In vivo transfection of cytokine genes into tumor cells using a synthetic vehicle promotes antitumor immune responses in a visceral tumor model.

The tumor microenvironment (TME) strongly affects the clinical outcomes of immunotherapy. This study aimed to activate the antitumor immune response by manipulating the TME by transfecting genes encoding relevant cytokines into tumor cells using a synthetic vehicle, which is designed to target tumor cells and promote the expression of transfected genes. Lung tumors were formed by injecting CT26.WT intravenously into BALB/c mice. Upon intravenous injection of the green fluorescent protein-coding plasmid encapsulated in the vehicle, 14.2% tumor-specific expression was observed. Transfection of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and CD40 ligand (L)-plasmid combination and interferon gamma (IFNγ) and CD40L-plasmid combination showed 45.5% and 54.5% complete remission (CR), respectively, on day 60; alternate treatments with both the plasmid combinations elicited 66.7% CR, while the control animals died within 48 days. Immune status analysis revealed that the density of dendritic cells significantly increased in tumors, particularly after GM-CSF- and CD40L-gene transfection, while that of regulatory T cells significantly decreased. The proportion of activated killer cells and antitumoral macrophages significantly increased, specifically after IFNγ and CD40L transfection. Furthermore, the level of the immune escape molecule programmed death ligand-1 decreased in tumors after transfecting these cytokine genes. As a result, tumor cell-specific transfection of these cytokine genes by the synthetic vehicle significantly promotes antitumor immune responses in the TME, a key aim for visceral tumor therapy.

Generation, characterization, and differentiation of induced pluripotent stem-like cells in the domestic cat.

Induced pluripotent stem (iPS) cells are generated from somatic cells and can differentiate into various cell types. Therefore, these cells are expected to be a powerful tool for modeling diseases and transplantation therapy. Generation of domestic cat iPS cells depending on leukemia inhibitory factor has been reported; however, this strategy may not be optimized. Considering that domestic cats are excellent models for studying spontaneous diseases, iPS cell generation is crucial. In this study, we aimed to derive iPS cells from cat embryonic fibroblasts retrovirally transfected with mouse Oct3/4, Klf4, Sox2, and c-Myc. After transfection, embryonic fibroblasts were reseeded onto inactivated SNL 76/7 and cultured in a medium supplemented with basic fibroblast growth factor. Flat, compact, primary colonies resembling human iPS colonies were observed. Additionally, primary colonies were more frequently observed in the KnockOut Serum Replacement medium than in the fetal bovine serum (FBS) medium. However, enhanced maintenance and proliferation of iPS-like cells occurred in the FBS medium. These iPS-like cells expressed embryonic stem cell markers, had normal karyotypes, proliferated beyond 45 passages, and differentiated into all three germ layers in vitro. Notably, expression of exogenous Oct3/4, Klf4, and Sox2 was silenced in these cells. However, the iPS-like cells failed to form teratomas. In conclusion, this is the first study to establish and characterize cat iPS-like cells, which can differentiate into different cell types depending on the basic fibroblast growth factor.

Effects of the preservation medium and storage duration of domestic cat ovaries on the maturational and developmental competence of oocytes in vitro.

We examined the effectiveness of saline, Euro-Collins solution (EC), and ET-Kyoto solution (ET-K) as preservation media for the cold storage of feline ovaries. Ovaries were maintained in these media at 4°C for 24, 48, or 72 h until oocyte retrieval. The ET-K group exhibited a higher oocyte maturation rate than the saline group after 72 h of storage. Moreover, ET-K could sustain the competence of the feline oocytes to cleave after 48 h, and the morula formation rate of the ET-K group was higher than that of the other groups after 24 and 48 h. Furthermore, the ET-K group exhibited a higher blastocyst formation rate than the other groups after storage for 24 h, and only ET-K retained the developmental competence in blastocysts after 48 h of storage. In addition, regarding the cell numbers of the blastocysts, there was no significant difference among the tested groups. In conclusion, our results indicate that ET-K is a suitable preservation medium for feline ovaries.

Canine induced pluripotent stem cell maintenance under feeder-free and chemically-defined conditions.

Canine induced pluripotent stem cells (ciPSCs) provide a platform for regenerative veterinary medicine, disease modeling, and drug discovery. However, in the conventional method, ciPSCs are maintained using chemically-undefined media containing unknown animal components under on-murine embryonic fibroblast feeder conditions, which were reported to modify cell surface of iPSCs and increases the risk of immune rejection when the cells are transplanted into patients. Moreover, in the conventional method, ciPSCs are mechanically passaged, which requires much time and effort. Therefore, the large-scale expansion of ciPSCs is difficult, which should be resolved for using ciPSCs in clinical application and research. Here, it was shown that StemFit® AK02N and iMatrix-511 could maintain the pluripotency of ciPSCs using conventional culture method. Furthermore, it was demonstrated that the feeder-free and chemically-defined ciPSC culture systems using StemFit® AK02N and iMatrix-511 could stably maintain and allow the easy expansion of ciPSCs generated using N2B27 and StemFit® AK02N, without causing karyotype abnormalities. ciPSCs expressed several pluripotency markers and formed teratomas, including cells derived from three germ layers.

