A de novo nonsense variant in the DMD gene associated with X-linked dystrophin-deficient muscular dystrophy in a cat.

BACKGROUND: X-linked dystrophin-deficient muscular dystrophy (MD) is a form of MD caused by variants in the DMD gene. It is a fatal disease characterized by progressive weakness and degeneration of skeletal muscles. HYPOTHESIS/OBJECTIVES: Identify deleterious genetic variants in DMD by whole-genome sequencing (WGS) using a next-generation sequencer. ANIMALS: One MD-affected cat, its parents, and 354 cats from a breeding colony. METHODS: We compared the WGS data of the affected cat with data available in the National Center for Biotechnology Information database and searched for candidate high-impact variants by in silico analyses. Next, we confirmed the candidate variants by Sanger sequencing using samples from the parents and cats from the breeding colony. We used 2 genome assemblies, the standard felCat9 (from an Abyssinian cat) and the novel AnAms1.0 (from an American Shorthair cat), to evaluate genome assembly differences. RESULTS: We found 2 novel high-impact variants: a 1-bp deletion in felCat9 and an identical nonsense variant in felCat9 and AnAms1.0. Whole genome and Sanger sequencing validation showed that the deletion in felCat9 was a false positive because of misassembly. Among the 357 cats, the nonsense variant was only found in the affected cat, which indicated it was a de novo variant. CONCLUSION AND CLINICAL IMPORTANCE: We identified a de novo variant in the affected cat and next-generation sequencing-based genotyping of the whole DMD gene was determined to be necessary for affected cats because the parents of the affected cat did not have the risk variant.

Successful treatment of sclerosing encapsulating peritonitis in a cat using bioresorbable hyaluronate-carboxymethylcellulose membrane after surgical adhesiolysis and long-term prednisolone.

Case summary: An 8-year-old neutered male domestic shorthair indoor cat was presented with an 8-week history of intermittent vomiting, anorexia and weight loss that had been unresponsive to supportive treatment. Abdominal ultrasound revealed plication of the small intestine and fluid accumulation proximal to the lesion, and a linear foreign body was suspected. An exploratory celiotomy showed cocoon-like encapsulation of the entire intestine. Surgical adhesiolysis and full-thickness biopsy were performed, and histopathologic examination revealed mild thickening of the visceral peritoneum with fibrin deposition, as well as mild neutrophil and lymphocyte infiltration. These findings were compatible with sclerosing encapsulating peritonitis (SEP). The cat recovered well postoperatively and was discharged the next day. Prednisolone was administered for 7 weeks to prevent recurrence of SEP. Five months after surgery, the cat was re-presented with anorexia and chronic vomiting. Based on the clinical examination findings, recurrent SEP was suspected. At the second surgery, surgical adhesiolysis was repeated and a bioresorbable hyaluronate-carboxymethylcellulose membrane was used to cover the serosal surface and thus prevent adhesion formation. Histopathologic findings of the peritoneal biopsy specimen confirmed SEP. Long-term prednisolone treatment (1 mg/kg for the first dose and 0.5 mg/kg every 48 h for maintenance) was administered postoperatively. The cat survived for more than 1239 days without recurrence. Relevance and novel information: To our knowledge, this is the first report of SEP in a cat with long-term survival. The use of a bioresorbable hyaluronate-carboxymethylcellulose membrane and long-term prednisolone treatment may have prevented short-term and long-term recurrence, respectively, in this case.

Anaplastic oligodendroglioma with nasal invasion and systemic metastasis in a dog.

An 11-year-old spayed female French bulldog was referred on suspicion of nasal tumor. Anaplastic oligodendroglioma in the olfactory bulb that was suspected to have invaded the nasal cavity was diagnosed from imaging and histopathology. Metastasis to cervical lymph nodes was suspected, with no other metastases identified. The brain-to-nasal lesion and lymph nodes were treated with hypo-fractionated radiation therapy. Nasal congestion soon resolved. About 3 months later, follow-up computed tomography revealed multiple hepatic and splenic masses, which were cytologically suspected as metastatic oligodendroglioma. Nimustine, followed by toceranib phosphate, seemed to have no effect, and the dog died on day 167. Postmortem examination revealed the primary tumor disappearance and systemic metastases. Canine oligodendroglioma can grow outside the cranial vault, and systemically metastasize.

