Longitudinal Frequencies of Blood Leukocyte Subpopulations Differ between NOD and NOR Mice but Do Not Predict Diabetes in NOD Mice.

Immune phenotyping provides insight into disease pathogenesis and prognostic markers. Trajectories from age of 4 to 36 weeks were modeled for insulin autoantibodies and for leukocyte subpopulations in peripheral blood from female NOD (n = 58) and NOR (n = 22) mice. NOD mice had higher trajectories of insulin autoantibodies, CD4(+) and CD8(+) T lymphocytes, B lymphocytes, IgD(+)IgM(-) B lymphocytes, and NK cells and lower trajectories of CD4(+)CD25(+) T lymphocytes, IgM(+) B lymphocytes, granulocytes, and monocytes than NOR mice (all p < 0.001). Of these, only the increased IAA trajectory was observed in NOD mice that developed diabetes as compared to NOD mice that remained diabetes-free. Therefore, the profound differences in peripheral blood leukocyte proportions observed between the diabetes-prone NOD mice and the diabetes-resistant mice do not explain the variation in diabetes development within NOD mice and do not provide markers for diabetes prediction in this model.

Beta-cell autoimmunity.

Beta cell destruction in autoimmune diabetes is accompanied by the presence of autoantibodies and autoreactive T cells against beta cell antigens. Autoantibodies to insulin are predictive of future diabetes in man and in the non-obese diabetic mouse model. Furthermore, the detection of peripheral autoreactive CD8(+) T cells in this mouse model is indicative of beta cell killing and correlates with the development of diabetes. We describe two protocols that are helpful for the detection of beta-cell autoimmunity in mice. The first protocol describes the detection of insulin-specific autoantibodies using a radio-binding assay. The other is a general CD8(+) T cell ELISpot protocol for the detection of peptide-specific responses of CD8(+) T cells from secondary lymphoid organs or pancreatic islets.

Field evaluations of the efficacy and safety of Emodepside plus toltrazuril (Procox® oral suspension for dogs) against naturally acquired nematode and Isospora spp. infections in dogs.

Three controlled, blinded and randomised multicentre field studies evaluated the efficacy and safety of a new formulation containing emodepside plus toltrazuril (Procox® suspension for dogs) against naturally acquired parasite infections in dogs. In two studies dogs positive for gastrointestinal nematodes and/or Isospora spp. were treated with emodepside/toltrazuril suspension (at least 0.45 mg emodepside plus 9 mg toltrazuril per kg body weight) or a reference product containing either milbemycin oxime plus praziquantel (Milbemax®) or sulfadimethoxine (Kokzidiol SD®) at recommended dose rates. The third study investigated efficacy against prepatent natural Isospora spp. infections in comparison to an untreated control group by enrolling Isospora- negative dogs that were at risk to develop a patent infection during the study.No suspected adverse drug reactions were observed in any of the 403 dogs enrolled in the three studies including 234 dogs treated with emodepside/toltrazuril suspension. In dogs treated with emodepside/toltrazuril suspension against nematode infection faecal egg counts were reduced by 100 % (reference product: 99.7 %). Similarly, in the dogs that had been treated against patent Isospora spp. infection, faecal oocyst counts were reduced by 100 % (reference product: 99.0 %). In both studies, statistical analysis demonstrated non-inferiority and even superiority to the reference products (p ≤ 0.009). Dogs treated with emodepside/toltrazuril suspension during suspected prepatent Isospora spp. infection had 98.7 % lower faecal oocyst counts after treatment compared to untreated dogs (p < 0.0001).The studies demonstrated that emodepside/toltrazuril suspension is safe and highly efficacious against nematodes and Isospora spp. under field conditions.

An interferon-induced helicase (IFIH1) gene polymorphism associates with different rates of progression from autoimmunity to type 1 diabetes.