Generation of Footprint-Free Canine Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus Vector.

Using auto-erasable Sendai virus vector, we generated ciPSC line. After several passages, virus was not present in ciPSCs by RT-PCR. ciPSCs from canine PBMCs had pluripotent state, differentiated all three germ layers in vitro, and had normal 78 XX karyotype. These results proved that PBMCs were one of the good cell sources to generate ciPSC lines from companion and patient dogs.

Distribution of infectious endogenous retroviruses in mixed-breed and purebred cats.

Endogenous retroviruses of domestic cats (ERV-DCs) are members of the genus Gammaretrovirus that infect domestic cats (Felis silvestris catus). Uniquely, domestic cats harbor replication-competent proviruses such as ERV-DC10 (ERV-DC18) and ERV-DC14 (xenotropic and nonecotropic viruses, respectively). The purpose of this study was to assess invasion by two distinct infectious ERV-DCs, ERV-DC10 and ERV-DC14, in domestic cats. Of a total sample of 1646 cats, 568 animals (34.5%) were positive for ERV-DC10 (heterozygous: 377; homozygous: 191), 68 animals (4.1%) were positive for ERV-DC14 (heterozygous: 67; homozygous: 1), and 10 animals (0.6%) were positive for both ERV-DC10 and ERV-DC14. ERV-DC10 and ERV-DC14 were detected in domestic cats in Japan as well as in Tanzania, Sri Lanka, Vietnam, South Korea and Spain. Breeding cats, including Singapura, Norwegian Forest and Ragdoll cats, showed high frequencies of ERV-DC10 (60-100%). By contrast, ERV-DC14 was detected at low frequency in breeding cats. Our results suggest that ERV-DC10 is widely distributed while ERV-DC14 is maintained in a minor population of cats. Thus, ERV-DC10 and ERV-DC14 have invaded cat populations independently.

Development of feline embryos produced by Piezo-actuated intracytoplasmic sperm injection of elongated spermatids.

Piezo-actuated intracytoplasmic sperm injection (Piezo-ICSI) is used as an efficient in vitro fertilization method with various animals. With this method, elongated spermatids are collected from testicular tissues and are easier to obtain from animals that unexpectedly die than ejaculate sperm. Additionally, elongated spermatid injection often results in the development of embryos and offspring. To develop assisted reproductive techniques (ARTs) for domestic cats, we examined the effects of oocyte activation on cleavage and embryo development after Piezo-ICSI with motile sperm (experiment 1) and after Piezo-ICSI with either testicular sperm or elongated spermatids (experiment 2). In experiment 1, the proportions of cleaved embryos, morulas, and blastocysts following Piezo-ICSI with ethanol activation were significantly higher (P < 0.05) than in the non-activated groups. However, the proportion of blastocysts and the blastocyst quality did not differ significantly (P > 0.05) between the ethanol-activated and non-activated groups. In experiment 2, the cleavage frequencies of oocytes after Piezo-ICSI of testicular sperm or elongated spermatids and ethanol activation were higher (P < 0.05) than that of oocytes in the non-activated group, but the occurrence of blastocyst formation and quality of blastocysts did not differ between the activated and non-activated groups. In summary, cat embryos can be produced by Piezo-actuated microinjection of elongated spermatids. Ethanol activation increased the frequency of cleavage, but it affected neither the occurrence of blastocyst development nor the quality of blastocysts. These results represent an expansion in the repertoire of ARTs that are potentially applicable to both domestic and endangered species of cats.

Feeder-independent canine induced pluripotent stem cells maintained under serum-free conditions.

Canine induced pluripotent stem cells (ciPSCs) are an attractive source for regenerative veterinary medicine, and may also serve as a disease model for human regenerative medicine. Extending the application of ciPSCs from bench to bedside, however, requires resolving many issues. We generated ciPSCs expressing doxycycline-inducible murine Oct3/4 (Pou5f1), Sox2, Klf4, and c-Myc, which were introduced using lentiviral vectors. The resultant ciPSCs required doxycycline to proliferate in the undifferentiated state. Those ciPSC colonies exhibiting basic fibroblast growth factor (bFGF)-dependent proliferation were dissociated into single cells for passaging, and were maintained on a Matrigel-coated dish without feeder cells in a serum-free medium. The established ciPSCs had the ability to differentiate into three germ layers, via formation of embryoid bodies, as well as into cells expressing the same markers as mesenchymal stem cells. These ciPSCs may thus serve as a suitable source of pluripotent stem cell lines for regenerative veterinary medicine, with fewer concerns of contamination from unknown animal components.