A case of immature teratoma with nephroblastic components in the oral cavity of a cat.

A 2-month-old female mixed cat was emaciated due to dysphagia, and inspection of the mouth revealed a 2 cm pedunculated mass elongated from the palate, which occupied the oral cavity. The mass was surgically removed, and histopathological examination revealed that the tumor was composed of three germ cell layers: ectodermal (skin and skin appendages), mesodermal (cartilaginous and osseous structures), and endodermal (glandular and respiratory mucosa) tissues. An immature teratoma was diagnosed because of the presence of immature neuroectodermal tissues, and the presence of nephroblastic components was a characteristic finding in this case. This is the first report of an oropharyngeal teratoma in cats and the first case of an immature teratoma with nephroblastic components in a domestic species.

Impact of near-infrared fluorescence imaging with indocyanine green on the surgical treatment of pulmonary masses in dogs.

Objectives: To investigate the intraoperative identification and complete resection of pulmonary masses, and to evaluate lymph node metastasis of pulmonary malignant tumors in dogs using indocyanine green (ICG) fluorescence imaging. Methods: Forty dogs with pulmonary masses were included, all of which underwent surgical treatment. ICG fluorescence imaging was performed on pulmonary masses before lobectomy and the resection margins after lobectomy. In addition, ICG fluorescence of the excised masses and lymph nodes was evaluated in the shaded box. The fluorescence findings were compared with the histopathological diagnosis. Results: Of 44 nodules resected from 40 dogs, 32 nodules were histopathologically diagnosed as lung adenocarcinoma, five were histiocytic sarcoma, three were undifferentiated sarcoma, two were malignant epithelial tumor metastases, one was carcinosarcoma, and one was a non-neoplastic lesion. Fluorescence was observed in all nodules. In addition to the main lesion, other fluorescent nodules were found in four dogs. Regarding the diagnostic accuracy of complete resection based on ICG fluorescence, the sensitivity was 67.7% and the specificity was 60.0%. The sensitivity and specificity of ICG fluorescence for the diagnosis of lymph node metastasis were 100 and 75.0%, respectively. Conclusions: ICG fluorescence imaging might be a useful intraoperative diagnostic method to identify the location of tumors and lymph node metastasis, but not to evaluate complete tumor resection, in dogs with pulmonary malignant tumors.

Effectiveness of Brexpiprazole in a Patient With Bipolar Disorder and Comorbid Persistent Genital Arousal Disorder/Genito-Pelvic Dysesthesia: A Case Report.

Although the symptoms of persistent genital arousal disorder/genito-pelvic dysesthesia (PGAD/GPD) can have negative impacts on patients' lives, it is an under-recognized clinical entity. We describe the case of a 61-year-old Japanese female who suffered simultaneously from bipolar disorder and PGAD/GPD. She developed PGAD/GPD approx. 10 years after being diagnosed with bipolar disorder. Despite 20 years of various drug treatments, her bipolar disorder and PGAD/GPD symptoms showed little improvement. She had also undergone multiple sessions of cognitive behavioral therapy (CBT) and mindfulness, nerve block, botulinum toxin injections, and laser treatment for PGAD/GPD. Her PGAD/GPD symptoms remained with no significant improvement, and her bipolar disorder symptoms had also not responded well to medication. With the administration of brexpiprazole, she achieved remission of her bipolar disorder. Her PGAD/GPD symptoms also eventually improved. When PGAD/GPD is comorbid with bipolar disorder, the improvement of bipolar disorder may also lead to relief of PGAD/GPD symptoms. This case reveals that brexpiprazole, which has a unique profile, may be effective for PGAD/GPD.

Clinical impact of near-infrared fluorescence imaging with indocyanine green on surgical treatment for hepatic masses in dogs.

