Human Muse cells reduce myocardial infarct size and improve cardiac function without causing arrythmias in a swine model of acute myocardial infarction.

BACKGROUND: We recently reported that multilineage-differentiating stress enduring (Muse) cells intravenously administered after acute myocardial infarction (AMI), selectively engrafted to the infarct area, spontaneously differentiated into cardiomyocytes and vessels, reduced the infarct size, improved the left ventricular (LV) function and remodeling in rabbits. We aimed to clarify the efficiency of Muse cells in a larger animal AMI model of mini-pigs using a semi-clinical grade human Muse cell product. METHOD AND RESULT: Mini-pigs underwent 30 min of coronary artery occlusion followed by 2 weeks of reperfusion. Semi-clinical grade human Muse cell product (1x107, Muse group, n = 5) or saline (Vehicle group, n = 7) were intravenously administered at 24 h after reperfusion. The infarct size, LV function and remodeling were evaluated by echocardiography. Arrhythmias were evaluated by an implantable loop recorder. The infarct size was significantly smaller in the Muse group (10.5±3.3%) than in the Vehicle group (21.0±2.0%). Both the LV ejection fraction and fractional shortening were significantly greater in the Muse group than in the Vehicle group. The LV end-systolic and end-diastolic dimensions were significantly smaller in the Muse group than in the Vehicle group. Human Muse cells homed into the infarct border area and expressed cardiac troponin I and vascular endothelial CD31. No arrhythmias and no blood test abnormality were observed. CONCLUSION: Muse cell product might be promising for AMI therapy based on the efficiency and safety in a mini-pig AMI.

Urinary liver-type fatty acid-binding protein in two dogs with acquired Fanconi syndrome: A case report.

Background: Fanconi syndrome (FS) is defined as multiple defects of the proximal tubules and is diagnosed by clinical symptoms. However, in dogs with FS, the damage in the proximal tubules that is responsible for the clinical symptoms has not been evaluated. Among FS cases, tubular damage in acquired FS is reversible following the elimination of a causative factor. Liver-type fatty acid-binding protein (L-FABP) is a biomarker of tubular damage in various animals including dogs. Urinary L-FABP measurement may be useful for the diagnosis and follow-up evaluation in canine FS. Case Description: At the first visit, two Toy Poodles that had no remarkable findings on physical examination presented with glycosuria without hyperglycemia, hypokalemia, hyperchloremia, increased levels of plasma alkaline phosphatase, and metabolic acidosis. Considering all the factors involved, the dogs were clinically diagnosed with acquired FS. The owner reported that they routinely fed the dog with chicken jerky, a recently considered cause of acquired FS. Following the withdrawal of the jerky, abnormalities including glycosuria improved in both dogs. Moreover, urinary L-FABP levels, which were high at diagnosis, presented a decreasing trend during the follow-up. However, in one dog, the elevated urinary L-FABP level did not return to normal. Conclusion: Although the clinical symptoms of acquired FS in dogs could be improved by the elimination of a causative factor, the severity of tubular damage described by urinary L-FABP may not be necessarily linked to the degree of functional deterioration. Therefore, the evaluation of proximal tubular damage by L-FABP may be of clinical value during the follow-up of acquired FS in canines.

Effect of cyclooxygenase-2 inhibitors at therapeutic doses on body temperature during anesthesia in healthy dogs administered with amino acids.

In healthy dogs, amino acid infusion significantly attenuates the decrease in body temperature during anesthesia by facilitating insulin secretion, suggesting that such an increase in insulin secretion is related to increased heat production. In dogs, selective cyclooxygenase-2 (COX-2) inhibitors, which are used for pain relief in veterinary medicine, possess anti-pyretic action. And, in mice and humans, selective COX-2 inhibitors increase insulin secretion and sensitivity. Therefore, treatment with COX-2 inhibitors may negate or accelerate the attenuating effect on decreased body temperature during anesthesia by amino acid infusion. In the present study, influences on insulin secretion and body temperature by treatment with meloxicam or robenacoxib at therapeutic dose were evaluated in healthy dogs. Treatment with meloxicam or robenacoxib did not affect insulin secretion in the unanesthetized and anesthetized dogs, and did not affect body temperature and heart rate under the anesthetized condition with amino acid infusion. In conclusion, COX-2 inhibitors at therapeutic doses did not affect body temperature during anesthesia in dogs administered amino acids.

