小林 正典

Canine prostate cancer (cPCa) is a malignant neoplasm with no effective therapy. The BRAF V595E mutation, corresponding to the human BRAF V600E mutation, is found frequently in cPCa. Activating BRAF mutations are recognized as oncogenic drivers, and blockade of MAPK/ERK phosphorylation may be an effective therapeutic target against BRAF-mutated tumors. The aim of this study was to establish a novel cPCa cell line and to clarify the antitumor effects of MEK inhibitors on cPCa in vitro and in vivo. We established the novel CHP-2 cPCa cell line that was derived from the prostatic tissue of a cPCa patient. Sequencing of the canine BRAF gene in two cPCa cell lines revealed the presence of the BRAF V595E mutation. MEK inhibitors (trametinib, cobimetinib, and mirdametinib) strongly suppressed cell proliferation in vitro, and trametinib showed the highest efficacy against cPCa cells with minimal cytotoxicity to non-cancer COPK cells. Furthermore, we orally administered 0.3 or 1.0 mg/kg trametinib to CHP-2 xenografted mice and examined its antitumor effects in vivo. Trametinib reduced tumor volume, decreased phosphorylated ERK levels, and lowered Ki-67 expression in xenografts in a dose-dependent manner. Although no clear adverse events were observed with administration, trametinib-treated xenografts showed osteogenesis that was independent of dosage. Our results indicate that trametinib induces cell cycle arrest by inhibiting ERK activation, resulting in cPCa tumor regression in a dose-dependent manner. MEK inhibitors, in addition to BRAF inhibitors, may be a targeted agent option for cPCa with the BRAF V595E mutation. This article is protected by copyright. All rights reserved.

Vitamin D Receptor (VDR) expression is implicated in human Prostatic Carcinoma (PC), but its role in canine PC is unclear. To investigate how VDR expression is affected by age and castration in healthy dogs and how it changes with PC, we evaluated prostates from 8-month-old (n = 5) or 6-year-old (n = 8) intact males, 1-8-year-old healthy castrated males (n = 4) and 8-15-year-old castrated males (n = 7) with PC, by performing histopathology, immunohistochemistry, and ELISA. The results showed that VDR expression in canine prostate increased in an age-dependent manner and decreased after castration compared with intact dogs at 6 years of age. Castrated dogs with PC showed increased VDR expression compared with healthy castrated dogs and VDR expression in PC differed according to the pattern of tumor proliferation. The findings suggest that prostatic VDR expression may be a useful prognostic marker and therapeutic target for canine PC.

In this study, cauda epididymal sperm were collected from Amur leopard cats with various causes of death as well as Tsushima leopard cats that underwent castration surgery, and sperm quality was compared with that in domestic cats. A sufficient number of sperm similar to those in domestic cats could be collected from the cauda epididymis of Amur leopard cats. However, in old leopard cats, no or very few cauda epididymal sperm were recovered, although there were no differences in sperm motility and sperm abnormality. There were no significant differences in sperm quality immediately after collection and after freezing-thawing of cauda epididymal sperm compared with corresponding estimates in domestic cats.

Tolerance towards fetal alloantigens in the maternal immune system is essential for maintaining pregnancy. Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells characterized by their ability to suppress immune activity and maintain maternal-fetal immune tolerance. However, the mechanisms underlying MDSC induction have not been elucidated. Herein, we investigated the myeloid-derived suppressor cells (MDSCs) in the peripheral blood of pregnant canines and its induction mechanism. By analyzing the concentration of MDSCs in the peripheral blood of pregnant canines, elevation of MDSCs has been observed during pregnancy. In addition, MDSCs from pregnant canines inhibit T cell activation. These results suggest that the elevated MDSCs in canine pregnancy may contribute to reduces maternal immune activity. To clarify the cause of MDSCs elevation in canine pregnancy, we analyzed the relationship between pregnancy-related hormones (estradiol, progesterone, and relaxin) and MDSCs. Serum relaxin levels, but not estradiol and progesterone, were correlated with the ratio of monocyte MDSCs. Additionally, relaxin induced monocytic MDSCs as well as inhibited T cell activation in vitro. Therefore, relaxin contributes to the elevation of monocytic MDSCs in the peripheral blood of pregnant canines. Our findings highlight the novel role of relaxin in pregnancy and contribute to a better understanding of maternal-fetal immune tolerance.

