Clinical and clinicopathological features and outcomes of cats with suspected dietary induced pancytopenia.

BACKGROUND: After a strong epidemiological link to diet was established in an outbreak of pancytopenia in cats in spring 2021 in the United Kingdom, 3 dry diets were recalled. Concentrations of the hemato- and myelotoxic mycotoxins T-2, HT-2 and diacetoxyscirpenol (DAS) greater than the European Commission guidance for dry cat foods were detected in the recalled diets. OBJECTIVES: To describe clinical and clinicopathological findings in cats diagnosed with suspected diet induced pancytopenia. ANIMALS: Fifty cats presenting with pancytopenia after exposure to a recalled diet. METHODS: Multicenter retrospective case series study. Cats with known exposure to 1 of the recalled diets were included if presented with bi- or pancytopenia and underwent bone marrow examination. RESULTS: Case fatality rate was 78%. Bone marrow aspirates and biopsy examination results were available in 23 cats; 19 cats had a bone marrow aspirate, and 8 cats had a biopsy core, available for examination. Bone marrow hypo to aplasia-often affecting all cell lines-was the main feature in all 31 available core specimens. A disproportionately pronounced effect on myeloid and megakaryocytic cells was observed in 19 cats. Myelofibrosis or bone marrow necrosis was not a feature. CONCLUSION AND CLINICAL IMPORTANCE: Mycotoxin induced pancytopenia should be considered as differential diagnosis in otherwise healthy cats presenting with bi- or pancytopenia and bone marrow hypo- to aplasia.

Successful use of a benchtop fluorescent enzyme immunoassay analyzer to measure serum cortisol concentration as a screening test for hypoadrenocorticism in dogs.

OBJECTIVE: To assess the diagnostic performance of a benchtop fluorescent enzyme immunoassay analyzer (AIA-360; Tosoh Bioscience Inc) for the measurement of serum cortisol concentration as a screening test for hypoadrenocorticism in dogs. ANIMALS: 173 client-owned dogs (20 with hypoadrenocorticism and 153 with nonadrenal illness). PROCEDURES: Medical records of all dogs that underwent an ACTH stimulation test between June 2015 and October 2019 were reviewed retrospectively. Dogs were excluded if the ACTH stimulation test was performed on the basis of a suspicion of hypercortisolism, serum cortisol concentrations were measured using an analyzer other than the one assessed in the present study, or dogs had received medication known to affect the pituitary-adrenal axis in the 4 weeks1,2 preceding ACTH stimulation testing. The diagnostic performance of the benchtop analyzer was evaluated by calculating sensitivity, specificity, and likelihood ratios at various cutoff points. RESULTS: Serum resting cortisol cutoff point concentrations of 0.8 µg/dL (22 nmol/L), 1 µg/dL (28 nmol/L), and 2 µg/dL (55 nmol/L) had a sensitivity of 100%. An optimal serum resting cortisol cutoff point of 0.58 µg/dL (16 nmol/L) had a sensitivity, specificity, and positive and negative likelihood ratios of 100%, 97%, and 30.6 and 0.0, respectively. CLINICAL RELEVANCE: Findings indicated that previously derived cutoff points could be used with excellent sensitivity to exclude hypoadrenocorticism in this population of dogs when serum cortisol concentration was measured with the evaluated benchtop analyzer. An ACTH stimulation test may need to only be performed to diagnose hypoadrenocorticism if resting serum cortisol concentration is ≤ 0.58 µg/dL when measured with the evaluated benchtop analyzer.

Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts.

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-ß-cyclodextrin (methyl-ß-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

Behavior in dogs with spontaneous hypothyroidism during treatment with levothyroxine.

BACKGROUND: Thyroid hormone supplementation anecdotally has been described as a valid treatment option for dogs with aggression-related problems. However, prospective, controlled, and blinded trials evaluating behavior and neurohormonal status in hypothyroid dogs during treatment with levothyroxine are lacking. OBJECTIVE: Levothyroxine supplementation will have a significant influence on the behavior and neurohormonal status of dogs with spontaneous hypothyroidism. ANIMALS: Twenty client-owned dogs diagnosed with spontaneous hypothyroidism. METHODS: This prospective study was to evaluate the behavior of dogs, which was screened at initial presentation, and after 6 weeks, and 6 months of treatment with levothyroxine (starting dosage 10 µg/kg PO q12h) using the standardized Canine Behavioral Assessment and Research Questionnaire (C-BARQ). At each time period, circulating serotonin and prolactin (PRL) concentrations were evaluated using a commercially validated ELISA kit and heterologous radioimmunoassay, respectively. RESULTS: After 6 weeks of thyroid hormone supplementation, C-BARQ scores demonstrated a significant increase in activity of hypothyroid dogs (P < .01). No significant change in any of the behavioral signs was observed after 6 months of treatment. No significant difference in circulating concentrations of serotonin (P > .99 and P = .46) and PRL (P = .99 and P = .37) were noted between the 6-week and 6-month periods compared with baseline. CONCLUSIONS AND CLINICAL IMPORTANCE: The results of this study indicate increased activity of hypothyroid dogs after 6 weeks of thyroid hormone supplementation. None of the hypothyroid dogs in this cohort showed a significant change in any of the evaluated behavioral signs and neurohormonal status after 6 months of thyroid hormone supplementation.

Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged beta-actin (pEYFP-ACTIN) and the neurokinin type 1 receptor (NK1-R).

This study focused on establishing and making a comprehensive functional characterization of an HEK-293-transfected cell line that would coexpress the enhanced yellow fluorescent protein-actin (pEYFP-actin) construct and the neurokinin type 1 receptor (NK1-R), which is a member of the seven transmembrane (7TM) receptor family. In the initial selection procedure, the cloning ring technique was used alone, but failed to yield clones with homogenous pEYFP-actin expression. Flow cytometry sorting (FCS) was subsequently used to enrich the pEYFP-actin-expressing subpopulation of cells. The enzyme-linked immunosorbent assay (ELISA), FCS and quantitative real-time reverse transcription/polymerase chain reaction (RT-PCR) were then employed to monitor the passage-dependent effects on transgene expression and to estimate the total beta-actin/pEYFP-actin ratio. NK1-R was characterized via radioactive ligand binding and the second messenger assay. The suitability of the pEYFP-actin as a marker of endogenous actin was assessed by colocalizing pEYFP-actin with rhodamine-phalloidine-stained F-actin and by comparing receptor- and jasplakinolide-induced changes in the actin cytoskeleton organization. These experiments demonstrated that: i) both constructs expressed in the generated transfected cell line are functional; ii) the estimated pEYFP-actin: endogenous beta-actin ratio is within the limits required for the functional integrity of the actin filaments; and iii) pEYFP-actin and rhodamine-phalloidine-stained F-actin structures colocalize and display comparable reorganization patterns in pharmacologically challenged cells.