A case of prostate B-cell lymphoma recognized in the contents of an irreducible perineal hernia in a dog.

We present a case of an intact 14-year-old male dog with a prostate B-cell lymphoma recognized in the contents of an irreducible perineal hernia. The enlarged prostate was replaced after reducing its size by partial excision, and the perineal hernia was repaired using the tunica vaginalis communis. However, the pelvic cavity was largely occupied by the replaced prostate, and urinary retention developed. The prostate was resected on the next day via abdominal median incision, and a bladder-urethra anastomosis was performed. Lymphoma has rarely been demonstrated to develop in the prostate, with the lesion comprising the hernia contents. The present study suggests the necessity of early treatment for perineal hernias and the possibility that lymphoma may be present in the hernia contents.

Fluorescein sodium-guided resection of intracranial lesions in 22 dogs.

OBJECTIVE: To evaluate the safety of an intraoperative fluorescein sodium (FS) injection and elucidate the relationships between the MRI findings, pathological diagnoses, and intraoperative staining characteristics of intracranial lesions in 22 dogs. STUDY DESIGN: Prospective case series. ANIMALS: Twenty-two dogs with intracranial lesions. METHODS: FS was intravenously administered to 22 dogs undergoing craniotomy for suspected intracranial tumors to evaluate perioperative and postoperative adverse effects. The intensities and patterns of gadolinium (Gd) enhancement on preoperative magnetic resonance imaging and intraoperative FS staining were graded, and their relationship was evaluated. Intraoperative FS staining characteristics and pathological diagnoses were compared. RESULTS: The only adverse effect noted was repetitive vomiting in 2 dogs. The intensities and patterns between preoperative Gd enhancement and intraoperative FS staining appeared to agree. High-grade glioma and histiocytic sarcoma had more intense FS staining. Lesions with strong Gd enhancement, including meningiomas and choroid plexus tumors, also had intense FS staining. CONCLUSION: The intraoperative use of FS is a simple and safe technique to guide the resection of intracranial lesions in dogs. The findings on Gd enhancement, FS staining, and histopathology appeared to agree but require validation in a larger set of cases. CLINICAL SIGNIFICANCE: The intraoperative use of FS may improve the prognosis of dogs with brain tumors by guiding the resection of lesions.

Vertebral replacement for the treatment of vertebral osteosarcoma in a cat.

A 7-year-old cat was referred with pelvic limb ataxia. Radiography and CT revealed bone resorption of the L1 vertebral arch, and myelography identified a compressive extradural lesion. The mass was surgically removed and histopathologically diagnosed as giant cell osteosarcoma. Three years later, the recurrent tumor resection and vertebral fixation were performed. Six months later, vertebrectomy was performed to radically excise the recurrent mass and a titanium spinal cage was placed. The cat is alive approximately 5 years after the first surgery. This case report describes vertebrectomy and vertebral body replacement as a radical treatment for feline vertebral osteosarcoma.

Selection of broad-spectrum cephalosporin-resistant Escherichia coli in the feces of healthy dogs after administration of first-generation cephalosporins.

