Modulation of MHC expression by interferon-gamma and its influence on PBMC-mediated cytotoxicity in canine mast cell tumour cells.

Immunotherapy is a promising alternative treatment for canine mast cell tumour (MCT). However, evasion of immune recognition by downregulating major histocompatibility complex (MHC) molecules might decline treatment efficiency. Enhancing MHC expression through interferon-gamma (IFN-γ) is crucial for effective immunotherapy. In-house and reference canine MCT cell lines derived from different tissue origins were used. The impacts of IFN-γ treatment on cell viability, expression levels of MHC molecules, as well as cell apoptosis were evaluated through the MTT assay, RT-qPCR and flow cytometry. The results revealed that IFN-γ treatment significantly influenced the viability of canine MCT cell lines, with varying responses observed among different cell lines. Notably, IFN-γ treatment increased the expression of MHC I and MHC II, potentially enhancing immune recognition and MCT cell clearance. Flow cytometry analysis in PBMCs-mediated cytotoxicity assays showed no significant differences in overall apoptosis between IFN-γ treated and untreated canine MCT cell lines across various target-to-effector ratios. However, a trend towards higher percentages of late and total apoptotic cells was observed in the IFN-γ treated C18 and CMMC cell lines, but not in the VIMC and CoMS cell lines. These results indicate a variable response to IFN-γ treatment among different canine MCT cell lines. In summary, our study suggests IFN-γ's potential therapeutic role in enhancing immune recognition and clearance of MCT cells by upregulating MHC expression and possibly promoting apoptosis, despite variable responses across different cell lines. Further investigations are necessary to elucidate the underlying mechanisms and evaluate IFN-γ's efficacy in in vivo models.

Bilateral Common Carotid Artery Stenosis in Mice: A Model of Chronic Cerebral Hypoperfusion-Induced Vascular Cognitive Impairment.

Vascular cognitive impairment (VCI) is a syndrome defined as cognitive decline caused by vascular disease and is associated with various types of dementia. Chronic cerebral hypoperfusion (CCH) is one of the major contributors to VCI. Among the various rodent models used to study CCH-induced VCI, we have found the mouse bilateral common carotid artery stenosis (BCAS) model to be highly suitable. Here, we introduce the BCAS model of C57BL/6J mice generated using microcoils with an internal diameter of 0.18 mm. To produce the mouse BCAS model, the bilateral common carotid arteries are isolated from the adhering tissues and vagus nerves and twined around the microcoils. This model shows cognitive impairment and white matter lesions preceding neuronal dysfunction around postoperative day 28, which is similar to the human clinical picture. Overall, the mouse BCAS model will continue to be useful in studying CCH-induced VCI. Key features • This mouse BCAS model requires approximately 4 weeks to show phenotypes such as cognitive impairment and white matter injury.

Anti-tumor effect of proteasome inhibitor on canine urothelial carcinoma.

Canine urothelial carcinoma (cUC) is one of the most malignant tumors affecting dogs; however, its proliferative mechanism is yet to be fully elucidated. The ubiquitin-proteasome system (UPS) is an important metabolic pathway regulating protein degradation, and its dysfunction leads to apoptosis. We investigated the antitumor effect of the proteasome inhibitor bortezomib, which blocks the UPS. Bortezomib inhibited cell growth in cUC cell lines by inducing apoptosis in vitro. These findings suggest the potential of bortezomib as a novel therapeutic drug for dogs with cUC.

Surgical Orthodontic Treatment for Skeletal Maxillary Protrusion in Sturge-Weber Syndrome: A Case Report and Review of the Literature.

Sturge-Weber syndrome (SWS) is characterized by hemangiomas, glaucoma, and central nervous system disorders. Here, we report the case of a 15-year-old boy with SWS and upper-lip hypertrophy who underwent surgical orthodontic treatment for correction of a large overjet and deep overbite. In addition to the a large overjet and deep overbite, interdental spacing was observed in both the arches. The mandible was retrognathic and deviated to the right side. No maxillary occlusal canting or temporomandibular joint symptoms were observed. The patient was diagnosed with skeletal maxillary protrusion with spaced dentition and mandibular deviation to the right due to SWS. After presurgical orthodontic treatment using a multibracket appliance, we performed a sagittal split ramus osteotomy (SSRO) alone due to the presence of a hemangioma around the maxilla. No abnormal bleeding or cerebral hemorrhage due to increased blood pressure was observed during the SSRO. Postoperatively, the maxillary and mandibular arches were well-aligned, the deep overbite and excessive overjet improved, and bilateral angle class I molar and canine relationships were established. Furthermore, mandibular deviation improved, and the midlines of both arches approximately coincided with the facial midline. In conclusion, orthognathic surgery is feasible in patients with SWS after carefully evaluating the sites and sizes of the hemangiomas.

