Individual differences in in vitro and in vivo metabolic clearances of the antipsychotic drug olanzapine from non-smoking and smoking Japanese subjects genotyped for cytochrome P4502D6 and flavincontaining monooxygenase 3.

OBJECTIVE: The antipsychotic olanzapine is reportedly metabolized by inducible human cytochrome P450 (CYP) 1A2 and variable copy-number CYP2D6 and polymorphic flavin-containing monooxygenase 3 (FMO3) in different pathways. We investigated individual differences in the metabolite formation and clearance of olanzapine in vitro and in vivo. METHODS: Human liver microsomal olanzapine oxidation activities were evaluated, and plasma concentrations of olanzapine were determined in 21 Japanese patients (mean age: 50 years, range: 32-69 years, 14 male and 7 female, including 6 smokers) genotyped for CYP2D6 (*1, *5, and *10) and FMO3 (E158K, C197fsX, R205C, V257M, E308G, and R500X). RESULTS: Furafylline (a CYP1A2 inhibitor), quinidine (a CYP2D6 inhibitor), and heat treatment (inactivates FMO3) suppressed liver microsomal metabolic clearance of olanzapine by approximately 30%. Olanzapine N-demethylation and N-oxygenation were found to be catalyzed by CYP1A2 and CYP2D6 and by CYP2D6 and FMO3, respectively, in experiments using liver microsomes and recombinant enzymes. Plasma concentrations and clearance of olanzapine were not affected by CYP2D6 or FMO3 genotypes or smoking behavior. CONCLUSIONS: Olanzapine clearance was not affected by CYP2D6 or FMO3 genotypes or smoking behavior as a single factor under the present conditions because olanzapine clearance is mediated by multiple enzymes involved in two major and one minor pathways.

Comparison of polyclonal and monoclonal antibody assays for serum amyloid A in cats: a study based on an automated turbidimetric immunoassay in a primary care veterinary hospital.

OBJECTIVE: Comparing the utility of the anti-human serum amyloid A (SAA)-specific monoclonal and polyclonal antibodies assays (LZ-SAA) with the pure monoclonal anti-human antibody assays (VET-SAA) during clinical practice in primary care hospital populations by measuring SAA measurement in healthy and diseased domestic cats. ANIMALS: 52 healthy and 185 diseased client-owned cats. METHODS: SAA concentration was measured using different LZ-SAA and VET-SAA measurements for healthy and various diseased cats. Sensitivity, specificity, and accuracy were calculated for each disease. RESULTS: VET-SAA has higher sensitivity than LZ-SAA for the most common diseases presenting to primary care veterinary hospitals, including chronic kidney disease, tumors, and gingivostomatitis. Our results reveal the capability of detecting low SAA concentrations in healthy and diseased cats using VET-SAA in contrast to LZ-SAA, which found elevations of SAA concentrations only in diseased cats. CLINICAL RELEVANCE: Our findings indicate that switching to the new VET-SAA instead of the conventional LZ-SAA will likely enhance the diagnostic performance in primary care veterinary hospitals.

Comparison of survival times of cats with hyperthyroidism treated with thyroidectomy or methimazole at a primary care hospital in Japan.

OBJECTIVE: We identified the associated factors and compared the survival times of feline hyperthyroidism (FHT) between thyroidectomy and methimazole alone. METHODS: The medical records of 41 cats diagnosed with new-onset hyperthyroidism were retrospectively reviewed. The cats were categorized into the thyroidectomy (n = 15) and methimazole (26) treatment groups. Survival analyses using the Kaplan-Meier method, log-rank test, and Cox proportional hazards models were conducted to compare the time to the selected outcomes. RESULTS: Univariate analysis revealed that survival time was significantly longer with thyroidectomy than with methimazole (P < .001). Multivariate analyses revealed thyroidectomy as an independent prognostic factor for good outcomes (hazard ratio, 0.209; 95% CI, 0.073 to 0.601; P = .004). The recurrence rate was significantly lower in cats that underwent thyroidectomy than in those that received methimazole alone (P = .011). CLINICAL RELEVANCE: Compared with methimazole alone, thyroidectomy was associated with a longer survival time in FHT and can be considered an irreversible treatment modality in settings where radioisotopes are not available.

Complete remission of two canine cases with precursor-targeted immune-mediated anemia after combination therapy with prednisolone, cyclosporine, and oclacitinib.

Background: Precursor-targeted immune-mediated anemia (PIMA) has been described in dogs presenting with nonregenerative anemia and evidence of ineffective erythropoiesis. Although it has been suggested that its occurrence may be related to the immune targeting of erythroid precursors, this pathogenesis has not been established. PIMA is mainly treated with glucocorticoids, and in cases where glucocorticoids alone are not effective, immunosuppressants are also used as combination therapy. However, not all cases of PIMA go into remission after these treatments. Case Description: Two dogs with severe nonregenerative anemia diagnosed as PIMA based on the results of clinical pathological examinations, including bone marrow examination, were treated with whole-blood transfusion and immunosuppressive doses of prednisolone, mycophenolate mofetil, and cyclosporine. However, these treatments failed to achieve remission of PIMA. Therefore, concomitant administration of oclacitinib, which is a Janus kinase-1 inhibitor that has been applied recently to the treatment of immune-mediated diseases, was performed; this combined regimen improved the anemia and achieved complete remission of PIMA. Conclusion: Oclacitinib may be an option for the treatment of PIMA in dogs failing to achieve remission with conventional immunosuppressive therapy.

A distinct mammalian disome collision interface harbors K63-linked polyubiquitination of uS10 to trigger hRQT-mediated subunit dissociation.