Although antimicrobial products are essential for treating diseases caused by bacteria, antimicrobial treatment selects for antimicrobial-resistant (AMR) bacteria. The aim of this study was to determine the effects of administration of first-generation cephalosporins on development of resistant Escherichia coli in dog feces. The proportions of cephalexin (LEX)-resistant E. coli in fecal samples of three healthy dogs treated i.v. with cefazolin before castration and then orally with LEX for 3 days post-operation (PO) were examined using DHL agar with or without LEX (50 µg/mL). LEX-resistant E. coli were found within 3 days PO, accounted for 100% of all identified E. coli 3-5 days PO in all dogs, and were predominantly found until 12 days PO. LEX-resistant E. coli isolates on DHL agar containing LEX were subjected to antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) genotyping, ß-lactamase typing and plasmid profiling. All isolates tested exhibited cefotaxime (CTX) resistance (CTX minimal inhibitory concentration ≥4 µg/mL). Seven PFGE profiles were classified into five groups and three ß-lactamase combinations (blaCMY-4 -blaTEM-1 , blaTEM-1 -blaCTX-M-15 and blaTEM-1 -blaCTX-M-15 -blaCMY-4 ). All isolates exhibited identical PFGE profiles in all dogs on four days PO and subsequently showed divergent PFGE profiles. Our results indicate there are two selection periods for AMR bacteria resulting from the use of antimicrobials. Thus, continuing hygiene practices are necessary to prevent AMR bacteria transfer via dog feces after antimicrobial administration.

Changes in respiratory function in Pembroke Welsh Corgi dogs with degenerative myelopathy.

Canine degenerative myelopathy (DM) is characterized by progressive degeneration of the spinal cord. Although atrophic changes in the intercostal muscles were previously reported in the late stage of DM in Pembroke Welsh Corgis (PWCs), changes in respiratory function have not yet been examined. In the present study, we performed an arterial blood gas analysis and measured respiratory movements over progressive disease stages to document changes in respiratory function in DM-affected PWCs. We found that respiratory dysfunction progressed during the later stages of DM and correlated with a change in respiratory movement to the abdominal breathing pattern. These results suggested that hypoventilation occurred due to dysfunctional changes in the intercostal muscles and resulted in hypoxemia in the later stages of DM.

Intravenous infusion of amino acids in dogs attenuates hypothermia during anaesthesia and stimulates insulin secretion.

OBJECTIVE: To evaluate the effect of intravenous infusion of amino acids on the prevention of hypothermia during anaesthesia in dogs. STUDY DESIGN: Randomized experimental trial. ANIMALS: Seven healthy Beagle dogs. METHODS: Four concentrations of amino acids were prepared with a 10% amino acid solution and an acetated Ringer's solution, and dogs were infused with each of the solutions at 1 week intervals. Dogs were infused with amino acid solution at 12 mL kg(-1)  hour(-1) for 60 minutes before and for 60 minutes after induction of anaesthesia. Acetated Ringer's solution was infused at the same rate for the remaining 60 minutes of anaesthesia. The infusion treatments were: 1) A0, nutrient-free acetated Ringer's solution; 2) A6, 0.6 g kg(-1)  hour(-1) ; 3) A9, 0.9 g kg(-1)  hour(-1) ; and 4) A12, 1.2 g kg(-1) hour(-1) . Rectal temperature (RT), heart rate (HR), mean arterial pressure (MAP), blood insulin, glucose, urea nitrogen (BUN) and creatinine concentrations, and time to extubation were measured. RESULTS: Before anaesthesia, RT was not affected by amino acid infusion. RT decreased progressively during anaesthesia and the absolute values of RT from 30 to 120 minutes were significantly higher in A12 than in A0 (p < 0.05). Reductions in HR and MAP during anaesthesia were attenuated by amino acid infusion in a dose-dependent manner. Plasma insulin concentration was significantly higher in A12 than in A0 during amino acid infusion and the increase in insulin concentration was greater during than before anaesthesia. BUN increased during amino acid infusion in a dose- and time-dependent fashion. Time until extubation was shorter in A12 than in A0. CONCLUSIONS AND CLINICAL RELEVANCE: Amino acids infused at 1.2 g kg(-1)  hour(-1) in dogs attenuated the decrease in RT, HR, and MAP during anaesthesia, and induced a significant increase in plasma insulin concentration.