BACKGROUND: Near-infrared fluorescence imaging using indocyanine green (ICG) is clinically applied to intraoperatively identify hepatic masses in humans. In addition, it is reported to be effective for assessing complete resection in human hepatocellular carcinoma (HCC). However, there is limited information on ICG fluorescence imaging for canine HCC, and its clinical usefulness is still unclear. Therefore, the purpose of this study was to evaluate the intraoperative identification and status of surgical margin for canine hepatic masses using near-infrared ICG fluorescence imaging. This clinical study included 104 dogs with hepatic masses. Between 12 and 24 h prior to surgery, ICG solution was injected intravenously at a dose of 0.5 mg/kg. The fluorescence intensity and pattern of each hepatic mass was investigated using an infrared camera before resection. After resection, the fluorescence intensity of the resection margin was also investigated. The resected masses were histopathologically diagnosed and compared using ICG fluorescence imaging. RESULTS: One hundred and twenty-two masses obtained from 104 dogs included 76 HCCs, 16 hepatocellular adenomas, 12 focal nodular hyperplasias, and 18 other lesions. Of the 122 masses, 106 (94 partial, 9 whole, and 3 ring fluorescence patterns), 7, and 9 masses showed increased, the same, or decreased fluorescence compared to the normal liver tissue, respectively. The fluorescence intensity and pattern were not significantly related to the histopathological diagnosis. The sensitivity and specificity of the margin evaluation in the 47 dogs were 100% and 77.3%, respectively. The median survival times in cases of HCC with complete and incomplete resection were 914 and 254 days, respectively. The median survival time of patients with a complete resection was significantly longer than that of patients with a incomplete resection (p = 0.043). CONCLUSION: ICG fluorescence imaging has potential clinical value for the identification and margin evaluation of canine hepatic masses. Although it is difficult to use fluorescence imaging for the differential diagnosis of liver tumours, it may be useful for assessing complete resection in cases of hepatic masses demonstrating increased fluorescence in dogs, and complete resection of HCC could have a survival benefit.

Adenomatous Hyperplasia of Bowman's Capsule Epithelium in a Dog with Metastatic Nasal and Hepatic Neuroendocrine Carcinoma.

A 12-year-old spayed Shiba dog with a nasal neuroendocrine carcinoma and multiple hepatic nodules was necropsied. Histologically, proliferated blast cells with a monolayer or multilayered structure were observed in the kidney. This blast cell proliferation extended from Bowman's capsule epithelium to the proximal tubule in approximately 3% of nephrons. Immunohistochemistry revealed that blast cells were positive for vimentin, Wilm's tumour protein 1 (WT1), paired box 2 (PAX2) and CD10, but negative for cytokeratin (CK) AE1/AE3, CK19, CAM5.2, synaptophysin and chromogranin A. On the basis of these findings, adenomatous hyperplasia of Bowman's capsule epithelium was diagnosed. Multiple yellowish‒white nodules (1-3 cm) were found in the liver and diagnosed as neuroendocrine carcinoma with metastases to the lungs, adrenal glands and pancreaticoduodenal lymph nodes.

Clinical, diagnostic, and pathologic features and surgical outcomes of combined hepatocellular-cholangiocarcinoma in dogs: 14 cases (2009-2021).

OBJECTIVE: To describe the clinical, diagnostic, and pathological features and postoperative prognosis of canine combined hepatocellular-cholangiocarcinoma (cHCC-CCA). ANIMALS: 14 privately owned dogs that underwent surgical treatment. PROCEDURES: The medical records, including signalment, clinical signs, blood test, urine analysis, computed tomography (CT) findings, intraoperative findings, and pathological findings, were retrospectively reviewed in the dogs with cHCC-CCA. RESULTS: Of 306 dogs that underwent surgical removal of hepatic masses, 14 dogs (4.6%) were pathologically confirmed to have cHCC-CCA. Median age and body weight were 11.3 years and 7.3 kg, respectively. There were no specific clinicopathological findings for cHCC-CCA. CT revealed a massive hepatic mass in all dogs and the inclusion of cyst-like lesions within the mass in 13 dogs. Intrahepatic metastases were found at time of surgery in 2 dogs (14.3%). Of the residual 12 dogs, 1 dog showed postoperative formation of intrahepatic nodules suggestive of metastases and another had intrahepatic and pulmonary nodules and a forelimb skin mass, suggesting postoperative metastases. The median survival time of the patients with cHCC-CCA was 700 days (range, 10 to 869 days) after surgery. CLINICAL RELEVANCE: To the authors' knowledge, this is the first study to describe the clinical, diagnostic, and pathological features and postoperative prognosis of canine cHCC-CCA. The clinical and diagnostic features of canine cHCC-CCA might be more similar to those of HCC rather than to those of CCA, but the preoperative diagnosis differentiating between HCC and cHCC-CCA was challenging. Our study suggests that the postoperative prognosis of canine patients with cHCC-CCA is similar to that of dogs with HCC.