Oxidative stress owing to an imbalance between reactive oxygen species and antioxidants, such as coenzyme Q10 (CoQ10), is a major contributor to male infertility. We investigated the effects of the reduced form of CoQ10 (ubiquinol) supplementation on semen quality in dogs with poor semen quality. Three dogs received 100 mg of ubiquinol orally once daily for 12 weeks. Semen quality, serum testosterone, and seminal plasma superoxide dismutase (SOD) activity were examined at 2-week intervals from 2 weeks before ubiquinol supplementation to 4 weeks after the treatment. Ubiquinol improved sperm motility, reduced morphologically abnormal sperm, and increased seminal plasma SOD activity; however, it had no effect on testosterone level, semen volume, and sperm number. Ubiquinol supplementation could be used as a non-endocrine therapy for infertile dogs.

In this study, we aimed to elucidate the utility of transrectal ultrasonography (TRUS) and blood lactate concentration (bLac) measurement to diagnose cows with severe uterine torsion. We investigated the association of TRUS and bLac measurement with macroscopic findings on laparotomy for severe uterine torsion in nine cows. We found that an increased ultrasonographic cross-sectional thickness (15-25 mm) and multiple hypoechogenic areas corresponded to macroscopic vascular compromise in the uterus on laparotomy. In addition, bLac was elevated (≥5.0 mmol/l) in cows showing uterine necrosis on laparotomy. A combined diagnostic approach with TRUS and bLac measurement enables assessment of the uterine vascular status and has utility for selecting the treatment option (including laparotomy) and predicting the outcomes.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

Contents In this study, sperms collected from the right and left cauda epididymis were grouped into having canine prostatic fluid (PF) sensitization or not diluted with egg yolk Tris-fructose citrate extender, and stored at 4 degrees C. The necessity of canine PF in cooled preservation was determined by elucidating the sperm quality after the storage. As a result, while there was no difference among all groups up to 48hr of storage, after storage for 96hr and more, a significantly lower sperm motility was observed in the group without being sensitized to PF than the groups with being sensitized to PF (p<.05, p<.01). Although sperm abnormality increased in all groups with increased storage time, the group without being sensitized to PF showed significantly higher sperm abnormality than did the groups with being sensitized to PF after storage for 24hr and more (p<.01). From these findings, we concluded that PF was necessary for the cooled preservation of the canine sperm because these sperms were protected from any effects of low temperatures by being sensitized to PF.

Canine prostate cancer (cPCa) is an untreatable malignant neoplasm resulting in local tissue invasion and distant metastasis. MicroRNAs (miRs) are small non-coding RNAs that function as oncogenes or tumor suppressors. The purpose of this study was to characterize the expression of miRs that are altered in cPCa tissue. The expression levels of 277 mature miRs in prostatic tissue (n=5, respectively) were compared between the non-tumor and tumor groups using real-time PCR. Five miRs (miR-18a, 95, 221, 222 and 330) were up-regulated, but 14 miRs (miR-127, 148a, 205, 299, 329b, 335, 376a, 376c, 379, 380, 381, 411, 487b and 495) were down-regulated specifically in cPCa (P<0.05). These miRs have potential use as early diagnosis markers for cPCa and in miR-based therapy.

