Loss of intra-islet heparan sulfate is a highly sensitive marker of type 1 diabetes progression in humans

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstratedthat heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shownto be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intraislet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxideinduced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/ heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.: This work was supported by a National Health and Medical Research Council of Australia (NHMRC; https://www.nhmrc.gov.au/)/Juvenile Diabetes Research Foundation (JDRF) Special Program Grant in Type 1 Diabetes (#418138), The Canberra Hospital Private Practice Fund (http:// www.health.act.gov.au/research-publications/research/ppf-major-grants), JDRF nPOD Research Grant (#25-2010-716; http://www.jdrf.org), JDRF Research Grant (#47-2012-746) and NHMRC Project Grant (#1043284

Priming with a Recombinant Pantothenate Auxotroph of Mycobacterium bovis BCG and Boosting with MVA Elicits HIV-1 Gag Specific CD8+ T Cells

A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔpanCD) that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag) and BCGΔpanCD[pHS400] (BCGpan-Gag) were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag). After the boost high frequencies of predominantly Gag-specific CD8+ T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4+ T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge

Efficacy and safety of statin therapy in older people: a meta-analysis of individual participant data from 28 randomised controlled trials

Background: Statin therapy has been shown to reduce major vascular events and vascular mortality in a wide range of individuals, but there is uncertainty about its efficacy and safety among older people. We undertook a meta-analysis of data from all large statin trials to compare the effects of statin therapy at different ages. Methods: In this meta-analysis, randomised trials of statin therapy were eligible if they aimed to recruit at least 1000 participants with a scheduled treatment duration of at least 2 years. We analysed individual participant data from 22 trials (n=134 537) and detailed summary data from one trial (n=12 705) of statin therapy versus control, plus individual participant data from five trials of more intensive versus less intensive statin therapy (n=39 612). We subdivided participants into six age groups (55 years or younger, 56–60 years, 61–65 years, 66–70 years, 71–75 years, and older than 75 years). We estimated effects on major vascular events (ie, major coronary events, strokes, and coronary revascularisations), cause-specific mortality, and cancer incidence as the rate ratio (RR) per 1·0 mmol/L reduction in LDL cholesterol. We compared proportional risk reductions in different age subgroups by use of standard χ2 tests for heterogeneity when there were two groups, or trend when there were more than two groups. Findings: 14 483 (8%) of 186 854 participants in the 28 trials were older than 75 years at randomisation, and the median follow-up duration was 4·9 years. Overall, statin therapy or a more intensive statin regimen produced a 21% (RR 0·79, 95% CI 0·77–0·81) proportional reduction in major vascular events per 1·0 mmol/L reduction in LDL cholesterol. We observed a significant reduction in major vascular events in all age groups. Although proportional reductions in major vascular events diminished slightly with age, this trend was not statistically significant (ptrend=0·06). Overall, statin or more intensive therapy yielded a 24% (RR 0·76, 95% CI 0·73–0·79) proportional reduction in major coronary events per 1·0 mmol/L reduction in LDL cholesterol, and with increasing age, we observed a trend towards smaller proportional risk reductions in major coronary events (ptrend=0·009). We observed a 25% (RR 0·75, 95% CI 0·73–0·78) proportional reduction in the risk of coronary revascularisation procedures with statin therapy or a more intensive statin regimen per 1·0 mmol/L lower LDL cholesterol, which did not differ significantly across age groups (ptrend=0·6). Similarly, the proportional reductions in stroke of any type (RR 0·84, 95% CI 0·80–0·89) did not differ significantly across age groups (ptrend=0·7). After exclusion of four trials which enrolled only patients with heart failure or undergoing renal dialysis (among whom statin therapy has not been shown to be effective), the trend to smaller proportional risk reductions with increasing age persisted for major coronary events (ptrend=0·01), and remained non-significant for major vascular events (ptrend=0·3). The proportional reduction in major vascular events was similar, irrespective of age, among patients with pre-existing vascular disease (ptrend=0·2), but appeared smaller among older than among younger individuals not known to have vascular disease (ptrend=0·05). We found a 12% (RR 0·88, 95% CI 0·85–0·91) proportional reduction in vascular mortality per 1·0 mmol/L reduction in LDL cholesterol, with a trend towards smaller proportional reductions with older age (ptrend=0·004), but this trend did not persist after exclusion of the heart failure or dialysis trials (ptrend=0·2). Statin therapy had no effect at any age on non-vascular mortality, cancer death, or cancer incidence. Interpretation: Statin therapy produces significant reductions in major vascular events irrespective of age, but there is less direct evidence of benefit among patients older than 75 years who do not already have evidence of occlusive vascular disease. This limitation is now being addressed by further trials. Funding: Australian National Health and Medical Research Council, National Institute for Health Research Oxford Biomedical Research Centre, UK Medical Research Council, and British Heart Foundation

Denial of long-term issues with agriculture on tropical peatlands will have devastating consequences

Non peer reviewe

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Nod-factor attachment, calcium-fluxes, and lipid-transfer proteins in symbiotic signal transduction