Required Elements in tRNA for Methylation by the Eukaryotic tRNA (Guanine-N2-) Methyltransferase (Trm11-Trm112 Complex).

The Saccharomyces cerevisiae Trm11 and Trm112 complex (Trm11-Trm112) methylates the 2-amino group of guanosine at position 10 in tRNA and forms N2-methylguanosine. To determine the elements required in tRNA for methylation by Trm11-Trm112, we prepared 60 tRNA transcript variants and tested them for methylation by Trm11-Trm112. The results show that the precursor tRNA is not a substrate for Trm11-Trm112. Furthermore, the CCA terminus is essential for methylation by Trm11-Trm112, and Trm11-Trm112 also only methylates tRNAs with a regular-size variable region. In addition, the G10-C25 base pair is required for methylation by Trm11-Trm112. The data also demonstrated that Trm11-Trm112 recognizes the anticodon-loop and that U38 in tRNAAla acts negatively in terms of methylation. Likewise, the U32-A38 base pair in tRNACys negatively affects methylation. The only exception in our in vitro study was tRNAValAAC1. Our experiments showed that the tRNAValAAC1 transcript was slowly methylated by Trm11-Trm112. However, position 10 in this tRNA was reported to be unmodified G. We purified tRNAValAAC1 from wild-type and trm11 gene deletion strains and confirmed that a portion of tRNAValAAC1 is methylated by Trm11-Trm112 in S. cerevisiae. Thus, our study explains the m2G10 modification pattern of all S. cerevisiae class I tRNAs and elucidates the Trm11-Trm112 binding sites.

Granulomatous polyarthritis caused by Talaromyces georgiensis in a dog.

A 6-y-old, 3.5-kg, spayed female Toy Poodle was presented with left forelimb lameness of 2-d duration. Two months before the initial presentation, radiography showed osteolysis of the medial epicondyle of the left humerus, and the left forelimb was amputated. Grossly, the articular villi of the elbow joint were markedly thickened, and the articular cartilage surfaces of the distal humerus and proximal radius had partial erosion. Histologically, granulomatous arthritis and osteomyelitis characterized by the presence of abundant macrophages containing numerous fungi were observed. ITS and ß-tubulin sequences amplified from the isolate from the specimen were 100% and 99% identical to type strain UTHSC D16-145T of Talaromyces georgiensis, respectively. Canine osteoarthritis caused by T. georgiensis has not been reported previously, to our knowledge.

Fibrocartilaginous embolism of the cervical spinal cord and cerebellum in a cat.

A 9-year-old castrated male cat presented with sudden onset of paralysis in four limbs. Magnetic resonance imaging revealed an intramedullary lesion at C3, and lesions in the vermis and right hemisphere of the cerebellum, which were hyperintense on T2-weighted imaging. The cat was euthanized, and postmortem examination was performed. Grossly, transverse sectioning of the spinal cord revealed areas of malacia within the gray matter extending from C2 to C3. Histologically, the ventral spinal artery, the spinal intramedullary arteries and the arterioles of arachnoid in the cerebellum contained fibrocartilaginous emboli confirmed by metachromatic staining with toluidine blue. To our knowledge, this is the first report of fibrocartilaginous embolism involving both the cerebellum and cervical spinal cord in a cat.

Clinical significance of the two-base insertion mutation in the TP53 gene in canine histiocytic sarcoma.

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from dendritic cells or macrophages. We previously reported high incidence of the two-base (AT) insertion mutation (insAT) in the tumor protein p53 (TP53) gene in dogs with HS, and the aim of this study was to investigate the clinical significance of insAT in canine HS. The present study established a sensitive digital PCR-based assay for detecting insAT and examined its associations with clinical variables and survival time. The mutation was detected in 26 of 64 dogs (41%), and the mean mutant allele frequency was 1.9% (range, 0.014-35%), indicating that not all tumor cells harbor insAT. The incidence of insAT was significantly higher in dogs with metastatic lesions than in those without metastatic lesions. However, the existence of insAT was not associated with survival time or response to chemotherapy with lomustine or nimustine. This study suggested that HS cells might acquire insAT in the TP53 gene during development of metastasis, but insAT was not a prognostic factor in canine HS. Further studies are needed to investigate the contribution of insAT to the development of metastatic lesions of canine HS.