In order to determine blood lactate concentrations (bLac) and their validity as a diagnostic marker in bovine uterine torsion, blood samples were taken from 54 Holstein cows with uterine torsion before the correction of torsion. bLac in a group of cows with and without uterine necrosis were 15.0 and 3.0 mmol/l, respectively (P<0.01). Moreover, bLac in a group of dead or culled dams and in that of survived dams were 10.2 and 3.1 mmol/l, respectively (P<0.01). Furthermore, the proposed diagnostic cutoffs for bLac based on ROC analysis for detection of uterine necrosis and poor prognosis in dams were set at >5.0 and >6.5 mmol/l, respectively. These findings suggest that in dairy cows with uterine torsion, an increase in bLac is a diagnostic predictor of uterine necrosis as well as poor prognosis in dams.

Equine and human chorionic gonadotropins were administered to two female Amur leopard cats to induce estrus and ovulation during non-breeding season. Fresh semen collected from male cats was surgically inseminated into the uterine horn of the females. In one animal, two fetal sacs without heartbeats were observed on abdominal ultrasonography 31 days after insemination, which indicated that embryo death had occurred. In the other animal, fetal heartbeats were detected in two fetal sacs 29 days after insemination, which confirmed as pregnancy. This animal delivered two newborns 68 days after insemination; the one of the kittens was assumed to be stillbirth, and the other grew normally. In this study, we successfully obtained a kitten from an Amur leopard cat by artificial breeding for the first time in Japan.

Canine embryos (8-cell to blastocyst stages) frozen-thawed using the slow-freezing method with glycerol (four recipients) or dimethyl sulfoxide (three recipients) as a cryoprotectant and vitrified-warmed using the Cryotop method (five recipients) were surgically transferred into the unilateral uterine horn of recipient bitches. As a result, the morphology of embryos frozen-thawed using the slow freezing method was judged to be normal, but no conception occurred in any of the recipient bitches. Two of the five bitches that received transferred embryos (morula to early blastocyst stages) vitrified-warmed using the Cryotop method became pregnant and produced normal pups (1/9 embryos, 11.1% and 1/6 embryos, 17.0%). It was concluded that the Cryotop method was more appropriate for canine embryo cryopreservation than the slow-freezing method, which is used for the cryopreservation of embryos of other mammalian species.

We collected semen from a male Amur leopard cat using the transrectal electroejaculation method and investigated the semen qualities for about four years. In addition, the influence of the season on the spermatogenic function of the Amur leopard cat was investigated with regard to the semen qualities, testicular volume and serum testosterone level. As a result, we could collect semen with good sperm qualities that would be useable for artificial insemination. Some seasonality was noted in the testicular volume and serum testosterone level. We clarified that the semen qualities were favorable before and during the female breeding season compared with those after the breeding season.

OBJECTIVE To histologically evaluate and compare features of myofibers within the elongated soft palate (ESP) of brachycephalic and mesocephalic dogs with those in the soft palate of healthy dogs and to assess whether denervation or muscular dystrophy is associated with soft palate elongation. SAMPLE Soft palate specimens from 24 dogs with ESPs (obtained during surgical intervention) and from 14 healthy Beagles (control group). PROCEDURES All the soft palate specimens underwent histologic examination to assess myofiber atrophy, hypertrophy, hyalinization, and regeneration. The degrees of atrophy and hypertrophy were quantified on the basis of the coefficient of variation and the number of myofibers with hyalinization and regeneration. The specimens also underwent immunohistochemical analysis with anti-neurofilament or anti-dystrophin antibody to confirm the distribution of peripheral nerve branches innervating the palatine myofibers and myofiber dystrophin expression, respectively. RESULTS Myofiber atrophy, hypertrophy, hyalinization, and regeneration were identified in almost all the ESP specimens. Degrees of atrophy and hypertrophy were significantly greater in the ESP specimens, compared with the control specimens. There were fewer palatine peripheral nerve branches in the ESP specimens than in the control specimens. Almost all the myofibers in the ESP and control specimens were dystrophin positive. CONCLUSIONS AND CLINICAL RELEVANCE These results suggested that palatine myopathy in dogs may be caused, at least in part, by denervation of the palatine muscles and not by Duchenne-or Becker-type muscular dystrophy. These soft palate changes may contribute to upper airway collapse and the progression of brachycephalic airway obstructive syndrome.

