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.

An evolutionary medicine perspective on the cetacean pulmonary immune system - The first identification of SP-D and LBP in the bottlenose dolphin (Tursiops truncatus).

Evolutionary medicine expresses the present status of biomolecules affected by past evolutionary events. To clarify the whole picture of cetacean pneumonia, which is a major threat to cetaceans, their pulmonary immune system should be studied from the perspective of evolutionary medicine. In this in silico study, we focused on cetacean surfactant protein D (SP-D) and lipopolysaccharide-binding protein (LBP) as two representative molecules of the cetacean pulmonary immune system. Sequencing and analyzing SP-D and LBP in the bottlenose dolphin (Tursiops truncatus) lung and liver tissue collected post-mortem elucidated not only basic physicochemical properties but also their evolutionary background. This is the first study to report the sequences and expression of SP-D and LBP in the bottlenose dolphin. Besides, our findings also suggest the direction of an evolutionary arms race in the cetacean pulmonary immune system. These results have important positive implications for cetacean clinical medicine.

Agarose gel electrophoresis pattern of serum creatine kinase and lactate dehydrogenase isoenzymes in zoo-managed Asian elephants (Elephas maximus).

Serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH) isoenzymes were evaluated in nine zoo-managed Asian elephants (Elephas maximus) using a commercial agarose gel electrophoresis (AGE) kit. CK was separated into two major fractions, CK-BB and CK-MM, along with a small fraction of macroenzyme-CK type 2 (mCK2); CK-MM was the largest fraction. LDH was separated into five fractions (LDH1-5); LDH3 was the largest fraction. Age was negatively and positively correlated with the percentages of CK-BB and CK-MM, respectively, and negatively correlated with CK-BB and mCK2 activities. These results indicate that an AGE kit can be used to evaluate CK and LDH isoenzymes. Routine isoenzyme testing may enable early detection of disease and physiological changes.

Distribution of enrofloxacin within the bronchoalveolar region of healthy pigs.

This study aimed to elucidate the distribution of enrofloxacin (ERFX) within the bronchoalveolar region of pigs. Six clinically healthy pigs were allocated to intramuscular treatments with either a single dose of 5 mg/kg or 7.5 mg/kg ERFX. Samples of plasma and bronchoalveolar lavage fluid (BALF) were obtained from each pig 0 (before administration), 3, 8, and 24 hr after ERFX administration. The ERFX concentrations in pulmonary epithelial lining fluid (ELF) and BALF cells showed a similar pattern during the experimental period, whereby ERFX concentrations in both ELF and BALF cells were higher than those in the plasma. These results suggest that intramuscularly injected ERFX is well-distributed in the bronchoalveolar region.

Genetic characterization of bovine respiratory syncytial viruses in Japan between 2017 and 2019.

Bovine respiratory syncytial virus (BRSV) strains that were detected in Kagoshima prefecture and isolated in Hokkaido between 2017 and 2019, together with a BRSV vaccine strain, were subjected to full-genome sequencing. The BRSV strains identified in Japan were found to be genetically close to each other but distant from the vaccine strains. The deduced amino acids at positions 206 and 208 of the glycoprotein (G protein), which form one of the major epitopes of the recent Japanese BRSV strains, were different from those of the vaccine strains. Therefore, the recent Japanese BRSV strains might be antigenically different from the BRSV vaccine strains.

The serum activities of alkaline phosphatase isoenzymes measured using two approved methods in zoo-managed Asian elephants (Elephas maximus).

The approved Japanese measurement method of circulating alkaline phosphatase (ALP) has changed from that of the Japan Society of Clinical Chemistry (JSCC) to that of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). We measured the serum levels of total ALP (t-ALP) and those of the isoenzymes ALP2 and ALP3 in 50 Asian elephant (Elephas maximus) specimens using both methods. The activities determined by the IFCC method were roughly one-third lower than those determined by the JSCC method. We present conversion formulae. Our results enable comparisons of historical and current data on serum ALP activities in endangered, zoo-managed Asian elephants.

Flow cytometric analysis of bronchoalveolar lavage fluid immune dynamics in calves.