Translational stalling events that result in ribosome collisions induce Ribosome-associated Quality Control (RQC) in order to degrade potentially toxic truncated nascent proteins. For RQC induction, the collided ribosomes are first marked by the Hel2/ZNF598 E3 ubiquitin ligase to recruit the RQT complex for subunit dissociation. In yeast, uS10 is polyubiquitinated by Hel2, whereas eS10 is preferentially monoubiquitinated by ZNF598 in human cells for an unknown reason. Here, we characterize the ubiquitination activity of ZNF598 and its importance for human RQT-mediated subunit dissociation using the endogenous XBP1u and poly(A) translation stallers. Cryo-EM analysis of a human collided disome reveals a distinct composite interface, with substantial differences to yeast collided disomes. Biochemical analysis of collided ribosomes shows that ZNF598 forms K63-linked polyubiquitin chains on uS10, which are decisive for mammalian RQC initiation. The human RQT (hRQT) complex composed only of ASCC3, ASCC2 and TRIP4 dissociates collided ribosomes dependent on the ATPase activity of ASCC3 and the ubiquitin-binding capacity of ASCC2. The hRQT-mediated subunit dissociation requires the K63-linked polyubiquitination of uS10, while monoubiquitination of eS10 or uS10 is not sufficient. Therefore, we conclude that ZNF598 functionally marks collided mammalian ribosomes by K63-linked polyubiquitination of uS10 for the trimeric hRQT complex-mediated subunit dissociation.

Subclinical wandering spleen in a cat with gastrointestinal lymphoma.

We accidentally detected a subclinical wandering spleen on preoperative ultrasonography in a cat with gastrointestinal lymphoma. If the spleen is not in the normal position, the wandering spleen should be considered.

Expression of microRNAs in plasma and in extracellular vesicles derived from plasma for dogs with glioma and dogs with other brain diseases.

OBJECTIVE: To measure expression of microRNAs (miRNAs) in plasma and in extracellular vesicles (EVs) derived from plasma for dogs with glioma and dogs with other brain diseases. SAMPLE: Plasma samples from 11 dogs with glioma and 19 control dogs with various other brain diseases. PROCEDURES: EVs were isolated from plasma samples by means of ultracentrifugation. Expression of 4 candidate reference miRNAs (let-7a, miR-16, miR-26a, and miR-103) and 4 candidate target miRNAs (miR-15b, miR-21, miR-155, and miR-342-3p) was quantified with reverse transcription PCR assays. Three software programs were used to select the most suitable reference miRNAs from among the 4 candidate reference miRNAs. Expression of the 4 target miRNAs was then calculated relative to expression of the reference genes in plasma and EVs, and relative expression was compared between dogs with glioma and control dogs with other brain diseases. RESULTS: The most suitable reference miRNAs were miR-16 for plasma and let-7a for EVs. Relative expression of miR-15b in plasma and in EVs was significantly higher in dogs with glioma than in control dogs. Relative expression of miR-342-3p in EVs was significantly higher in dogs with glioma than in control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that miR-15b and miR-342-3p have potential as noninvasive biomarkers for differentiating glioma from other intracranial diseases in dogs. However, more extensive analysis of expression in specific glioma subtypes and grades, compared with expression in more defined control populations, will be necessary to assess their clinical relevance.

Primary malignant peripheral nerve sheath tumors arising from the spinal canal invading the abdominal cavity in a dog.

A 9-year-old neutered male Wire Fox Terrier presented with an 1-month history of hindlimb paresis. Magnetic resonance imaging revealed a contrast-enhanced mass at the level of the L2 vertebral canal. The dog became paraplegic with no deep perception of the hindlimbs, and the mass was surgically removed. The histopathological diagnosis was of a malignant peripheral nerve sheath tumor (MPNST). The dog suffered a relapse of right hindlimb ataxia at 225 days after the surgery. The dog died 434 days after the surgery. Necropsy found a large mass in the abdominal cavity invading from the L2-nerve. This is the first report of MPNST invading the abdominal cavity through the nerve root.

Identification of reference genes for microRNAs of extracellular vesicles isolated from plasma samples of healthy dogs by ultracentrifugation, precipitation, and membrane affinity chromatography methods.

OBJECTIVE: To compare ultracentrifugation, precipitation, and membrane affinity chromatography methods for isolation of extracellular vesicles (EVs) from canine plasma samples and to identify suitable reference genes for incorporation into a quantitative reverse transcription PCR assay of microRNA expression in plasma EVs of healthy dogs. ANIMALS: 6 healthy Beagles. PROCEDURES: Plasma samples were obtained from each dog, and EVs were isolated from 0.3 mL of these samples via ultracentrifugation, precipitation, and membrane-affinity chromatographic methods. Nanoparticle tracking analysis was performed to determine the concentration and size distribution of EVs isolated by the ultracentrifugation method. Expression levels (cycle threshold values) of 4 microRNAs (let-7a, miR-16, miR-26a, and miR-103) were then compared by means of quantitative reverse transcription PCR assay. Three statistical programs were used to identify the microRNAs most suitable for use as reference genes. RESULTS: Results indicated that ultracentrifugation was the most stable of all 3 methods for isolating microRNAs from 0.3 mL of plasma. Nanoparticle tracking revealed that EV samples obtained by the ultracentrifugation method contained a mean ± SD of approximately 1.59 × 1010 vesicles/mL ± 4.2 × 108 vesicles/mL. Of the 4 microRNAs in plasma EVs isolated by ultracentrifugation, miR-103 was the most stable. CONCLUSIONS AND CLINICAL RELEVANCE: The ultracentrifugation method has potential as a stable method for isolating EVs from canine plasma samples with a high recovery rate, and miR-103 may provide the most stable reference gene for normalizing microRNA expression data pertaining to plasma EVs isolated by ultracentrifugation.