Four dogs with poor semen quality, low seminal plasma superoxide dismutase (SOD) activity and low blood plasma testosterone (T) levels were orally administered one vitamin E tablet containing 50 mg alpha-tocopheryl acetate per dog daily for 4 weeks. The mean values of semen quality were temporarily improved after the start of vitamin E treatment and the values of 4, and 5 weeks after that were significantly different from those before the treatment (P<0.05-0.001). The mean blood plasma T and seminal plasma SOD activity values slightly increased in the 4 dogs after the treatment. The results of the present study indicate that poor semen quality in dogs with low seminal plasma SOD can be improved by vitamin E treatment.

Canine epididymal sperm was collected from the cauda epididymis using 2 different methods (flushing and mincing) to compare the qualities (the percentage of progressively motile, viable, morphologically abnormal, immature and intact acrosomes) before and after freezing and thawing. No significant difference was noted in the quality of the cauda epididymal sperm immediately after collection and after freezing-thawing between the collection methods, although the mean levels of sperm quality with the flushing method were slightly better than that of the mincing method. The flushing method is simple and free of blood contamination, although the vas deferens was too small to be perfused in only 1 dog, and our results suggest that the flushing method is preferable to the mincing method for collecting sperm from the canine cauda epididymis.

Contents Human chorionic gonadotropin (hCG) is a glycoprotein used in the treatment of spermatogenic dysfunction. However, previous studies performed in dogs show that repeated administration of large doses of hCG produces antibodies against hCG. In this study, we examined the efficacy of low-dose injections of hCG in four male dogs with spermatogenic dysfunction and low plasma testosterone (T) levels. We administered 100IU hCG per animal, five times at 3-day intervals, and evaluated the changes in semen quality and plasma T levels. The total number of sperm in ejaculate, the percentage of progressively motile sperm and the plasma T levels had increased by 3-5weeks after the first injection of hCG in three of the four dogs, but were unchanged in the fourth dog. These findings indicate that temporary improvement of the semen quality of dogs with spermatogenic dysfunction and low plasma T levels is possible after five low-dose injections of hCG.

Superoxide dismutase (SOD) activity in flushings from oviducts and uterine horns of 8 anestrous, 5 estrous and 7 diestrous bitches was measured. SOD activity in oviductal fluid in estrous bitches was significantly higher than that in anestrous and diestrous bitches (P<0.01). SOD activity in uterine fluid of diestrous bitches was, however, significantly higher than that in anestrous and estrous bitches (P<0.01). Additionally, sperm collected from normal dogs were incubated in MEM and in MEM containing SOD (SOD-MEM) for 24 hr. The percentages of sperm with viability, motility and hyperactivation in SOD-MEM were higher than those in MEM. SOD produced in oviduct and uterus may be able to maintain or improve sperm quality and fertility in the dog.

The objective of the present study was to determine an optimum temperature and extender for short-term transport of canine ejaculated semen. There was no significant difference in the qualities of semen diluted with two kinds of extender, egg yolk Tris-citrate fructose (EYT-FC) or glucose (EYT-GC) extender, between the 2, 8 or 12 and the 4°C control groups during storage for up to 48 hr, while the 16-24°C groups showed decreased sperm motility during storage for 48 hr. However, the 2°C group showed slightly lower sperm motility and slightly higher sperm abnormality than the 4°C group. Therefore, we concluded that semen qualities can be maintained for up to 48 hr when canine semen samples are extended with EYT-FC or EYT-GC and stored at a temperature in the range of 4-12°C. © 2014 The Japanese Society of Veterinary Science.