Understanding the immune dynamics in the respiratory mucosa of calves is necessary for a good management of bovine respiratory disease. Immune dynamics in the respiratory mucosa in humans and experimental animals has been assessed by flow cytometric analysis of bronchoalveolar lavage fluid (BALF); however, few reports have addressed this subject in calves. The aim of this study was to establish a universal method to analyze bronchoalveolar lavage fluid (BALF) by flow cytometry and to obtain basic knowledge of bovine respiratory mucosal immune dynamics. We investigated the immune cell populations in BALF and evaluated the surface antigen expression of alveolar macrophages in calves using flow cytometer. To further analyze the surface antigen variation observed in alveolar macrophages in detail, stimulation assays were performed in vitro. BALF cells were separated into three distinct populations based on their light scatter plot, which were considered to be macrophages, lymphocytes, and neutrophils. In most individuals, most of the BALF immune cells were alveolar macrophages, but an increased proportion of lymphocytes and neutrophils was observed in some individuals. Analysis of each surface antigen expression in alveolar macrophages showed that CD21 and MHC class II expression changed in response to changes in the leukocyte population. Moreover, when alveolar macrophages were stimulated with interferon-γ in vitro, the expression of CD21 was drastically reduced and MHC class II was increased, suggesting that functional changes in alveolar macrophages themselves are involved in the immune dynamics.

Pharmacokinetics in plasma and alveolar regions of a healthy calf intramuscularly administered a single dose of orbifloxacin.

This study analyzed the pharmacokinetics of orbifloxacin (OBFX) in plasma, and its migration and retention in epithelial lining fluid (ELF) and alveolar cells within the bronchoalveolar lavage fluid (BALF). Four healthy calves received a single dose of OBFX (5.0 mg/kg) intramuscularly. Post-administration OBFX dynamics were in accordance with a non-compartment model, including the absorption phase. The maximum concentration (Cmax) of plasma OBFX was 2.2 ± 0.1 µg/ml at 2.3 ± 0.5 hr post administration and gradually decreased to 0.3 ± 0.2 µg/ml at 24 hr following administration. The Cmax of ELF OBFX was 9.3 ± 0.4 µg/ml at 3.0 ± 2.0 hr post administration and gradually decreased to 1.2 ± 0.1 µg/ml at 24 hr following administration. The Cmax of alveolar cells OBFX was 9.3 ± 2.9 µg/ml at 4.0 hr post administration and gradually decreased to 1.1 ± 0.2 µg/ml at 24 hr following administration. The half-life of OBFX in plasma, ELF, and alveolar cells were 6.9 ± 2.2, 7.0 ± 0.6, and 7.8 ± 1.6 hr, respectively. The Cmax and the area under the concentration-time curve for 0-24 hr with OBFX were significantly higher in ELF and alveolar cells than in plasma (P<0.05). These results suggest that OBFX is distributed and retained at high concentrations in ELF and alveolar cells at 24 hr following administration. Hence, a single intramuscular dose of OBFX (5.0 mg/kg) may be an effective therapeutic agent against pneumonia.

Osteochondral regeneration of the femoral medial condyle by using a scaffold-free 3D construct of synovial membrane-derived mesenchymal stem cells in horses.

BACKGROUND: Medical interventions for subchondral bone cysts in horses have been extensively studied. This study investigated the regeneration of articular cartilage and subchondral bone with scaffold-free three-dimensional (3D) constructs of equine synovial membrane-derived mesenchymal stem cells (SM-MSCs) isolated from three ponies and expanded until over 1.0 × 107 cells at passage 2 (P2). RESULTS: SM-MSCs were strongly positive for CD11a/CD18, CD44, and major histocompatibility complex (MHC) class I; moderately positive for CD90, CD105, and MHC class II; and negative for CD34 and CD45 on flow cytometry and differentiated into osteogenic, chondrogenic, and adipogenic lineages in the tri-lineage differentiation assay. After culturing SM-MSCs until P3, we prepared a construct (diameter, 6.3 mm; height, 5.0 mm) comprising approximately 1920 spheroids containing 3.0 × 104 cells each. This construct was confirmed to be positive for type I collagen and negative for type II collagen, Alcian blue, and Safranin-O upon histological analysis and was subsequently implanted into an osteochondral defect (diameter, 6.8 mm; depth, 5.0 mm) at the right femoral medial condyle. The contralateral (left femoral) defect served as the control. At 3 and 6 months after surgery, the radiolucent volume (RV, mm3) of the defects was calculated based on multiplanar reconstruction of computed tomography (CT) images. Magnetic resonance (MR) images were evaluated using a modified two-dimensional MR observation of cartilage repair tissue (MOCART) grading system, while macroscopic (gross) and microscopic histological characteristics were scored according to the International Cartilage Repair Society (ICRS) scale. Compared to the control sites, the implanted defects showed lower RV percentages, better total MOCART scores, higher average gross scores, and higher average histological scores. CONCLUSIONS: Implantation of a scaffold-free 3D-construct of SM-MSCs into an osteochondral defect could regenerate the original structure of the cartilage and subchondral bone over 6 months post-surgery in horses, indicating the potential of this technique in treating equine subchondral bone cysts.

