Recombinant canine single chain insulin analogues: Insulin receptor binding capacity and ability to stimulate glucose uptake

Virtually all diabetic dogs require exogenous insulin therapy to control their hyperglycaemia. In the UK, the only licensed insulin product currently available is a purified porcine insulin preparation. Recombinant insulin is somewhat problematic in terms of its manufacture, since the gene product (preproinsulin) undergoes substantial post-translational modification in pancreatic β cells before it becomes biologically active. The aim of the present study was to develop recombinant canine single chain insulin (SCI) analogues that could be produced in a prokaryotic expression system and which would require minimal processing. Three recombinant SCI constructs were developed in a prokaryotic expression vector, by replacing the insulin C-peptide sequence with one encoding a synthetic peptide (GGGPGKR), or with one of two insulin-like growth factor (IGF)-2 C-peptide coding sequences (human: SRVSRRSR; canine: SRVTRRSSR). Recombinant proteins were expressed in the periplasmic fraction of Escherichia coli and assessed for their ability to bind to the insulin and IGF-1 receptors, and to stimulate glucose uptake in 3T3-L1 adipocytes. All three recombinant SCI analogues demonstrated preferential binding to the insulin receptor compared to the IGF-1 receptor, with increased binding compared to recombinant canine proinsulin. The recombinant SCI analogues stimulated glucose uptake in 3T3-L1 adipocytes compared to negligible uptake using recombinant canine proinsulin, with the canine insulin/cIGF-2 chimaeric SCI analogue demonstrating the greatest effect. Thus, biologically-active recombinant canine SCI analogues can be produced relatively easily in bacteria, which could potentially be used for treatment of diabetic dogs

An Age-Associated Decline in Thymic Output Differs in Dog Breeds According to Their Longevity

The age associated decline in immune function is preceded in mammals by a reduction in thymic output. Furthermore, there is increasing evidence of a link between immune competence and lifespan. One approach to determining thymic output is to quantify signal joint T cell receptor excision circles (sj-TRECs), a method which has been developed and used in several mammalian species. Life expectancy and the rate of aging vary in dogs depending upon their breed. In this study, we quantified sj-TRECs in blood samples from dogs of selected breeds to determine whether there was a relationship between longevity and thymic output. In Labrador retrievers, a breed with a median expected lifespan of 11 years, there was an age-associated decline in sj-TREC values, with the greatest decline occurring before 5 years of age, but with sj-TREC still detectable in some geriatric animals, over 13 years of age. In large short-lived breeds (Burnese mountain dogs, Great Danes and Dogue de Bordeaux), the decline in sj-TREC values began earlier in life, compared with small long-lived breeds (Jack Russell terriers and Yorkshire terriers), and the presence of animals with undetectable sj-TRECs occurred at a younger age in the short-lived breeds. The study findings suggest that age-associated changes in canine sj-TRECs are related to breed differences in longevity, and this research highlights the use of dogs as a potential model of immunosenescence

Autoantibodies against cytochrome P450 side-chain cleavage enzyme in dogs (Canis lupus familiaris) affected with hypoadrenocorticism (Addison’s Disease)

Canine hypoadrenocorticism likely arises from immune-mediated destruction of adrenocortical tissue, leading to glucocorticoid and mineralocorticoid deficiency. In humans with autoimmune Addison's disease (AAD) or autoimmune polyendocrine syndrome (APS), circulating autoantibodies have been demonstrated against enzymes associated with adrenal steroid synthesis. The current study investigates autoantibodies against steroid synthesis enzymes in dogs with spontaneous hypoadrenocorticism. Coding regions of canine CYP21A2 (21-hydroxylase; 21-OH), CYP17A1 (17-hydroxylase; 17-OH), CYP11A1 (P450 side-chain cleavage enzyme; P450scc) and HSD3B2 (3β hydroxysteroid dehydrogenase; 3βHSD) were amplified, cloned and expressed as 35S-methionine radiolabelled recombinant protein. In a pilot study, serum samples from 20 dogs with hypoadrenocorticism and four unaffected control dogs were screened by radio-immunoprecipitation assay. There was no evidence of reactivity against 21-OH, 17-OH or 3βHSD, but five dogs with hypoadrenocorticism showed immunoreactivity to P450scc compared with controls. Serum samples were subsequently obtained from 213 dogs diagnosed with hypoadrenocorticism and 110 dogs from a hospital control population. Thirty control dogs were randomly selected to establish a threshold for antibody positivity (mean + 3 × standard deviation). Dogs with hypoadrenocorticism were more likely to be P450scc autoantibody positive than hospital controls (24% vs. 1.2%, respectively; p = 0.0016). Sex was significantly associated with the presence of P450scc autoantibodies in the case population, with 30% of females testing positive compared with 17% of males (p = 0.037). Significant associations with breed (p = 0.015) and DLA-type (DQA1*006:01 allele; p = 0.017) were also found. This cross-sectional study indicates that P450scc autoantibodies are present in a proportion of dogs affected with hypoadrenocorticism