Identification of hypoxia-induced metabolism-associated genes in canine tumours.

Canine tumours including urothelial carcinoma, lung adenocarcinoma, mammary gland tumour, squamous cell carcinoma, and melanoma have been identified as causes of death, but effective therapies are limited due to insufficient knowledge of the molecular mechanisms involved. Within the tumour microenvironment, hypoxia activates hypoxia-inducible factor 1α (HIF1α) in tumour cells. High HIF1α expression correlates with enhanced glycolysis and poorer outcomes in human cancers. However, the molecular mechanisms underlying hypoxic tumour cells remain elusive in dogs. In our study, we investigated upregulated genes in a canine malignant melanoma cell line during hypoxia using RNA-sequencing analysis. Glycolysis and HIF1 signalling pathways were upregulated in hypoxic melanoma cells. HIF1α knockout melanoma cells revealed that the glycolysis marker MCT4 is regulated by HIF1α activation. Hypoxia induces high lactate secretion due to enhanced glycolysis in canine melanoma cells. Furthermore, we examined monocarboxylate transporter 4 (MCT4) expression in malignant melanoma and eight other types of canine tumour tissues using immunohistochemistry (IHC). Membrane-localized MCT4 protein was mostly detected in urothelial carcinoma and lung adenocarcinoma rather than malignant melanoma. We conclude that canine MCT4 protein plays a role in lactic acid efflux from glycolytic cells and may serve as a marker for hypoxia and glycolysis in canine tumours. These findings could inform future therapeutic strategies targeting MCT4.

Anesthetic management of a patient with Sturge-Weber syndrome in sagittal split ramus osteotomy surgery.

Sturge-Weber syndrome (SWS) is a neurocutaneous syndrome characterized by angiomas. This report presents airway management using submental intubation in sagittal split ramus osteotomy under general anesthesia and aimed to explore better anesthetic management for avoiding the rupture of angiomas in a patient with SWS.

Effects of trastuzumab emtansine on canine urothelial carcinoma cells in vitro and in vivo.

Urothelial carcinoma (UC) is the most common malignancy of the urinary tract in dogs and has aggressive behaviour. Although human epidermal growth factor receptor 2 (HER2) is a known therapeutic target with evidence in canine UC, the efficacy of anti-HER2 antibody drugs remains unknown. This study aimed to investigate the effects of anti-HER2 antibody drugs including trastuzumab and trastuzumab emtansine (T-DM1) on canine UC cell lines in vitro and in vivo. Four canine UC cell lines (Nene, TCCUB, Love, and Sora) were used. In western blotting, HER2 protein expression was observed in all the cell lines. Although both trastuzumab and T-DM1 showed dose-dependent growth inhibitory activity in the cell lines, T-DM1 showed much stronger activity than that of trastuzumab. In flow cytometry analyses with the canine UC cell line (Sora), T-DM1 but not trastuzumab significantly increased the percentages of early and late apoptotic cells in annexin V apoptotic assays and the sub-G1 phase fraction in cell cycle analyses. For the in vivo experiment, the canine UC cells (Sora) were subcutaneously injected into nude mice. Four days after inoculation, trastuzumab, T-DM1, or vehicle was administered intraperitoneally once a week for three times. Tumour volumes were significantly smaller in the T-DM1 group compared to the trastuzumab and vehicle control groups. These findings indicate that T-DM1 exerts a stronger antitumour effect than that of trastuzumab on canine UC cells in vitro and in vivo, possibly by inducing apoptosis due to DM1.

Upregulation and functional roles of miR-450b in canine oral melanoma.