Interferon gamma, lipopolysaccharide, and modified-live viral vaccines stimulation alter the mRNA expression of tumor necrosis factor α, inducible nitric oxide synthase, and interferon ß in bovine alveolar macrophages.

To understand the pathogenesis of bovine respiratory disease (BRD), it is necessary to elucidate the mechanisms of alveolar macrophage regulation by cytokines and pathogen-associated molecular patterns (PAMPs). Moreover, "non-specific effects (NSEs)" an innate immune regulatory mechanism in response to vaccines containing PAMPs, has recently attracted attention. It may be applied to BRD control, but there is limited knowledge in bovine. To investigate this, we stimulated alveolar macrophages in vitro with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid sodium salt (Poly I:C), interferon gamma (IFN-γ), and modified-live viral (MLV) vaccines, respectively, and analyzed changes in tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), and interferon beta (IFN-ß) mRNA expression levels. mRNA expression levels of TNF-α, iNOS, and IFN-ß were significantly increased in bovine alveolar macrophages stimulated by IFN-γ and MLV vaccine; LPS, IFN-γ, and MLV vaccine; and MLV vaccine only, respectively. Additionally, all MLV vaccine-stimulated mRNA expression increases were observed in a concentration-dependent manner. These results revealed in part, the mechanism of bovine alveolar macrophage regulation by cytokines and PAMPs. Understanding the regulatory mechanisms of alveolar macrophages will contribute to understanding the pathogenesis of BRD and preventive and therapeutic BRD management based on NSEs.

Primary sinonasal malignant melanoma with systemic metastasis in a non-gray horse.

A 27-y-old Anglo-Arabian gelding with bay coat color was presented with a swelling of the left maxillary region. Fenestration on the left maxilla revealed that the left maxillary sinus was filled with black-red tissue. A portion of the tissue was excised and diagnosed histologically as malignant melanoma. Genotyping of the STX17 gene for gray coat color revealed that the horse did not have the "gray" factor. The horse was euthanized ~3 mo after first presentation. During autopsy, a black-to-gray mass extended from the left nasal cavity to the surrounding paranasal sinus and invaded the hard palate, cribriform plate, and the cranial portion of the left olfactory bulb. Moreover, identical black nodules were present in lymph nodes from the mandible to the larynx, and in the spleen, liver, kidney, and adrenal glands. However, masses were not found in the skin, perineal region, or pelvic cavity. All of the black-to-gray nodules were malignant melanomas that were histologically identical to the initial biopsy; tumor emboli were also found in the kidney. Sinonasal mucosal melanoma is a rare disease in horses.

Effect of Beta-Carotene Supplementation on the Peripheral Blood Leukocyte Population in Japanese Black Calves.

The purpose of this study was to determine the effect of beta-carotene supplementation to Japanese Black calves on the peripheral blood leukocyte population. Twenty-two Japanese Black calves were alternately assigned to two groups. Eleven calves received 20 mg/d of beta-carotene orally from 2 to 8 wk of age (BC group), and the other 11 calves did not receive (control group). The serum beta-carotene concentration in the BC group at 4, 8 and 12 wk of age were significantly higher than those in the control group (p<0.05). The numbers of CD4+ cells in the BC group were significantly higher than those in the control group at 4 wk of age (p<0.05). These results confirmed that beta-carotene supplementation to Japanese Black calves affected the peripheral blood leukocyte population.

Pharmacokinetics in plasma and alveolar regions of healthy calves subcutaneously administered a single dose of enrofloxacin.