OBJECTIVE: Genome-wide association studies have identified gene regions associated with the development of type 1 diabetes. The aim of this study was to determine whether these associations are with the development of autoimmunity and/or progression to diabetes. RESEARCH DESIGN AND METHODS: Children (n = 1,650) of parents with type 1 diabetes were prospectively followed from birth (median follow-up 10.20 years) for the development of islet autoantibodies, thyroid peroxidase antibodies, tissue transglutaminase antibodies, and diabetes. Genotyping for single-nucleotide polymorphisms of the PTPN22, ERBB3, PTPN2, KIAA0350, CD25, and IFIH1 genes was performed using the MassARRAY system with iPLEX chemistry. RESULTS: Islet autoantibodies developed in 137 children and diabetes developed in 47 children. Type 1 diabetes risk was associated with the IFIH1 rs2111485 single-nucleotide polymorphism (hazard ratio 2.08; 95% CI 1.16-3.74; P = 0.014). None of the other genes were significantly associated with diabetes development in this cohort. IFIH1 genotypes did not associate with the development of islet autoantibodies (P = 0.80) or autoantibodies against thyroid peroxidase (P = 0.55) and tissue transglutaminase (P = 0.66). Islet autoantibody-positive children with the IFIH1 rs2111485 GG genotype had a faster progression to diabetes (31% within 5 years) than children with the type 1 diabetes protective GA or AA genotypes (11% within 5 years; P = 0.006). CONCLUSIONS: The findings indicate that IFIH1 genotypes influence progression from autoimmunity to diabetes development, consistent with the notion that protective genotypes downregulate responses to environmental insults after initiation of autoimmunity.

A simplified method to assess affinity of insulin autoantibodies.

Insulin autoantibodies (IAA) precede type 1 diabetes, but not all IAA-positive children develop other islet autoantibodies and disease. Diabetes risk can be stratified by laborious IAA affinity measurement using competition with multiple ligand concentrations. Here, we identify a single competitor concentration that discriminates low- and high-affinity IAA. Discrimination was achieved among 122 IAA-positive sera using 7.0 nM competitor which is 54-fold that of the assay radioligand concentration. Relative-binding <60% at this competitor concentration identified all 85 sera with affinities ≥1.0×108 L/mol and none with lower affinities (P<0.0001), and 45 (96%) of 47 multiple islet autoantibody-positive sera (P<0.0001). IAA competition was further tested in a second set of 119 IAA-positive sera. Of these, 99 fulfilled high-affinity competition criteria of <60% relative-binding at 7.0 nM competitor including 89 (94%) of 95 sera with multiple islet autoantibodies (P<0.0001). Thus, increased IAA specificity can be achieved with simple modification to existing assays.

Harmonization of glutamic acid decarboxylase and islet antigen-2 autoantibody assays for national institute of diabetes and digestive and kidney diseases consortia.

BACKGROUND/RATIONALE: Autoantibodies to islet antigen-2 (IA-2A) and glutamic acid decarboxylase (GADA) are markers for diagnosis, screening, and measuring outcomes in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) consortia studies. A harmonization program was established to increase comparability of results within and among these studies. METHODS: Large volumes of six working calibrators were prepared from pooled sera with GADA 4.8-493 World Health Organization (WHO) units/ml and IA-2A 2-235 WHO units/ml. Harmonized assay protocols for IA-2A and GADA using (35)S-methionine-labelled in vitro transcribed and translated antigens were developed based on methods in use in three NIDDK laboratories. Antibody thresholds were defined using sera from patients with recent onset type 1 diabetes and healthy controls. To evaluate the impact of the harmonized assay protocol on concordance of IA-2A and GADA results, two laboratories retested stored TEDDY study sera using the harmonized assays. RESULTS: The harmonized assays gave comparable but not identical results in the three laboratories. For IA-2A, using a common threshold of 5 DK units/ml, 549 of 550 control and patient samples were concordantly scored as positive or negative, specificity was greater than 99% with sensitivity 64% in all laboratories. For GADA, using thresholds equivalent to the 97th percentile of 974 control samples in each laboratory, 1051 (97.9%) of 1074 samples were concordant. On the retested TEDDY samples, discordance decreased from 4 to 1.8% for IA-2A (n = 604 samples; P = 0.02) and from 15.4 to 2.7% for GADA (n = 515 samples; P < 0.0001). CONCLUSION: Harmonization of GADA and IA-2A is feasible using large volume working calibrators and common protocols and is an effective approach to ensure consistency in autoantibody measurements.

No effect of the 1alpha,25-dihydroxyvitamin D3 on beta-cell residual function and insulin requirement in adults with new-onset type 1 diabetes.

OBJECTIVE: To determine whether daily intake of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] is safe and improves beta-cell function in patients with recently diagnosed type 1 diabetes. RESEARCH DESIGN AND METHODS: Safety was assessed in an open study of 25 patients aged 18-39 years with recent-onset type 1 diabetes who received 0.25 microg 1,25(OH)(2)D(3) daily for 9 months. An additional 40 patients were randomly assigned to 0.25 microg 1,25(OH)(2)D(3) or placebo daily for 9 months and followed for a total of 18 months for safety, beta-cell function, insulin requirement, and glycemic control. RESULTS: Safety assessment showed values in the normal range in nearly all patients, regardless of whether they received 1,25(OH)(2)D(3) or placebo. No differences in AUC C-peptide, peak C-peptide, and fasting C-peptide after a mixed-meal tolerance test between the treatment and placebo groups were observed at 9 and 18 months after study entry, with approximately 40% loss for each parameter over the 18-month period. A1C and daily insulin requirement were similar between treatment and placebo groups throughout the study follow-up period. CONCLUSIONS: Treatment with 1,25(OH)(2)D(3) at a daily dose of 0.25 microg was safe but did not reduce loss of beta-cell function.