Molecular cloning of feline resistin and the expression of resistin, leptin and adiponectin in the adipose tissue of normal and obese cats.

Resistin, one of the adipokines that has a cycteine-rich C-terminus, is considered to relate to the development of insulin resistance in rats. However, in cats, there is little knowledge regarding resistin. In this study, we cloned the feline resistin cDNA from adipose tissue by RT-PCR. The feline resistin clone contained an entire open reading frame encoding 107 amino acids that had 72.8%, 75.4%, 50.9% and 51.8% homology with bovine, human, mouse and rat homologues, respectively. In both subcutaneous and visceral adipose tissues, the transcription levels of feline resistin mRNA were significantly higher in obese cats than normal cats, and those of feline adiponectin mRNA were significantly lower in obese cats than normal cats. However, there was no difference in the expression of feline leptin between normal and obese cats. On the other hand, in both normal and obese cats, there were no significant differences in resistin, leptin and adiponectin mRNA levels between subcutaneous and visceral adipose tissues. In cats, the altered expression of resistin and adiponectin mRNA with obesity may contribute to the pathogenesis of insulin resistance and subsequent diabetes mellitus. In addition to feline adiponectin, the feline resistin cDNA clone obtained in this study will be useful for further investigation of the pathogenesis of obesity in cats.

Gallbladder lymphoma in a miniature dachshund.

A 7-year-old, miniature dachshund was referred for examination and treatment of persistent anorexia, deep yellow-coloured urine and leucocytosis. The clinical sign of jaundice, results from a serum biochemistry profile and ultrasonographic images suggested a biliary tract obstruction. A cholecystectomy was performed to remove the obstruction. Histopathological assessment of the resected gallbladder and partial common bile duct indicated diffuse large B-cell lymphoma. Twelve days after the initial operation, a second procedure was performed due to bile leakage into the abdominal cavity. Chemotherapy was administered twice after the second operation but discontinued, because the dog showed adverse effects. The dog is still alive 24 months after the surgery. To the authors' knowledge, this is the first description of canine gallbladder lymphoma.

Body and major organ sizes of young mature microminipigs determined by computed tomography.

To understand the anatomical characteristics of microminipigs, one of the smallest miniature pigs, as a large animal model, we measured the body and organ sizes of four-, five-, six-, and seven-month-old microminipigs (n = 4, females) using computed tomography. In addition, the results were compared with those of young mature beagles (10 months old, two males and three females), which have been widely used as a large animal model. The microminipigs at 4-6 months of age were much smaller than the beagles. However, when the microminipigs reached seven months of age, their overall size was similar to that of the beagles. The thoracic cavity volume of the seven-month-old microminipigs was less than half that of the beagles, and the cavity was largely filled by the heart. The liver size of the seven-month-old microminipigs was approximately half of that of the beagles. Moreover, the spleen of the seven-month-old microminipigs was different in morphology, but not different in size from that of the beagles. In addition, although their volumes were the same, the kidneys of the seven-month-old microminipigs, unlike those of the beagles, were flattened in shape. Collectively, the major abdominal organs of the seven-month-old microminipigs were either the same size or smaller than those of the beagles, but the abdominal cavity volume of the seven-month-old microminipigs was larger than that of the beagles. Thus, the abdominal cavity of microminipigs is assumed to be filled with the gastrointestinal tract. The anatomical characteristics of the young mature microminipigs revealed in our study suggest that microminipigs could have great potential as a large animal model for biomedical research.

Involvement of nuclear factor of activated T cells in granulocyte-macrophage colony-stimulating factor production in canine keratinocytes stimulated with a cysteine protease.