This study aimed to analyze the pharmacokinetics of enrofloxacin (ERFX) and its metabolite ciprofloxacin (CPFX) in plasma, as well as their migration to, and retention in, the epithelial lining fluid (ELF) and alveolar cells within the bronchoalveolar fluid (BALF). Four healthy calves were subcutaneously administered a single dose of ERFX (5 mg/kg). ERFX and CPFX dynamics post-administration were analyzed via a non-compartment model, including the absorption phase. The Cmax of plasma ERFX was 1.6 ± 0.4 µg/ml at 2.3 ± 0.5 hr post-administration and gradually decreased to 0.14 ± 0.03 µg/ml at 24 hr following administration. The mean residence time between 0 and 24 hr (MRT0-24) in plasma was 6.9 ± 1.0 hr. ERFX concentrations in ELF and alveolar cells peaked at 3.0 ± 2.0 hr and 4.0 ± 2.3 hr following administration, respectively, and gradually decreased to 0.9 ± 0.8 µg/ml and 0.8 ± 0.5 µg/ml thereafter. The plasma half-life (t1/2) of ERFX was 6.5 ± 0.7 hr, while that in ELF and alveolar cells was 6.5 ± 3.6 and 7.4 ± 4.3 hr, respectively. The Cmax and the area under the concentration-time curve for 0-24 hr for ERFX were significantly higher in alveolar cells than in plasma (P<0.05). These results suggest that ERFX is distributed at high concentrations in ELF and is retained at high concentrations in alveolar cells after 24 hr in the BALF region; hence, ERFX may be an effective therapeutic agent against pneumonia.

Distribution of marbofloxacin within the bronchoalveolar region of healthy pigs.

The purpose of this study was to clarify the distribution of marbofloxacin (MBFX) within the bronchoalveolar region of pigs. Four clinically healthy pigs were intramuscularly injected with a single dose of MBFX (2 mg/kg). Samples of plasma and bronchoalveolar lavage fluid (BALF) were obtained for each pig at 0 (before administration), 3, 8 and 24 hr after administration of MBFX. As a result, the MBFX concentrations in pulmonary epithelial lining fluid (ELF) and in alveolar cells showed a similar pattern of concentrations during the experimental period. The MBFX concentrations both in ELF and alveolar cells were higher than in plasma. These results suggest that intramuscularly injected MBFX was well distributed in the bronchoalveolar region.

Distribution of marbofloxacin in the bronchoalveolar region in healthy calves.

The purpose of this study was to clarify the distribution of marbofloxacin (MBFX) within the bronchoalveolar region of calves. Four clinically healthy calves were intramuscularly injected with a single dose of MBFX (2 mg/kg). Samples of plasma and bronchoalveolar lavage fluid (BALF) were obtained for each calf at 0 (before administration), 1, 2, 6 and 24 hr after injection of MBFX. The injections and series of sample collections were conducted and repeated again after two weeks. The results show that the MBFX concentrations in the pulmonary epithelial lining fluid (ELF) were significantly higher than that in plasma and in alveolar cells at 2 hr after injection (P<0.05). For concentrations of MBFX within the ELF, the mean area under the MBFX concentration curve calculated during the 0 to 24 hr timeframe (AUC0-24) was significantly higher than the mean determined from samples collected from the plasma (P<0.05). These results suggest that intramuscularly injected MBFX was well distributed in the bronchoalveolar region.

Seroprevalence of Cryptosporidium parvum and Neospora caninum in cattle in the southern Kyushu region of Japan.

Cryptosporidium parvum and Neospora caninum are common parasites in domesticated cattle worldwide, including in Japan. We carried out a serological survey to detect C. parvum and N. caninum infection among cattle in the southern Kyushu region of Japan-including the small islands-by indirect enzyme-linked immunosorbent assay based on recombinant antigens. We found that total seropositivity in 570 Japanese black cattle was 96.3% for C. parvum and 18.4% for N. caninum. Although seroprevalence was correlated with cattle age, differences in the seroprevalence of C. parvum among age groups were not statistically significant. On the other hand, N. caninum seroprevalence increased with age, suggesting horizontal transmission through ingestion of food or water contaminated with oocysts. These findings underscore the importance of monitoring C. parvum and N. caninum in cattle and implementing measures to prevent the spread of infection to other livestock and to humans.

Genetic analyses for conservation of the traditional Tokara horse using 31 microsatellite markers.