German new onset diabetes in the young incident cohort study: DiMelli study design and first-year results.

BACKGROUND: Diabetes incidence in childhood and youth is increasing worldwide, including autoimmune and non-autoimmune cases. Recent findings suggest that there is a larger than expected proportion of type 2 diabetes in youth, and potential cases of intermediate diabetes phenotypes. Most pediatric diabetes registries focus on type 1 diabetes. Also, there is an absence of reliable data on type 2 diabetes incidence in youth. AIMS: The DiMelli study aims to establish a diabetes incidence cohort registry of patients in Germany, diagnosed with diabetes mellitus before age 20 years. It will be used to characterize diabetes phenotypes by immunologic, metabolic, and genetic markers. DiMelli will assess the contribution of obesity and socio-demographic factors to the development of diabetes in childhood and youth. METHODS: Recruitment of patients started in 2009, and is expected to continue at a rate of 250 patients per year. RESULTS: 84% of the 216 patients recruited within the first year were positive for multiple islet autoantibodies, 12% for one islet autoantibody, and 4% were islet autoantibody-negative. Patients with multiple islet autoantibodies were younger and had lower fasting C-peptide levels, compared to islet autoantibody-negative patients (median age 10.0 vs. 14.1 years, p < 0.01). CONCLUSIONS: Results from the first year of the study show that DiMelli will help to reveal new knowledge on the etiology of diabetes, and the contribution of genetic predisposition and environmental risk factors to the different types of diabetes.

[Bacteriological and virological status in upper respiratory tract infections of cats (cat common cold complex)]. / Bakteriologischer und virologischer Status bei Katzen mit Erkrankungen der oberen Atemwege (Katzenschnupfenkomplex).

Between October 2002 and January 2005,460 bacteriological samples from cats with an acute upper respiratory tract infection were analysed in clinical field studies in two accredited laboratories in Germany. Oropharyngeal swabs were taken from these cats and sent to the laboratories for routine diagnostics. In the swab samples of 460 cats 382 bacteria strains were isolated. The following bacteria were isolated most frequently: Pasteurella spp. (32.5 %), Staphylococcus spp. (18.5 %), Escherichia coli (17.0 %), Streptococcus spp. (9.1 %), Pseudomonas spp. (6.9 %) and Klebsiella spp. (3.0 %). Bordetella bronchiseptica was found in 0.4 % of the animals To evaluate possible regional and time influences, the animals were split into three populations: 1: Germany, laboratory A; 2: Germany, laboratory B; 3: France and Belgium, laboratory B. In population 1 an 2 Pasteurella spp. were found most frequently with 42.2 % and 36.5 %, respectively. The second most frequently isolated bacterial species were Staphylococcus spp. with 14.1 % and 21.4 % and E. coli with 13.6 % and 17.5 % respectively. In population 3 Staphylococcus spp., E. coli (20 % each) and Pasteurella spp. (18.5 %) were isolated at almost the same frequency. Virological parameter were additionally analysed in 328 cats (population 2 and 3). Serum samples were analysed for antibodies specific for Feline Calicivirus (FCV) and Feline Immunodeficiency Virus (FIV) and for Feline Leukaemia Virus (FeLV) antigen. Oropharyngeal swabs were analysed for Feline Herpesvirus (FHV) by using PCR. Calicivirus-specific antibodies were found in 99.6 % of the cats of population 2 and in 100 % of the animals in population 3. Herpesvirus was detected in 15.3 % and 23.3 % of the cats, respectively. FeLV-Antigen was found in 0.4 % of the animals in population 2 and in 10.1 % of the cats in population 3, while FIV-antibodies were identified in 8.7 % of the animals of population 2 and in 6.1 % of the cats of population 3. In total FHV was found in 19.3 % and FCV-specific antibodies in 99.7 % of the animals. 5.3 % of the cats carried FeLV-Antigen, and 7.4 % FIV-specific antibodies. The results of the bacteriological analysis as well as the results of the virological examination confirm previously published data. In this study Pasteurella spp. were most frequently isolated (32.5 %).