Canine oral melanoma (COM) is a common and highly aggressive disease with the potential to model human melanomas. Dysregulated microRNAs represent an interesting line of research for COM because they are implicated in tumor progression. One example is miR-450b, which has been investigated for its molecular mechanisms and biological functions in multiple human cancers, but not human or canine melanoma. Here, we aimed to investigate miR-450b as a potential diagnostic biomarker of COM and its functional roles in metastatic and non-metastatic forms of the disease. We investigated the expression of miR-450b and its target mRNA genes in clinical (tumor tissue and plasma) samples and metastatic and primary-tumor cell lines. Knockdown and overexpression experiments were performed to determine the influence of miR-450b on cell proliferation, migration, colony formation, and apoptosis. miR-450b was significantly upregulated in COM and differentiated between metastatic and non-metastatic tumors, and its potential as a biomarker of metastatic and non-metastatic COM was further confirmed in ROC analysis. miR-450b knockdown promoted cell proliferation, migration, and clonogenicity and inhibited apoptosis, whereas its overexpression yielded the reverse pattern. miR-450b directly binds 3' UTR of PAX9 mRNA and modulates its function leading to BMP4 downregulation and MMP9 upregulation at the transcript level. Furthermore, we surmised that miR-450b activates the Wnt signaling pathway based on gene ontology and enrichment analyses. We concluded that miR-450b has the potential as a diagnostic biomarker and could be a target candidate for COM treatment.

Impact of collagen matrix on reconstructive material selection and postoperative complications in endoscopic endonasal skull base surgery.

OBJECTIVE: The aim of this study was to investigate the impact of collagen matrix on reconstructive material selection and postoperative complications in endoscopic endonasal skull base surgery. METHODS: The authors retrospectively reviewed the data of consecutive patients who underwent purely endoscopic endonasal skull base surgery from January 2015 to March 2023. Intraoperative CSF leakage was classified according to the Esposito grade, and skull base repair was tailored to the leakage grade. The patients were divided into two groups: before (group A) and after (group B) collagen matrix implementation. The rates of autologous graft harvesting (fat, fascia, and nasoseptal flap), postoperative CSF leakage, and donor-site complications were compared between the two groups. RESULTS: In total, 270 patients were included. Group A included 159 patients and group B included 111 patients. There were no differences in patient characteristics, including age, pathology, and Esposito grade, between the two groups. The overall fat usage rate was significantly higher in group A (63.5%) than in group B (39.6%) (p = 0.0001), and the fascia usage rate was also significantly higher in group A (25.8%) than in group B (4.5%) (p < 0.0001). The nasoseptal flap usage rate did not differ between group A (32.7%) and group B (30.6%) (p = 0.79). Postoperative CSF leakage was similar between the two groups (0.63% in group A vs 1.8% in group B, p = 0.57), and the overall rate of CSF leakage was 1.1%. Donor-site complications occurred in 3 patients in group A, including 1 abdominal hematoma, 1 delayed abdominal infection, and 1 fluid collection after fascia lata harvesting. CONCLUSIONS: Collagen matrix implementation significantly decreased autologous graft harvesting without increasing postoperative CSF leakage, contributing to less invasive surgery.

Pan-tumour analysis of COX-2 expression in dogs.

Cyclooxgenase-2 (COX-2) is associated with inflammatory microenvironment and tumour progression. COX-2 expression was reported in canine tumours, and anti-COX treatment showed therapeutic effects in selected tumour types. Currently, direct comparisons between different tumour types or reports were impossible due to varying evaluation protocols. Additionally, COX-2 expression in relatively uncommon tumours were yet to be evaluated. Here, we analysed COX-2 expression across various tumour types in dogs in a consistent protocol, aiming to revisit accumulated evidence in the field and report novel candidate tumours for anti-COX therapy. COX-2 expression in 32 histological types of tumours, which consisted of 347 samples in total, was investigated using immunohistochemistry followed by the Belshaw's method scoring (range: 0-12). More than the half of the samples expressed COX-2 in mast cell tumours, transitional cell carcinoma in the urinary tract, squamous cell carcinoma, liposarcoma, and melanoma, with COX-2 median scores ranging from 1-8. On the other hand, <20% tissues expressed COX-2 in the half of tumour types investigated. Overall COX-2 positive rate was 27%. In conclusion, the results confirmed COX-2 expression in the well-known COX-2-expresing tumour types and suggested novel candidate tumours for anti-COX-2 therapy. At the same time, overall COX-2 expression was low, and inter- and intra-histology heterogeneity was apparent. This study will provide a foundation reference for future research in canine tumours.