© Medwell Journals, 2014. The purpose of this study is to reveal the effect of estrogen-inducing SOD activity on the endometrium of domestic rabbits. Researchers measured Superoxide Dismutase (SOD) activity to investigate the reason of occurrence of popular uterine adenocarcinoma in rabbits in estradiol-17β-stimulated endometrium epithelial cell culture. Then, we also examined the distribution of cells expressing Estrogen Receptor α (ERα)-like Immunoreactivites (ERα-like-IR) in the uterus prone to developing adenocarcinoma. The addition of estradiol-17β to the primary culture of endometrial epithelial cells obviously decreased SOD activity. Moreover, the areas of endometrium in which uterine glands were confined in control rabbits and notably larger in rabbits with glandular hyperplasia of the uterus compared in normal dog uterus that the occurrence of uterine adenocarcinoma is rare. ERα-like-IR cells were found in all proliferating glands in rabbits with hyperplasia.

Superoxide dismutase (SOD) has a key role in protecting1 cells from Reactive Oxygen Species (ROS). Cancer cells produce more ROS involved in proliferation and survival of cancer cells. We, therefore, investigated the percentage of castrated dogs with Prostatic Adenocarcinoma (PA) and the relation between the occurrence of PA in the dog and the prostatic SOD activity after castration. Between 2005 and 2012, diagnosis of Prostatic Adenocarcinoma (PA) was made in 24 dogs at the Veterinary Teaching Hospital of Nippon Veterinary and Life Science University. The percentage of castrated dogs in the 24 dogs with PA and the relation between the occurrence of PA in the dog and the prostatic superoxide dismutase (SOD) activity after castration were examined. Prostatic parenchyma specimens were collected from 6 healthy dogs just before castration and 6 months after castration to measure prostatic SOD activity with a SOD assay kit. Nineteen (79.2%) of the 24 dogs diagnosed with PA had been castrated at 1-6 (2.7±1.7) years of age. Their prostatic SOD activity was significantly lower after castration than before castration (p&lt 0.01). The results of this study indicated that castration could not prevent PA from occurring and the reduction of prostatic SOD activity after castration might lead to oncogenesis in PA of the dog. © 2014 Academic Journals Inc.

In healthy male dogs, peripheral plasma testosterone (T), prostaglandin E-2 (PGE(2)) and seminal plasma PGE(2) levels were measured before, during and after ejaculation, and semen quality was examined after oral administration of PGE(2). Plasma T and PGE(2) levels did not change during these periods, but the seminal plasma PGE(2) level of combined the first and second fractions was significantly higher than those at 0-5 and 5-10 min after the start of ejaculation of the third fraction. Semen volume but not quality increased after PGE(2) administration. In conclusion, large amounts of PGE(2) are released from the prostate gland during the early part of ejaculation, and PGE(2) plays an essential role in secretion of seminal plasma.

The molecular mechanism of muscle degeneration in a lethal muscle disorder Duchene muscular dystrophy (DMD) has not been fully elucidated. The dystrophic dog, a model of DMD, shows a high mortality rate with a marked increase in serum creatine kinase (CK) levels in the neonatal period. By measuring serum CK levels in cord and venous blood, we found initial pulmonary respiration resulted in massive diaphragm damage in the neonates and thereby lead to the high serum CK levels. Furthermore, molecular biological techniques revealed that osteopontin was prominently upregulated in the dystrophic diaphragm prior to the respiration, and that immediate-early genes (c-fos and egr-1) and inflammation/immune response genes (IL-6, IL-8, COX-2, and selectin E) were distinctly overexpressed after the damage by the respiration. Hence, we segregated dystrophic phases at the molecular level before and after mechanical damage. These molecules could be biomarkers of muscle damage and potential targets in pharmaceutical therapies.

Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6-8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD.

