Feline Adipose Derived Multipotent Stromal Cell Transdifferentiation Into Functional Insulin Producing Cell Clusters

Diabetes mellitus (DM) is one of the most prevalent feline endocrinopathies, affecting up to 1% of pet cats. De novo generation of functional insulin producing cell (IPC) clusters transdifferentiation of feline adipose-derived multipotent stromal cells (ASCs) may not only provide a viable, functional cell therapy for feline DM, but may also serve as a platform for developing a comparable human treatment given feline and human DM similarities. Cells were induced to form IPCs with a novel, three-stage culture process with stromal or differentiation medium under static and dynamic conditions. Clusters were evaluated for intracellular zinc, viability, intracellular insulin, glucagon, and somatostatin, ultrastructure, glucose stimulated insulin secretion in the presence or absence of theophylline, and protein and gene expression. Isolated cells were multipotent, and cell clusters cultured in both media had robust cell viability. Those cultured in differentiation medium contained zinc and mono- or polyhormonal α-, β-, and δ-like cells based on immunohistochemical labeling and Mallory-Heidenhan Azan-Gomori\u27s staining. Ultrastructurally, cell clusters cultured in differentiation medium contained insulin granules within vesicles, and clusters had a concentration-dependent insulin response to glucose in the presence and absence of theophylline which increased both insulin secretion and intracellular content. Expression of NK6.1, Pax6, Isl1, Glut2, RAB3A, glucagon, insulin, and somatostatin increased with differentiation stage for both sexes, and expression of nestin at stages 1 and 2 and Neurod1 at stage 2 was higher in cells from female donors. The cluster insulin secretion responses and endocrine and oncogene gene expression profiles were inconsistent with insulinoma characteristics. A total of 180 proteins were upregulated in differentiated clusters, and the majority were associated with biological regulation, metabolic processes, or stimulus response. Dynamic culture of IPC clusters resulted in clusters composed of cells primarily expressing insulin that released higher insulin with glucose stimulation than those in static culture. Collectively, the results of this study support generation of functional IPC clusters using feline ASCs isolated from tissues removed during routine sterilization. Further, cluster functionality is enhanced with dynamic, motion-driven shear stress. This work establishes a foundation for development of strategies for IPC therapy for short or long-term diabetes treatment and may represent an option to study prevention and treatment of diabetes across species

Genetic diversity of Trypanosoma cruzi parasites infecting dogs in southern Louisiana sheds light on parasite transmission cycles and serological diagnostic performance.

BackgroundChagas disease is a neglected zoonosis of growing concern in the southern US, caused by the parasite Trypanosoma cruzi. We genotyped parasites in a large cohort of PCR positive dogs to shed light on parasite transmission cycles and assess potential relationships between parasite diversity and serological test performance.Methodology/principal findingsWe used a metabarcoding approach based on deep sequencing of T. cruzi mini-exon marker to assess parasite diversity. Phylogenetic analysis of 178 sequences from 40 dogs confirmed the presence of T. cruzi discrete typing unit (DTU) TcI and TcIV, as well as TcII, TcV and TcVI for the first time in US dogs. Infections with multiple DTUs occurred in 38% of the dogs. These data indicate a greater genetic diversity of T. cruzi than previously detected in the US. Comparison of T. cruzi sequence diversity indicated that highly similar T. cruzi strains from these DTUs circulate in hosts and vectors in Louisiana, indicating that they are involved in a shared T. cruzi parasite transmission cycle. However, TcIV and TcV were sampled more frequently in vectors, while TcII and TcVI were sampled more frequently in dogs.Conclusions/significanceThese observations point to ecological host-fitting being a dominant mechanism involved in the diversification of T. cruzi-host associations. Dogs with negative, discordant or confirmed positive T. cruzi serology harbored TcI parasites with different mini-exon sequences, which strongly supports the hypothesis that parasite genetic diversity is a key factor affecting serological test performance. Thus, the identification of conserved parasite antigens should be a high priority for the improvement of current serological tests