Detection of α-defensin in eosinophils in helminth-infected mouse model.

α-defensin is a potent antimicrobial peptide secreted from intestinal mucosal epithelial cells, such as Paneth cells, and affects not only bacteria but also parasites and fungi. Recently, human eosinophils have also been shown to produce α-defensin, but no studies have been done on other animals. In this study, we attempted to detect α-defensin protein in mouse eosinophils infiltrating the intestinal mucosa during a helminth infection using Zamboni fixation and immunohistochemistry. Most of the eosinophils infiltrating the intestinal mucosa during helminth infection were positive for α-defensin. The expression level of α-defensin mRNA was 50 fold that in the control. Meanwhile, the number of Paneth cells was doubled, and their α-defensin fluorescence intensity was increased. These results suggested that eosinophils are also important producers of α-defensin, such as Paneth cells in mice, and that α-defensin produced from eosinophils might be involved in defensive mechanisms against helminths. Moreover, the experimental system used in this study is a good model to study the generation of α-defensin by eosinophils.

A retrospective study of disease incidence in African pygmy hedgehogs (Atelerix albiventris).

The African pygmy hedgehog (Atelerix albiventris) is becoming a popular pet in Japan. The aim of this study was to determine the incidence of various diseases in African pygmy hedgehogs. We histologically investigated 105 samples from 100 privately-owned pet African pygmy hedgehogs that were submitted to two laboratories (North Lab and Patho Labo) between 2012 and 2017. Tissues submitted for this study were taken from female reproductive organs (33 cases; 31.43%), skin (20 cases; 19.05%), and the oral mucosa (19 cases; 18.1%). The most common histological diagnoses included endometrial stromal nodules identified as benign uterine neoplasia (14 cases; 13.33%); endometrial polyps identified as non-neoplastic polyps (7 cases; 6.67%), gingival hyperplasia and chronic suppurative inflammation in the oral mucosa (11 cases; 10.48%), fibrosarcomas in the skin (8 cases; 7.62%), and mammary tumors (8 cases; 7.62%). In this study, lymphoma and oral squamous cell carcinoma were less common than in the previous reports. The present study revealed the disease prevalence in captive African pygmy hedghogs that were histopathologically examined.

Eosinophil depletion suppresses radiation-induced small intestinal fibrosis.

Radiation-induced intestinal fibrosis (RIF) is a serious complication after abdominal radiotherapy for pelvic tumor or peritoneal metastasis. Herein, we show that RIF is mediated by eosinophil interactions with α-smooth muscle actin-positive (α-SMA+) stromal cells. Abdominal irradiation caused RIF especially in the submucosa (SM) of the small intestine, which was associated with the excessive accumulation of eosinophils in both human and mouse. Eosinophil-deficient mice showed markedly ameliorated RIF, suggesting the importance of eosinophils. After abdominal irradiation, chronic crypt cell death caused elevation of extracellular adenosine triphosphate, which in turn activated expression of C-C motif chemokine 11 (CCL11) by pericryptal α-SMA+ cells in the SM to attract eosinophils in mice. Inhibition of C-C chemokine receptor 3 (CCR3) by genetic deficiency or neutralizing antibody (Ab) treatment suppressed eosinophil accumulation in the SM after irradiation in mice, suggesting a critical role of the CCL11/CCR3 axis in the eosinophil recruitment. Activated α-SMA+ cells also expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) to activate eosinophils. Transforming growth factor-ß1 from GM-CSF-stimulated eosinophils promoted collagen expression by α-SMA+ cells. In translational studies, treatment with a newly developed interleukin-5 receptor α-targeting Ab, analogous to the human agent benralizumab, depleted intestinal eosinophils and suppressed RIF in mice. Collectively, we identified eosinophils as a crucial factor in the pathogenesis of RIF and showed potential therapeutic strategies for RIF by targeting eosinophils.

A newly developed ultraminiature wearable electromyogram system useful for analyses of masseteric activity during the whole day.