BACKGROUND: A previous study demonstrated that the cysteine protease of Dermatophagoides farinae induced production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a canine epidermal keratinocyte progenitor cell line (CPEK); however, the molecular mechanism has not been elucidated. HYPOTHESIS/OBJECTIVES: Given that the transcription of GM-CSF mRNA in human lymphocytes is mainly regulated by the nuclear factor of activated T cells (NFAT), it is hypothesized that NFAT also contributes to GM-CSF production in canine keratinocytes stimulated with a cysteine protease. METHODS: Nuclear translocation of NFAT was evaluated in CPEK cells in the absence or presence of the cysteine protease papain. We also investigated whether blockade of NFAT could inhibit GM-CSF production. RESULTS: Papain-induced nuclear translocation of NFAT, producing GM-CSF, was partly inhibited by ciclosporin. CONCLUSIONS AND CLINICAL IMPORTANCE: The results suggest that GM-CSF production mediated by the cysteine protease is regulated not only by NFAT but also by unknown signalling pathways in canine keratinocytes.

Efficacy of fosfomycin on Escherichia coli isolated from bitches with pyometra.

The aim of this study was to determine the antimicrobial resistance of Escherichia coli isolated from the uteri of bitches with pyometra, and 38 E. coli isolates were used. The antimicrobials used were ampicillin (ABPC), amoxicillin/clavulanic acid, gentamicin, minocycline, cefazolin, levofloxacin (LVFX), trimethoprim-sulfamethoxazole (ST) and fosfomycin (FOM). Resistance to ABPC occurred most frequently, followed by LVFX and ST. Multi-drug resistance, defined as resistance against 3 or more classes of antimicrobials, was found in 23.7% of all isolates. Nine out of 13 resistant strains were multi-drug resistant, but no strain was found to be resistant to FOM. This suggests that FOM should be administered for E. coli from pyometra.

Portal vein aneurysm in a dog.

Portal vein aneurysm (PVA) is a rare abnormal dilatation of the portal vein, which has not been reported in dogs. We describe the findings of ultrasound and computed tomography in a case of PVA in a young male toy poodle, with the final diagnosis established by explorative surgical observation. The dog had an aneurysmal fusiform dilatation in the extrahepatic portal vein with portal hypertension and multiple portsystemic shunts. This is the first report of canine PVA.

Production of GM-CSF mediated by cysteine protease of Der f in canine keratinocytes.

House dust mite (HDM) allergens are the most common allergens for induction of IgE-mediated hypersensitivity. Recently, epicutaneous sensitization with HDM allergens has been emphasized in the development of atopic dermatitis (AD) by producing various soluble factors in keratinocytes. Among the soluble factors, GM-CSF is a key molecule that activates Langerhans cells, antigen-presenting cells in the epidermis. In the present study, we investigated the effects of Dermatophagoides farinae (Der f) on GM-CSF production in a canine keratinocyte cell line, CPEK. CPEKs were found to produce GM-CSF upon stimulation by Der f. The GM-CSF production was suppressed by addition of a cysteine protease inhibitor. The present results suggest that cysteine protease-derived Der f may be an initiator of allergic inflammation by inducing the production of GM-CSF in keratinocytes.

Gene transcription analysis in lesional skin of canine epitheliotropic cutaneous lymphoma using quantitative real-time RT-PCR.

Canine epitheliotropic cutaneous lymphoma (cECL) is characterized by infiltration of neoplastic lymphocytes in the skin with a specific tropism for the epidermis. Migration of lymphocytes is strictly controlled by interactions between chemokines and chemokine receptors, which may be involved in the pathogenesis of cECL. In this study, we investigated mRNA transcription levels of several chemokines (CCL17, CCL19, CCL21, CCL22, CCL27, CCL28 and CXCL10) and chemokine receptors (CCR4, CCR7, CCR10 and CXCR3) in lesional skin of cECL by quantitative real-time RT-PCR. To examine the subsets of accumulating neoplastic lymphocytes, we also investigated transcription levels of type-1 (IFN-γ, IL-12p35, IL-12p40 and LT-α) and type-2 (IL-4 and IL-13) cytokines and cytotoxic markers (perforin and granzyme B). We found that the lesional skin had higher mRNA transcription of CCL19, CXCL10, CCR4, CCR7, CCR10 and CXCR3 and lower transcription of CCL27 than healthy dog skin (p<0.05). In addition, transcription levels of type-1 cytokine and cytotoxic markers in lesional skin were significantly higher than those in healthy dog skin. These results indicate that the transcription of some chemokines and chemokine receptors, which are necessary for skin-homing, epitheliotropism and peripheral segregation of T-cells, is upregulated in the lesional skin of cECL. In addition, our results also indicate that the subset of neoplastic lymphocytes in cECL is most likely type-1 cytotoxic T-cells.