Teaching veterinary surgical skills: Comparison of massed versus spaced instruction

OBJECTIVE: To determine the effect of massed instruction (MI) versus spaced instruction (SI) of veterinary surgical skills on students\u27 cognitive load and skill retention. STUDY DESIGN: Prospective randomized cohort study STUDY POPULATION: First-year veterinary students from Louisiana State University (LSU; n = 47) and Lincoln Memorial University (LMU; n = 101). METHODS: Students were randomized to MI (two skills in a single session of twice the duration) or SI (one skill per session on two consecutive days). Instructors, instructional ratio, and total educational time was equivalent. Following instruction, students completed a cognitive load questionnaire and underwent a structured assessment immediately after (LMU only), 1 day after, and 3-4 weeks after learning the second skill. Students completed two supervised practice sessions one and 2 weeks after the initial laboratory session(s). RESULTS: Overall cognitive load did not differ between groups (p \u3e .05), although LMUs MI group reported higher physical and time demands, effort, and frustration. At initial assessment, SI students scored higher than MI students for the first skill at both LSU (mean checklist score = 27.7 vs. mean = 24; p = .004) and LMU (mean global rating score = 4.76 vs. mean = 4.55; p = .029). Differences between groups were no longer evident by 3-4 weeks after instruction. CONCLUSION: SI may lead to improved immediate performance; however, supervised practice was sufficient to overcome the initial disparity. CLINICAL SIGNIFICANCE: SI may be beneficial for initial skill performance. However, SI and MI students had similar performance after 3 weeks, suggesting the more convenient curricular design of MI may be sufficient as long as practice sessions are incorporated

Impact of heat treatment on Dirofilaria immitis antigen detection in shelter dogs

Abstract Background The diagnosis and management of canine heartworm disease is a growing concern for shelter veterinarians. Although the accuracy of commercial antigen test kits has been widely studied, recent reports have renewed interest in antigen blocking as a causative factor for false “no antigen detected” results. The objectives of this study were to determine the prevalence of false “no antigen detected” results in adult dogs entering shelters in northern, southern, and western regions of the country and to identify historical and clinical risk factors for such results. Methods Serum samples were evaluated for Dirofilaria immitis antigen using a commercially available point-of-care ELISA; samples in which no antigen was detected underwent a heat treatment protocol and repeat antigen testing. Whole blood samples underwent Knott testing to identify the presence of microfilariae. Historical and clinical findings were analyzed using exact logistic regression. Results A total of 616 samples were analyzed. Overall prevalence of positive antigen test results (prior to heat treatment) was 7.3% and frequency of false “no antigen detected” results due to antigen blocking (ie, samples with no antigen detected prior to heat treatment and positive after heat treatment) was 5.2%. Among dogs that had no detectable antigen on the initial tests, dogs that had microfilariae detected via modified Knott testing (OR = 32.30, p-value = 0.013) and dogs that previously received a heartworm preventive (OR = 3.81, p-value = 0.016) had greater odds of antigen blocking than dogs without these factors. Among dogs that were heartworm positive, those without microfilariae detected had greater odds of antigen blocking than dogs with this factor (OR = 11.84, p-value = 0.0005). Geographic region of origin was significantly associated with occurrence of antigen blocking (p = 0.0036); however, blocking occurred in all regions sizably contributing to heartworm diagnoses. Of the 74 dogs found to be infected with heartworms in this study, 39.2% (29) had no detectable antigen prior to heat treatment. Conclusions Heat treatment of serum samples should be considered to improve diagnostic test accuracy, particularly in dogs that reportedly received a heartworm preventive prior to antigen testing regardless of region of origin

Additional file 3: of Impact of heat treatment on Dirofilaria immitis antigen detection in shelter dogs

Univariable exact logistic regression for antigen blocking in dogs that were heartworm positive with a point-of-care ELISA with and without heat treatment (n = 74). (DOCX 20 kb