PURPOSE: We describe the characteristics of a new data-logger-type ultraminiature electromyogram (EMG) system (FLA-500-SD) and methods used for recording and we show its potential in clinical applications by presenting an example of a clinical case. METHOD: FLA contains electrodes, an amplifier, 12-bit analog-to-digital (A/D) converter at a sampling frequency of 1kHz, 16-bit CPU, a 3.7-V coin-shaped lithium battery, and a micro SD card. The size of FLA is 37.0×23.5×8.6mm, and its weight is 6g (9g with a battery inserted). The device is wearable and patients can attach the device and operate it by themselves in daily life. Data recorded in the micro SD card are transferred to a personal computer and analyzed. Although the device is ultraminiature and wearable, it has the capacity for recording a precise and clear masseteric surface electromyogram that is not inferior to that recorded by conventional stationary-type EMG recording systems. CONCLUSIONS: To our knowledge, the device is the smallest and lightest device with capacity for the longest consecutive measuring time as a data-logger-type electromyograph with built-in electrodes and memory. The device is useful for analyses of masseteric activity during the whole day. In the future, it is expected that applications of the device will expanded to observation, evaluation and diagnosis of normal or abnormal gnathic functions, e.g., assessment of sleep and awake bruxism and observation of the chewing state in daily life.

Elimination of eosinophils using anti-IL-5 receptor alpha antibodies effectively suppresses IL-33-mediated pulmonary arterial hypertrophy.

Interleukin (IL)-5 is a critical regulator of eosinophils and a therapeutic target for asthma. The administration of anti-IL-5 or anti-IL-5 receptor (IL-5R) antibodies has been shown to reduce eosinophil counts and ameliorate asthmatic symptoms in studies on animal models of allergy as well as in human clinical trials. In order to explore other potential clinical uses of IL-5R antibodies, we used an animal model of IL-33-mediated pulmonary arterial hypertrophy. We first generated chimeric monoclonal antibodies against the mouse IL-5 receptor α chain (IL-5Rα), which comprised an Fc region from human IgG1 and a Fab region from a previously established anti-mouse IL-5Rα monoclonal antibody. To investigate the role of antibody-dependent cell-mediated cytotoxicity (ADCC), chimeric antibodies that lacked ADCC were prepared. These antibodies recognized IL-5Rα to the same extent as the ADCC-sufficient antibodies. Administration of chimeric antibodies with ADCC resulted in the elimination of eosinophils from the lung and thus suppressed the development of arterial hypertrophy. This effect was attenuated in mice treated with antibodies lacking ADCC. Taken together, the results of this study provided a potential use for anti-IL-5Rα antibodies in the treatment of arterial hypertrophy, which leads to pulmonary hypertension.

Experiments on laser cleaning of sooted optical windows.

To develop laser-ignition technology, transparent glass plates were artificially sooted and irradiated repetitively by laser from the front (sooted) and back sides separately. Generally, the back-side irradiation was more effective at soot removal. However, the cleaning effect was saturated after thousands of laser shots. Although the saturated soot quantity was a decreasing function of the laser fluence per pulse, its magnitude remained the same for both front-side and back-side irradiations. In examining several soot-removal mechanisms proposed so far, it was found that the aerodynamic force produced by the flow induced by the laser heating of the soot was the most plausible mechanism.

Rhythm and amplitude of rhythmic masticatory muscle activity during sleep in bruxers - comparison with gum chewing.

OBJECTIVES: The aim of this study was to elucidate characteristics of rhythmic masticatory muscle activity (RMMA) during sleep by comparing masseteric EMG (electromyogram) activities of RMMA with gum chewing. METHOD: The parts of five or more consecutive phasic bursts in RMMA of 23 bruxers were analyzed. Wilcoxon signed-rank test for matched pairs and Spearman's correlation coefficient by the rank test were used for statistical analysis. RESULTS: Root mean square value of RMMA phasic burst was smaller than that during gum chewing, but correlates to that of gum chewing. The cycle of RMMA was longer than that of gum chewing due to the longer burst duration of RMMA, and variation in the cycles of RMMA was wider. DISCUSSION: These findings suggest that the longer but smaller EMG burst in comparison with gum chewing is one of the characteristics of RMMA. The relation between size of RMMA phasic bursts and gum chewing is also suggested.