EBF1 Limits the Numbers of Cochlear Hair and Supporting Cells and Forms the Scala Tympani and Spiral Limbus during Inner Ear Development.

Early B-cell factor 1 (EBF1) is a basic helix-loop-helix transcription factor essential for the differentiation of various tissues. Our single-cell RNA sequencing data suggest that Ebf1 is expressed in the sensory epithelium of the mouse inner ear. Here, we found that the murine Ebf1 gene and its protein are expressed in the prosensory domain of the inner ear, medial region of the cochlear duct floor, otic mesenchyme, and cochleovestibular ganglion. Ebf1 deletion in mice results in incomplete formation of the spiral limbus and scala tympani, increased number of cells in the organ of Corti and Kölliker's organ, and aberrant course of the spiral ganglion axons. Ebf1 deletion in the mouse cochlear epithelia caused the proliferation of SOX2-positive cochlear cells at E13.5, indicating that EBF1 suppresses the proliferation of the prosensory domain and cells of Kölliker's organ to facilitate the development of appropriate numbers of hair and supporting cells. Furthermore, mice with deletion of cochlear epithelium-specific Ebf1 showed poor postnatal hearing function. Our results suggest that Ebf1 is essential for normal auditory function in mammals.

Pharmacokinetics of monoclonal antibodies locally-applied into the middle ear of guinea pigs.

Countless therapeutic antibodies are currently available for the treatment of a broad range of diseases. Some target molecules of therapeutic antibodies are involved in the pathogenesis of sensorineural hearing loss (SNHL), suggesting that SNHL may be a novel target for monoclonal antibody (mAb) therapy. When considering mAb therapy for SNHL, understanding of the pharmacokinetics of mAbs after local application into the middle ear is crucial. To reveal the fundamental characteristics of mAb pharmacokinetics following local application into the middle ear of guinea pigs, we performed pharmacokinetic analyses of mouse monoclonal antibodies to FLAG-tag (FLAG-mAbs), which have no specific binding sites in the middle and inner ear. FLAG-mAbs were rapidly transferred from the middle ear to the cochlear fluid, indicating high permeability of the round window membrane to mAbs. FLAG-mAbs were eliminated from the cochlear fluid 3 h after application, similar to small molecules. Whole-body autoradiography and quantitative assessments of cerebrospinal fluid and serum demonstrated that the biodistribution of FLAG-mAbs was limited to the middle and inner ear. Altogether, the pharmacokinetics of mAbs are similar to those of small molecules when locally applied into the middle ear, suggesting the necessity of drug delivery systems for appropriate mAb delivery to the cochlear fluid after local application into the middle ear.

Hypoxia-related Y RNA fragments as a novel potential biomarker for distinguishing metastatic oral melanoma from non-metastatic oral melanoma in dogs.

Hypoxia may promote tumor progression, and hypoxically altered noncoding RNA (ncRNA) expression may play a role in metastasis. Canine oral melanoma (COM) frequently metastasizes, and ncRNA expression under hypoxia may be clinically significant. We aimed to elucidate ncRNA fragments whose expression is altered by hypoxia in COM-derived primary KMeC and metastatic LMeC cell lines using next-generation sequencing to validate these results in qRT-PCR, and then compare expression between metastatic and non-metastatic COM. The NGS analysis and subsequent qRT-PCR validation were performed using hypoxic and normoxic KMeC and LMeC cells, and clinical samples [tumor tissue, plasma, and plasma-derived extracellular vesicles] obtained from dogs with metastatic or non-metastatic melanoma were analyzed with qRT-PCR. Y RNA was significantly decreased in metastatic LMeC cells versus primary KMeC cells in hypoxic and normoxic conditions. The expression of Y RNA was decreased in dogs with metastatic melanoma versus those with non-metastatic melanoma for all clinical sample types, reflecting the pattern found with hypoxia. Receiver operating characteristic analysis demonstrated that Y RNA level is a promising biomarker for discriminating metastatic from non-metastatic melanoma in plasma [area under the curve (AUC) = 0.993, p < 0.0001] and plasma-derived extracellular vesicles (AUC = 0.981, p = 0.0002). Overall, Y RNA may be more resistant to hypoxic stress in the metastatic than the non-metastatic state for COM. However, further investigation is required to elucidate the biological functions of Y RNA under hypoxic conditions.