Polymorphisms in the CTLA4 promoter sequence are associated with canine hypoadrenocorticism

Canine hypoadrenocorticism is an immune-mediated endocrinopathy that shares both clinical and pathophysiological similarities with Addison’s disease in humans. Several dog breeds are overrepresented in the disease population, suggesting that a genetic component is involved, although this is likely to be polygenic. Previous research has implicated CTLA4 as a potential susceptibility gene. CTLA4 is an important regulator of T cell function and polymorphisms/mutations in CTLA4 have been associated with a number of autoimmune phenotypes in both humans and rodent models of autoimmunity. The aim of the current study was to undertake a case:control association study of CTLA4 promotor polymorphisms in three dog breeds, cocker spaniels, springer spaniels and West Highland white terriers (WHWT)

Breed differences in development of anti-insulin antibodies in diabetic dogs and investigation of the role of dog leukocyte antigen (DLA) genes

Administration of insulin for treatment of diabetes mellitus in dogs can stimulate an immune response, with a proportion of animals developing anti-insulin antibodies (AIA). For an IgG antibody response to occur, this would require B cell presentation of insulin peptides by major histocompatibility complex (MHC) class II molecules, encoded by dog leukocyte antigen (DLA) genes, in order to receive T-cell help for class switching. DLA genes are highly polymorphic in the dog population and vary from breed to breed. The aim of the present study was to evaluate AIA reactivity in diabetic dogs of different breeds and to investigate whether DLA genes influence AIA status. Indirect ELISA was used to determine serological reactivity to insulin in diabetic dogs, treated with either a porcine or bovine insulin preparation. DLA haplotypes for diabetic dogs were determined by sequence-based typing of DLA-DRB1, -DQA1 and -DQB1 loci. Significantly greater insulin reactivity was seen in treated diabetic dogs (n = 942) compared with non-diabetic dogs (n = 100). Relatively few newly diagnosed diabetic dogs (3/109) were found to be AIA positive, although this provides evidence that insulin autoantibodies might be involved in the pathogenesis of the disease in some cases. Of the diabetic dogs treated with a bovine insulin preparation, 52.3% (182/348) were AIA positive, compared with 12.6% (75/594) of dogs treated with a porcine insulin preparation, suggesting that bovine insulin is more immunogenic. Breeds such as dachshund, Cairn terrier, miniature schnauzer and Tibetan terrier were more likely to develop AIA, whereas cocker spaniels were less likely to develop AIA, compared with crossbreed dogs. In diabetic dogs, DLA haplotype DRB1*0015--DQA1*006--DQB1*023 was associated with being AIA positive, whereas the haplotype DLA-DRB1*006--DQA1*005--DQB1*007 showed an association with being AIA negative. These research findings suggest that DLA genes influence AIA responses in treated diabetic dogs

Yellow and Red Supergiants in the Large Magellanic Cloud

Due to their transitionary nature, yellow supergiants provide a critical challenge for evolutionary modeling. Previous studies within M31 and the SMC show that the Geneva evolutionary models do a poor job at predicting the lifetimes of these short-lived stars. Here we extend this study to the LMC while also investigating the galaxy's red supergiant content. This task is complicated by contamination by Galactic foreground stars that color and magnitude criteria alone cannot weed out. Therefore, we use proper motions and the LMC's large systemic radial velocity (\sim278 km/s) to separate out these foreground dwarfs. After observing nearly 2,000 stars, we identified 317 probable yellow supergiants, 6 possible yellow supergiants and 505 probable red supergiants. Foreground contamination of our yellow supergiant sample was \sim80%, while that of the the red supergiant sample was only 3%. By placing the yellow supergiants on the H-R diagram and comparing them against the evolutionary tracks, we find that new Geneva evolutionary models do an exemplary job at predicting both the locations and the lifetimes of these transitory objects.Comment: Accepted for publication in the Ap

Syndromic surveillance: two decades experience of sustainable systems – its people not just data!