Suitable reference genes for quantitative real-time rt-pcr in total RNA extracted from canine whole blood using the PAXgene™ system.

Since blood cells produce various soluble factors like cytokines or chemokines, gene expression analysis in whole blood could be important to investigate disease pathogenesis. In gene expression analysis with quantitative real-time RT-PCR, accurate determination of relative mRNA transcription levels requires appropriate reference genes. To identify the optimal reference gene in canine whole blood, we compared transcription levels of twelve candidate reference genes in total RNA extracted using the PAXgene system. The stability of the reference gene was evaluated by three different statistical programs, GeNorm, Normfinder and Bestkeeper. The results indicated that SDHA, CG14980 and TBP were the most stably expressed genes, which can be used as optimal reference genes for gene expression analysis in canine whole blood.

Effect of recombinant canine interferon-γ on granulocyte-macrophage colony-stimulating factor, transforming growth factor-ß and CC chemokine ligand 17 mRNA transcription in a canine keratinocyte cell line (CPEK).

Recombinant canine interferon-γ (rCaIFN-γ) produced by a baculovirus expression system has therapeutic efficacy against atopic dermatitis in dogs. Although the mechanism of action of rCaIFN-γ is not completely understood, rCaIFN-γ is thought to downregulate the activity of interleukin-4- and interleukin-5-producing T helper 2 cells. However, rCaIFN-γ may also act directly on canine keratinocytes by inhibiting the release of inflammatory mediators. In this study, we investigated the effects of rCaIFN-γ on cytokine and chemokine mRNA transcription in a canine keratinocyte cell line, CPEK. It was found that granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA transcription was significantly inhibited after treatment with rCaIFN-γ (P<0.001), whereas transforming growth factor-ß and CC chemokine ligand 17 mRNA levels were unchanged. This study suggests that rCaIFN-γ may suppress GM-CSF production from canine keratinocytes, although further studies are required to confirm this.

Identification of the signaling pathway of TNF-α-induced CCL17/TARC transcription in a canine keratinocyte cell line.

A CC chemokine, CCL17/TARC, has been shown to be a factor in the immunopathogenesis of canine atopic dermatitis (cAD). In canine keratinocytes, the transcription of CCL17 mRNA is preferentially induced by tumor necrosis factor-alpha (TNF-α); however, its regulatory mechanism has not been elucidated. The aim of the present study is to clarify the regulatory mechanism of TNF-α-induced CCL17 mRNA transcription in canine keratinocytes leading to the development of a chemokine-targeted therapy for cAD. In a cell line of canine epidermal keratinocyte, CPEK, stimulation with TNF-α induced not only the activation of nuclear factor-kappa B (NF-κB) but also the phosphorylation of c-Jun-N-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38). Extracellular signal-regulated kinase (ERK) was found to be constitutively phosphorylated, which was temporarily augmented by TNF-α. Results of the inhibition assay indicated that the CCL17 mRNA transcription level was significantly decreased by p38 inhibitors but was not altered by either JNK or NF-κB inhibitors. Surprisingly, the ERK inhibitor increased the transcription level of CCL17 mRNA. Stimulation with epidermal growth factor (EGF), an ERK activator, suppressed the transcription of CCL17 mRNA. The present results suggest that TNF-α-induced CCL17 mRNA transcription in CPEK is positively regulated by p38 but negatively controlled by ERK.