A Beagle dog (3 years old) that ejaculated high percentages (mean +/- SE: 29.1 +/- 1.2%) of sperm with a knobbed acrosome abnormality and a low number of sperm and that also had a low plasma testosterone (T) level was given 10 subcutaneous injections of 1 mu g of gonadotropin-releasing hormone analogue (GnRH-A) at 3-day intervals. The plasma T level and number of sperm increased 12-14 weeks after the first injection. Although the percentages of sperm with knobbed acrosome abnormality did not change after the GnRH-A therapy, the number of sperm and percentage of actively motile sperm increased after the therapy, and a bitch gave birth to 5 healthy puppies after intravaginal artificial insemination with fresh semen collected 14 weeks after the first injection.

The fertility was compared between ejaculated and cauda epididymal sperm sensitized with prostatic fluid in dog after freeze-thawing using the fertility of ova from the contralateral ovary after injection (2 x 10(8) sperm) into dog uterus on the unilateral ovariectomized side, on the basis of the presence or absence of conception. No significant difference was observed in sperm quality after freeze-thawing between the two groups and conception rates were equivalent and low. Therefore, to achieve a high fertility by intrauterine insemination of canine frozen-thawed ejaculated and cauda epididymal sperm, intrauterine insemination on both sides is recommended, rather than insemination with a lot of sperm of the uterine horn on one side.

A spontaneous epileptic model of cats has not been described previously. Recently, we identified familial epileptic cats and investigated their clinical features. These epileptic cats are healthy except for the presence of recurrent seizures that are typically a focal limbic seizure with secondary generalization. Furthermore, generalized seizures were induced by vestibular stimulation in some cats. This spontaneous epileptic cat strain may be a valuable model for idiopathic/genetic epilepsy. (C) 2010 Elsevier B.V. All rights reserved.

Hippocampal atrophy, which is a component of hippocampal sclerosis and recognized commonly in human intractable epilepsy, is controversial in canine epilepsy. We examined the hippocampal volume in 58 epileptic dogs and 35 control dogs using magnetic resonance (MR) images, and calculated the relative hippocampal volume asymmetry of the right and left hippocampus. Subjectively, there were visible MR imaging abnormalities in seven of the 58 epileptic dogs (12%). The hippocampel volume asymmetric ratio of epileptic dogs (5.84 +/- 4.47%) was significantly greater than that of control dogs (1.62 +/- 0.88%). Using a cutoff threshold asymmetric ratio of 6% that is indicated in human epilepsy, 28 epileptic dogs (48%) were characterized as having unilateral hippocampus atrophy. The hippocampal volume asymmetry ratio cannot be used to detect bilateral atrophy. In conclusion, although less frequent than that observed in human epilepsy patients, hippocampal atrophy may occur in canine epilepsy. (C) 2010 Veterinary Radiology & Ultrasound, Vol. 51, No. 5, 2010, pp 485-490.

Duchenne muscular dystrophy (DMID) is a devastating muscle disorder that is characterized by progressive muscle necrosis, fibrosis, and fatty infiltration. To examine the temporospatial pathological changes, a noninvasive evaluation method such as magnetic resonance imaging (MRI) is needed. The aim of this study was to precisely assess muscle necrosis and inflammation based on a sequence of T2-weighted imaging (T2WI), gadolinium-enhanced imaging, and selective fat suppression, chemical shift selective T2-weighted imaging (CHESS-T2WI), on a 3.0-Tesla MRI unit in 3-month-old and 7-year-old dogs with canine X-linked muscular dystrophy (CXMDJ), a suitable animal model for DMD. The results show that CHESS-T2WI was more sensitive and useful from the early to late stages of CXMDJ than T2WI or contrast enhancement imaging in the evaluation of muscle necrosis, because these latter sequences can be influenced by fatty infiltration or interstitial connective tissues. Muscle Nerve 40: 815-826, 2009