Elevated expression of miR-301a and its functional roles in canine oral melanoma.

miR-301a is one of numerous dysregulated microRNAs (miRNAs) in canine oral melanoma (COM), one of which is miR-301a (upregulated). Its biological role has been described in various human cancer types, including malignant melanoma, but not in COM. Accordingly, in this study, we investigated miR-301a expression in COM in greater detail to ascertain whether it could serve as a diagnostic biomarker, elucidate its functional roles in this cancer, and predict the possible pathways by which it exerts its effects. Relative expression of miR-301a was investigated in clinical oral tissue and plasma samples and COM cell (KMeC and LMeC) lines using qRT-PCR. Knockdown of miR-301a was also validated for KMeC and LMeC cells using qRT-PCR. We performed CCK-8 assays to assess cell proliferation, monolayer wound-healing, and transwell migration assays to assess cell migration, a colony-formation assay to assess clonogenicity, a TUNEL assay and flow cytometry to assess apoptosis-related effects, and gene enrichment analyses to predict possible related pathways. miR-301a was markedly upregulated in COM oral tissue and plasma clinically, suggesting its potential as a diagnostic biomarker for COM diagnosis. In vitro assays demonstrated that miR-301 significantly inhibited apoptosis in COM cells while promoting cell migration, proliferation, and clonogenicity. We also predicted that miR-301 exerts cancer-promoting effects through the Wnt signalling pathway for COM. Our findings suggest that miR-301a is a COM oncomiR that regulates several oncogenic phenotypes with the potential to be a diagnostic biomarker.

PMab-301: An Anti-Giraffe Podoplanin Monoclonal Antibody for Immunohistochemistry.

Immunohistochemistry staining is an essential method in pathological diagnoses. Podoplanin (PDPN) is a specific maker of alveolar epithelium, lymphatic vessels, and glomeruli. In this study, we established a novel anti-giraffe PDPN (girPDPN) mAb, PMab-301, using the Cell-Based Immunization and Screening (CBIS) method. PMab-301 (mouse IgG1, kappa) detected girPDPN in various applications, such as flow cytometry, western blot, and immunohistochemistry. PMab-301 specifically stained type-I alveolar cells using formalin-fixed paraffin-embedded giraffe lung tissues. Our findings suggest the potential usefulness of PMab-301 for the pathophysiological analyses of giraffe tissues.

Evaluation of semitendinosus muscle transposition for treatment of perineal hernias in 33 small-breed dogs.

Little is known about the perioperative complications and treatment outcomes of repairing perineal hernias using semitendinosus muscle transposition (SMT) in small-breed dogs. The objective of this study was to retrospectively evaluate the complications and prognosis of perineal hernia repair using SMT in 33 small, male dogs (weighing < 15 kg). Before the study began, the medical records of all small-breed dogs that underwent SMT from March 2013 to June 2022 at the Veterinary Medical Center, University of Tokyo were analyzed, and telephone interviews were conducted with their owners or referring physicians. Short-term complications were observed in 42.4% of the patients. Lameness was the most common complication, although all cases were resolved within a few days. The recurrence rate during the study period (median: 265 d) was 9.1%. The results of this study showed that transposition of the semitendinosus muscle is more invasive than other surgical procedures for perineal hernias. It is, however, an effective alternative when repair with internal obturator muscle transposition alone is expected to be difficult, such as severe atrophy of the internal obturator muscle or herniation in the ventral direction, and in cases with a history of surgery by transposition of the internal obturator muscle.