Syndromic surveillance is a form of surveillance that generates information for public health action by collecting, analysing and interpreting routine health-related data on symptoms and clinical signs reported by patients and clinicians rather than being based on microbiologically or clinically confirmed cases. In England, a suite of national real-time syndromic surveillance systems (SSS) have been developed over the last 20 years, utilising data from a variety of health care settings (a telehealth triage system, general practice and emergency departments). The real-time systems in England have been used for early detection (e.g. seasonal influenza), for situational awareness (e.g. describing the size and demographics of the impact of a heatwave) and for reassurance of lack of impact on population health of mass gatherings (e.g. the London 2012 Olympic and Paralympic Games).We highlight the lessons learnt from running SSS, for nearly two decades, and propose questions and issues still to be addressed. We feel that syndromic surveillance is an example of the use of ‘big data’, but contend that the focus for sustainable and useful systems should be on the added value of such systems and the importance of people working together to maximise the value for the public health of syndromic surveillance services

The Star-Forming Galaxy Contribution to the Cosmic MeV and GeV Gamma-Ray Background

While star-forming galaxies could be major contributors to the cosmic GeV $\gamma$-ray background, they are expected to be MeV-dim because of the "pion bump" falling off below ~100 MeV. However, there are very few observations of galaxies in the MeV range, and other emission processes could be present. We investigate the MeV background from star-forming galaxies by running one-zone models of cosmic ray populations, including Inverse Compton and bremsstrahlung, as well as nuclear lines (including $^{26}$Al), emission from core-collapse supernovae, and positron annihilation emission, in addition to the pionic emission. We use the Milky Way and M82 as templates of normal and starburst galaxies, and compare our models to radio and GeV--TeV $\gamma$-ray data. We find that (1) higher gas densities in high-z normal galaxies lead to a strong pion bump, (2) starbursts may have significant MeV emission if their magnetic field strengths are low, and (3) cascades can contribute to the MeV emission of starbursts if they emit mainly hadronic $\gamma$-rays. Our fiducial model predicts that most of the unresolved GeV background is from star-forming galaxies, but this prediction is uncertain by an order of magnitude. About ~2% of the claimed 1 MeV background is diffuse emission from star-forming galaxies; we place a firm upper limit of <~10% based on the spectral shape of the background. The star-formation contribution is constrained to be small, because its spectrum is peaked, while the observed background is steeply falling with energy through the MeV-GeV range.Comment: Published in ApJ, 27 pages, emulateapj format. Readers may be interested in the concurrent paper by Chakraborty and Fields (arXiv:1206.0770), a calculation of the Inverse Compton background from star-forming galaxie

Polymorphisms in the Tlr4 and Tlr5 Gene Are Significantly Associated with Inflammatory Bowel Disease in German Shepherd Dogs

Inflammatory bowel disease (IBD) is considered to be the most common cause of vomiting and diarrhoea in dogs, and the German shepherd dog (GSD) is particularly susceptible. The exact aetiology of IBD is unknown, however associations have been identified between specific single-nucleotide polymorphisms (SNPs) in Toll-like receptors (TLRs) and human IBD. However, to date, no genetic studies have been undertaken in canine IBD. The aim of this study was to investigate whether polymorphisms in canine TLR 2, 4 and 5 genes are associated with IBD in GSDs. Mutational analysis of TLR2, TLR4 and TLR5 was performed in 10 unrelated GSDs with IBD. Four non-synonymous SNPs (T23C, G1039A, A1571T and G1807A) were identified in the TLR4 gene, and three non-synonymous SNPs (G22A, C100T and T1844C) were identified in the TLR5 gene. The non-synonymous SNPs identified in TLR4 and TLR5 were evaluated further in a case-control study using a SNaPSHOT multiplex reaction. Sequencing information from 55 unrelated GSDs with IBD were compared to a control group consisting of 61 unrelated GSDs. The G22A SNP in TLR5 was significantly associated with IBD in GSDs, whereas the remaining two SNPs were found to be significantly protective for IBD. Furthermore, the two SNPs in TLR4 (A1571T and G1807A) were in complete linkage disequilibrium, and were also significantly associated with IBD. The TLR5 risk haplotype (ACC) without the two associated TLR4 SNP alleles was significantly associated with IBD, however the presence of the two TLR4 SNP risk alleles without the TLR5 risk haplotype was not statistically associated with IBD. Our study suggests that the three TLR5 SNPs and two TLR4 SNPs; A1571T and G1807A could play a role in the pathogenesis of IBD in GSDs. Further studies are required to confirm the functional importance of these polymorphisms in the pathogenesis of this disease