteins such as ESAT-6 in combination with intracellular cytokine staining and flow cytometry has the potential to identify individuals with MTB infection

Loss of CD154 impairs the Th2 extrafollicular plasma cell response but not early T cell proliferation and interleukin-4 induction

Ligation of CD40 by CD4 T cells through CD154 is key both to germinal centre induction and follicular T-dependent Ig class switching, but its requirement for aspects of T cell priming and extrafollicular antibody responses is less clear. Here comparison of the T helper (Th) type 2 response in lymph nodes from wild-type mice and CD154-deficient mice after immunization with alum-precipitated antigen reveals selective effects of this immunodeficiency. The timing and magnitude of the early interleukin (IL)-4 induction and proliferation in T cells of the T zone were unaltered by CD154 deficiency. As expected, germinal centres were not induced. Additionally the T-dependent extrafollicular antibody response, which initially requires T cell help but expands without further T cell involvement, was severely curtailed. The median number of extrafollicular antigen-specific plasma cells was 370-fold lower in CD154-deficient mice. Of these plasma cells the median proportion that had switched to IgG1 was <5%, while in wild-type mice the proportion was 89%. Surprisingly, some CD154-deficient lymph nodes showed substantial switching to IgG1. Commensurately, increases in γ1 germline transcripts and Blimp-1 mRNA were observed, albeit significantly lower than in controls, but activation-induced cytidine deaminase mRNA was undetectable in CD154-deficient mice. These experiments demonstrate that the acquisition of some T cell priming characteristics can be CD154-independent; in contrast, T-dependent extrafollicular responses require CD154. Thus functional CD154 ligation during the first encounter of T cells and B cells in the T zone is critical for follicular and extrafollicular antibody responses

Deletion of the rodent malaria ortholog for falcipain-1 highlights differences between hepatic and blood stage merozoites

:© 2017 Hopp et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Proteases have been implicated in a variety of developmental processes during the malaria parasite lifecycle. In particular, invasion and egress of the parasite from the infected hepatocyte and erythrocyte, critically depend on protease activity. Although falcipain-1 was the first cysteine protease to be characterized in P. falciparum, its role in the lifecycle of the parasite has been the subject of some controversy. While an inhibitor of falcipain-1 blocked erythrocyte invasion by merozoites, two independent studies showed that falcipain-1 disruption did not affect growth of blood stage parasites. To shed light on the role of this protease over the entire Plasmodium lifecycle, we disrupted berghepain-1, its ortholog in the rodent parasite P. berghei. We found that this mutant parasite displays a pronounced delay in blood stage infection after inoculation of sporozoites. Experiments designed to pinpoint the defect of berghepain-1 knockout parasites found that it was not due to alterations in gliding motility, hepatocyte invasion or liver stage development and that injection of berghepain-1 knockout merosomes replicated the phenotype of delayed blood stage growth after sporozoite inoculation. We identified an additional role for berghepain-1 in preparing blood stage merozoites for infection of erythrocytes and observed that berghepain-1 knockout parasites exhibit a reticulocyte restriction, suggesting that berghepain-1 activity broadens the erythrocyte repertoire of the parasite. The lack of berghepain-1 expression resulted in a greater reduction in erythrocyte infectivity in hepatocyte-derived merozoites than it did in erythrocyte-derived merozoites. These observations indicate a role for berghepain-1 in processing ligands important for merozoite infectivity and provide evidence supporting the notion that hepatic and erythrocytic merozoites, though structurally similar, are not identical.This work was supported by the National Institutes of Health (R01 grant A1056840 to PS), a Johns Hopkins Malaria Research Institute fellowship (CSH) (malaria.jhsph.edu/opportunities/fellowships/) and the Netherlands Organization for Scientific Research Rubicon fellowship (WAvdL) (www.nwo.nl/funding-rubicon).info:eu-repo/semantics/publishedVersio