Objective: Duchenne muscular dystrophy (DMD) is caused by the inability to produce dystrophin protein at the myofiber membrane. A method to rescue dystrophin production by antisense oligonucleotides, termed exon-skipping, has been reported for the mdx mouse and in four DMD patients by local intramuscular injection. We sought to test efficacy and toxicity of intravenous oligonucleotide (morpholino)-induced exon skipping in the DMD dog model. Methods: We tested a series of antisense drugs singly and as cocktails, both in primary cell culture, and two in vivo delivery methods (intramuscular injection and systemic intravenous injection). The efficiency and efficacy of multiexon skipping (exons 6-9) were tested at the messenger RNA, protein, histological, and clinical levels. Results: Weekly or biweekly systemic intravenous injections with a three-morpholino cocktail over the Course of 5 to 22 weeks induced therapeutic levels of dystrophin expression throughout the body, with an average of about 26% normal levels. This was accompanied by reduced inflammatory signals examined by magnetic resonance imaging and histology, improved or stabilized timed running tests, and clinical symptoms. Blood tests indicated no evidence of toxicity. Interpretation: This is the First report of widespread rescue of dystrophin expression to therapeutic levels in the dog model of DMD. This study also provides a proof of concept for systemic multiexon-skipping therapy. Use of cocktails of morpholino, as shown here, allows broader application of this approach to a greater proportion of DMD patients (90%) and also offers the prospect of selecting deletions that optimize the Functionality of the dystrophin protein.

With the eventual goal of making zonisamide (ZNS), a relatively new antiepileptic drug, available for the treatment of epilepsy in cats, the pharmacokinetics after a single oral administration at 10 mg/kg and the toxicity after 9-week daily administration of 20 mg/kg/day of ZNS were Studied in healthy cats. Pharmacokinetic parameters obtained with a single administration of ZNS at 10 mg/day were as follows: C(max) = 13.1 mu g/ml; T(max) = 4.0 h; T(1/2) = 33.0 h; areas under the curves (AUCs) - 720.3 mu g/ml h (values represent the medians). The Study with daily administrations revealed that the toxicity of ZNS was comparatively low in cats, suggesting that it may be an available drug for cats. However, half of the cats that were administered 20 mg/kg/day daily showed adverse reactions Such as anorexia, diarrhoea, vomiting, somnolence and locomotor ataxia. (C) 2008 ESFM and AAFP. published by Elsevier Ltd. All rights reserved.

A male six-year-old Pomeranian showed recurrent seizures and progressive left hemiparesis. MRI revealed a mass in the right paramedian frontal-temporal region with hyperintensity on T1-weighted (T1W) and mixed-intensity on T2-weighted (T2W) images. After gadolinium enhancement, the mass was enhanced homogenously and demonstrated the dural tail sign. Surgical resection of mass was performed and its histological diagnosis was meningioma. The meningioma of this case had a high cellular density with some lipid contents and intra- and extra-tumor hemorrhage, however, calcification was not found. These cellular characteristics may contribute to a higher signal intensity on T1W imaging.

This case report documents clinical and molecular findings in two littermate kittens of the Japanese domestic cat with GM2 gangliosidosis variant 0. Analysis included detailed physical, magnetic resonance imaging, biochemical, pathological and genetic examinations. At first, these littermate kittens showed typical cerebellar signs at approximately 2 months of age. About 2 months later, they progressively showed other neurological signs and subsequently died at about 7 months of age. Magnetic resonance imaging just before the death showed an enlarged ventricular system, T1 hyperintensity in the internal capsule, and T2 hyperintensity in the white matter of the whole brain. Histological findings suggested a type of lysosomal storage disease. Biochemical studies demonstrated that the kittens were affected with GM2 gangliosidosis variant 0, and a DNA assay finally demonstrated that these animals were homozygous for the mutation, which the authors had identified in a different family of the Japanese domestic cat. The findings in the present cases provide useful information about GM2 gangliosidosis variant 0 in Japanese domestic cats.