On sait peu de choses sur les complications peropératoires et les résultats du traitement de la réparation des hernies périnéales à l'aide de la transposition du muscle semi-tendineux (SMT) chez les chiens de petite race. L'objectif de cette étude était d'évaluer rétrospectivement les complications et le pronostic de la réparation d'une hernie périnéale par SMT chez 33 petits chiens mâles (pesant < 15 kg). Avant le début de l'étude, les dossiers médicaux de tous les chiens de petite race ayant subi une SMT de mars 2013 à juin 2022 au Veterinary Medical Center de l'Université de Tokyo ont été analysés et des entrevues téléphoniques ont été menées avec leurs propriétaires ou médecins vétérinaires référents. Des complications à court terme ont été observées chez 42,4 % des patients. La boiterie était la complication la plus courante, bien que tous les cas aient été résolus en quelques jours. Le taux de récidive au cours de la période d'étude (médiane: 265 jours) était de 9,1 %. Les résultats de cette étude ont démontré que la transposition du muscle semi-tendineux est plus invasive que les autres interventions chirurgicales pour les hernies périnéales. Il s'agit cependant d'une alternative efficace lorsque la réparation par transposition du muscle obturateur interne seul s'annonce difficile, comme une atrophie sévère du muscle obturateur interne ou une hernie dans le sens ventral, et dans les cas d'antécédents chirurgicaux par transposition du muscle obturateur interne.(Traduit par Docteur Serge Messier).

Pretreatment with tadalafil attenuates cardiotoxicity induced by combretastatin A4 disodium phosphate in rats.

Combretastatin A4 disodium phosphate (CA4DP) is a prodrug of combretastatin A4 (CA4), a microtubule-disassembling agent that exhibits antitumor effects by inhibiting tumor cell proliferation and inducing morphological changes and apoptosis in vascular endothelial cells in tumors. However, cardiotoxicity induced by ischemia and hypertension is a severe adverse event. In this study, we focused on the fact that phosphodiesterase (PDE) 5 inhibitors dilate the heart and peripheral blood vessels and aimed to investigate whether co-administration of tadalafil, a PDE5 inhibitor, can attenuate cardiotoxicity without altering the antitumor effect of CA4DP. To investigate cardiotoxicity, CA4DP and/or tadalafil were administered to rats, and blood pressure, echocardiography, histopathology, and cGMP concentration in the myocardium were examined. Administration of CA4DP increased systolic blood pressure, decreased cardiac function, lowered cGMP levels in the myocardium, and led to necrosis of myocardial cells. Co-administration of tadalafil attenuated these CA4DP-induced changes. To investigate the antitumor effect, canine mammary carcinoma cell lines (CHMp-13a) and human umbilical vein endothelial cells were cultured with CA4 and/or tadalafil, and cell proliferation and endothelial vascular tube disruption were examined. CHMp-13a cells were transplanted into nude mice and treated with CA4DP and/or tadalafil. CA4-induced inhibition of cell proliferation and disruption of the endothelial vascular tube were not affected by co-treatment with tadalafil, and the antitumor effects of CA4DP in xenograft mice were not reduced by co-administration of tadalafil. These results revealed that myocardial damage induced by CA4DP was attenuated by co-administration of tadalafil while maintaining antitumor efficacy.

Inhibitors of the Mechanistic Target of Rapamycin Can Ameliorate Bortezomib-Induced Peripheral Neuropathy.

Bortezomib, an anticancer drug for multiple myeloma and mantle cell lymphoma, causes severe adverse events and leads to peripheral neuropathy. The associated neuropathy limits the use of bortezomib and could lead to discontinuation of the treatment; therefore, effective intervention is crucial. In the present study, we statistically searched for a drug that could alleviate bortezomib-induced peripheral neuropathy using adverse event self-reports. We observed that specific inhibitors of the mechanistic target of rapamycin (mTOR) lowered the incidence of bortezomib-induced peripheral neuropathy. These findings were experimentally validated in mice, which exhibited long-lasting mechanical hypersensitivity after repeated bortezomib treatment. This effect was inhibited for hours after a systemic injection with rapamycin or everolimus in a dose-dependent manner. Bortezomib-induced allodynia was accompanied by the activation of spinal astrocytes, and intrathecal injection of mTOR inhibitors or an inhibitor of ribosomal protein S6 kinase 1, a downstream target of mTOR, exhibited considerable analgesic effects in a dose-dependent manner. These results suggest that mTOR inhibitors, which are readily available to patients prescribed bortezomib, are one of the most effective therapeutics for bortezomib-induced peripheral neuropathy.