Temporal and spatial observations on the attachment of Nodfactors were made by biotinylating the reducing terminus of the lipo-chitooligosaccharides of the broad host-range Rhizobium sp. NGR234 with the fluorescent reagent 2-amino-(6-amidobiotinyl)pyridine. Complex formation between the biotinylated Nod-factors and fluorescent streptavidin allowed localisation of the binding-site. At concentrations of > 10⁻⁷ M, these fluorescently tagged moleculs bound rapidly and asymmetrically (≈ 1 min) to nodulation competent root-hairs but they did not bind to root-hairs of the nonhost Arabidopsis thaliana. Early cellular events within the root-hairs were studied by loading root-segments with the calcium indicators Fura-2 and Fluo-3. Fluorescence ratio imaging showed that addition of NodNGR factors provoked almost immediate, plateau-like increases in intra-cellular free calcium {[Ca²⁺]i} in root-hairs and epidermal cells. Confocal laser scanning microscopy (CLSM) revealed that calcium accumulation was concentrated at the tips and the sides of the responsive root-hairs. A gene encoding a lipid transfer-like protein (LPT2) was isolated from a Vigna unguiculata root-hair cDNA bank. Levels of the L TP2-transcript increased in root-hairs 24h after treatment with NGR234, or its Nod-factors. The L TP2-gene was cloned into the pMal™ expression vector and L TP2 purified by affinity chromatography. It was unable to transfer phospholipids between liposomes and mitochondria. Anti-sense analysis in which the LTP2 coding region was cloned between the 35S promoter and terminator sequences reduced nodulation when transformed into V. unguiculata

Loss of intra-islet heparan sulfate represents a novel marker for the progression of type 1 diabetes in humans

Background and aims: Human type 1 diabetes (T1D) is an autoimmune disease and multiple destructive mechanisms are likely. In addition, a residual beta cell mass can exist at diagnosis, highlighting the potential for rescue by novel therapies. We have previously demonstrated unusually high levels of the polysaccharide heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) inside mouse beta cells, a critical role for intracellular HS in beta cell survival, the progressive loss of islet HS during T1D progression in NOD mice and expression of the HS-degrading enzyme heparanase (Hpse) by islet-infiltrating leukocytes. Treatment of NOD mice with PI-88, a Hpse inhibitor/HS mimetic, reduced the incidence of T1D by 50% and preserved intra-islet HS, suggesting that Hpsemediated loss of beta cell HS contributes to T1D disease. In this study we examined the clinical relevance of HS for the viability of human beta cells and as a target for destruction in human T1D. Materials and methods: HS, HSPG core proteins (collagen type XVIII (Col18), syndecan-1 (Sdc1)), insulin, glucagon and Hpse were stained by immunohistochemistry/ immunofluorescence in paraffin sections (post-antigen retrieval) of normal (n=8) and T1D (n=8) human pancreases with insulin+ve islets, obtained from the JDRF Network for Pancreas Organ Donors with Diabetes (nPOD, USA); the stained islet area was quantified using Image J software. 10E4 anti-HS mAb was used to localise highly sulfated HS and HP130 mAb identified Hpse. Isolated human islets were dispersed into single cells (using Accutase) for flow cytometry analysis of HS/HSPGs in beta cells and of beta cell viability after culture with HS mimetics (heparin or PI-88) ± acute treatment with 30% hydrogen peroxide. Beta cells were identified using Newport Green (NG) and damaged cells were stained using 7AAD or Sytox Green. Results: Localisation of HS, Col18 and Sdc1 in normal human pancreas correlated with insulin-containing beta cells. For insulin-positive T1D islets, the insulin-stained islet area was 85% of normal islets. However, the area stained for HS, Col18 and Sdc1 was significantly reduced to 41% (P<0.0001), 55% (P<0.0001) and 42% (P<0.0001) of normal islets, respectively. Insulitis leukocytes showed cell surface staining for Hpse. Flow cytometry analyses showed that 84.1±3.3% of human islet cells were beta cells and contained intracellular HS and HSPG core protein. Uptake of HS mimetics during culture of human beta cells significantly improved beta cell viability by 1.6-fold (P<0.001) i.e., as HS replacers, significantly reduced the proportion of damaged/non-viable beta cells to 25-31% of controls (P<0.001) and significantly reduced hydrogen peroxide-induced death by 3-fold (P<0.001). Conclusion: These findings suggest that in human T1D, the loss of beta cell HS could be mediated by leukocyte-derived Hpse and result in increased susceptibility to oxidative damage. Dual activity Hpse inhibitors/HS replacers could therefore represent a novel class of T1D therapeutic

Isolation, tissue distribution, and chromosomal localization of a novel testis-specific human four-transmembrane gene related to CD20 and FcRI-b

CD20 and the beta subunit of the high affinity receptor for IgE (FcepsilonRIbeta) are related four-transmembrane molecules that are expressed on the surface of hematopoietic cells and play crucial roles in signal transduction. Herein, we report the identification and characterization of a human gene, TETM4, that encodes a novel four-transmembrane protein related to CD20 and FcepsilonRIbeta. The predicted TETM4 protein is 200 amino acids and contains four putative transmembrane regions, N- and C-terminal cytoplasmic domains, and three inter-transmembrane loop regions. TETM4 shows 31.0 and 23.2% overall identity with CD20 and FcepsilonRIbeta respectively, with the highest identity in the transmembrane regions, whereas the N- and C-termini and inter-transmembrane loops are more divergent. Northern blot and RT-PCR analysis suggest that TETM4 mRNA has a highly restricted tissue distribution, being expressed selectively in the testis. Using fluorescence in situ hybridization and radiation hybrid analysis, the TETM4 gene has been localized to chromosome 11q12. The genes for CD20 and FcepsilonRIbeta have also been mapped to the same region of chromosome 11 (11q12-13.1), suggesting that these genes have evolved by duplication to form a family of four-transmembrane genes. TETM4 is the first nonhematopoietic member of the CD20/FcepsilonRIbeta family, and like its hematopoietic-specific relatives, it may be involved in signal transduction as a component of a multimeric receptor complex