In order to promote conservation of the traditional Tokara horse in its remaining three breeding areas in Japan (Nakanoshima, Kaimondake, and Iriki), we genotyped 123 horses using 31 microsatellite markers and determined their genetic diversity. On average, the number of alleles (NA), observed heterozygosity (HO), expected heterozygosity (HE), and inbreeding coefficient (FIS) among all horses were 3.0, 0.424, 0.481, and 0.108, respectively. Compared with other endangered horse breeds, we found that, even though the size of the Tokara horse population has recently increased, the NA, HO, and HE of Tokara horses are still notably lower than those of other breeds. Neighbor-joining tree and STRUCTURE analysis showed that the current population of Tokara horses is divided into three subpopulations, corresponding to their respective feeding and breeding areas: Nakanoshima, Kaimondake, and Iriki. This subdivision was also reflected in the NA of microsatellite DNAs, with four, three, and four different loci showing single alleles in Nakanoshima, Kaimondake, and Iriki horses, respectively. These alleles are considered to have become fixed as a consequence of breeding within the limited number of horses in each area. Since Tokara horses are currently strongly divided into subpopulations, it is vitally important to exchange several horses among their different breeding units in order to maintain the genetic diversity of the Tokara horse as a unique breed. The data obtained in this study contribute toward explaining the history of Tokara horses and also provide important information for future monitoring of population diversity and guiding conservation measures for this endangered breed.

Isolation of equine peripheral blood stem cells from a Japanese native horse.

The sizes of Japanese native horses have drastically decreased, and protection of these populations is important for Japanese horse culture. Social trials as well as scientific attempts are necessary for maintaining the breed. Mesenchymal stem cells (MSCs) have potential as a cell source for various cell therapies. However, there have been no reports on MSCs of Japanese native horses. We aimed to isolate and characterize MSCs from a Japanese native horse, the Noma horse. Plastic-adherent and self-replicating cells were isolated from a Noma horse's peripheral blood (PB). The isolated cells had trilineage potential and a surface antigen of mesenchymal cells, so they fulfilled the minimal criteria of MSCs. Therefore, PB can be one source of MSCs for Japanese native horses.

Isolation and characterization of equine dental pulp stem cells derived from Thoroughbred wolf teeth.

Mesenchymal stem cells (MSCs) are adult multipotent stem cells that are capable of self-renewal and differentiation into multiple cell lineages. Methods for cell therapy using MSCs have been developed in equine medicine. Recently, human dental pulp stem cells (DPSCs) have drawn much attention owing to their trophic factor producing ability and minimally invasive collection methods. However, there have been no reports on equine dental pulp-derived cells (eDPCs). Therefore, the aim of this study was to isolate and characterize the eDPCs from discarded wolf teeth. Plastic-adherent spindle-shaped cells were isolated from wolf teeth. The doubling time of the isolated eDPCs was approximately 1 day. Differentiation assays using induction medium eDPCs differentiated into osteogenic, chondrogenic and adipogenic lineages. The eDPCs expressed mesenchymal makers (CD11a/18, CD44, CD90 CD105 and MHC class I and II), but did not express hematopoietic markers (CD34 and CD45). Taken together, the results show that eDPCs can be isolated from discarded wolf teeth, and they satisfy the minimal criteria for MSCs. Thus, these eDPCs can be referred to as equine DPSCs (eDPSCs). These eDPSCs may become a new source for cell therapy.

Oral administration of ß-carotene or lycopene prevents atopic dermatitis-like dermatitis in HR-1 mice.

Atopic dermatitis (AD) is a chronic relapsing eczematous skin disease. Certain populations of patients are resistant to standard therapies with topical steroids and/or calcineurin inhibitors, and require systemic medication, such as immunosuppressants. Recently, several reports have shed light on the anti-allergic effects of carotenoids. Therefore, we investigated the effect of p.o. administration of ß-carotene or lycopene on AD-like symptoms of HR-1 hairless mice fed with a low zinc/magnesium diet. Mice were divided into four groups: (i) fed with a standard diet (Co group); (ii) low zinc/magnesium diet (HR group); (iii) low zinc/magnesium and ß-carotene diet (HR-C group); and (iv) low zinc/magnesium and lycopene diet (HR-L group). They were then fed these diets for 8 weeks. Severities of dermatitis were assessed by their appearance, and histopathological and hematological observations. Mice in the HR group developed AD-like dermatitis both clinically and histologically. HR-C and HR-L group mice also developed xerosis and wrinkle-like skin changes, but they were milder than those of HR group mice. Histological analysis revealed that epidermis thickening and inflammatory cell infiltration in the skin of the HR-C and HR-L groups were both statistically less than those of the HR group. The concentration of thymus and activation regulated chemokine in the skin of the HR-L group and the concentration of CCL27 in the skin of the HR-C group were significantly lower than those of the HR group, respectively. In conclusion, p.o. administration of ß-carotene or lycopene prevents AD-like symptoms in association with a suppression of T-helper 2 chemokines in a murine model. Ingestion of carotenoids may be beneficial for patients with AD.