Analysis of IMP-1 type metallo-ß-lactamase-producing Acinetobacter radioresistens isolated from companion animals.

IMP-1 type metallo-ß-lactamase-producing (MBL-producing) Acinetobacter radioresistens was isolated from a dog with cystitis and a cat with conjunctivitis. The MBL-producing A. radioresistens isolates were resistant to all of the ß-lactam antibiotics used in the sensitivity tests, but were susceptible to gentamicin, amikacin, and minocycline. Also, one of the two strains of A. radioresistens was susceptible to ciprofloxacin and levofloxacin. These two cases were cured by administration of tetracyclines and fluoroquinolones, which elicited a positive result in the sensitivity tests. This report of the isolation of MBL-producing A. radioresistens in companion animals is the first in the world. To prevent the proliferation of MBL-producing bacteria, veterinary hospitals need to be aware of the behavior of MBL-producing organisms.

Expression and localization of epidermal growth factor, transforming growth factor-α and epidermal growth factor receptor in the canine testis.

Gene expression of epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and EGF receptor (EGF-R) and the localization of the corresponding proteins in the canine testis were studied. Levels of mRNA expressions were determined by semiquantitative reverse transcription polymerase chain reaction in the testes of the peripubertal (4-6 months), young adult (3-4 years), advanced adult (7-8 years) and senescent (11-16 years) groups. The EGF-R mRNA level in the testes of the peripubertal group was significantly higher than those in the other groups, whereas there was no difference in EGF and TGF-α mRNA levels among groups. Immunohistochemical stainings for EGF, TGF-α and EGF-R in the testis revealed that immunoreactivity in the seminiferous epithelium and Sertoli cell was weak and nonspecific for the stage of spermatogenesis, and distinct staining was found in Leydig cells. These results suggest that the EGF family of growth factors may be involved in testicular maturation and function in the dog.

Evaluation of serum phosphorylated neurofilament subunit NF-H as a prognostic biomarker in dogs with thoracolumbar intervertebral disc herniation.

OBJECTIVE: To investigate whether pNF-H is a prognostic biomarker of spinal cord injury (SCI) in paraplegic dogs with thoracolumbar intervertebral disc herniation (IVDH). STUDY DESIGN: Prospective, case-control clinical study ANIMALS: Dogs (n = 60) with SCI from IVDH and 6 healthy dogs. METHODS: Serum from 60 thoracolumbar IVDH dogs (Grade 4: 22 dogs; Grade 5: 38 dogs) collected 1-3 days after injury, and 6 control dogs, was analyzed using enzyme-linked immunosorbent assay (ELISA) against a phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H). Serum pNF-H levels were compared between different IVDH grades and their prognostic value was investigated. RESULTS: pNF-H levels were significantly greater in Grade 5 than Grade 4 dogs. There were significant differences in pNF-H levels between dogs that regained voluntarily ambulation and those that did not. All 8 dogs that had high pNF-H levels 1-3 days after injury did not regain the ability to walk after surgery. CONCLUSIONS: Serum pNF-H levels might be a biomarker for predicting prognosis of canine SCI.

Canine induced pluripotent stem cells can be successfully maintained in weekend-free culture systems.

Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs.

Generation of canine induced pluripotent stem cells under feeder-free conditions using Sendai virus vector encoding six canine reprogramming factors.

Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers in vitro and in vivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines.

Assessment of hypoxia-targeting therapy for gastrointestinal lymphoma in dogs: Preclinical test using murine models.

The transcription factor hypoxia-inducible factor 1α (HIF-1α) is expressed in several cancers under intratumoral hypoxic stress that arises during pathogenic processes, resulting in malignant progression. We previously reported that hypoxic stimulation enhances the growth potential of canine lymphoma cells by activating the HIF-1α signaling pathway. In contrast, evofosfamide (Evo) releases a DNA-alkylating moiety within hypoxic tumor regions, suggesting that Evo could serve as a hypoxia-targeting drug in canine lymphoma. This study aimed to use Evo to evaluate hypoxia-targeted therapy in dogs with gastrointestinal lymphoma (GIL) and investigate how Evo affects antitumor efficacy and adverse events in three type of murine xenograft models using T-cell GIL cells. In vitro tests, the sensitivity to Evo of three T-cell GIL cell lines under hypoxic culture was significantly higher than that under normoxic culture. Our metabolic analysis suggested that the three murine models might have high reproducibility as clinical cases in canine GIL. Our data showed that Evo showed significantly higher tumor growth potential and fewer adverse events in three type of murine models compared to lomustine; CeeNu (CCNU). Additionally, Evo suppressed the expression of HIF-1α protein in tumor tissues, suggesting that it may preferentially target and inhibit tumor cells in a hypoxic region. The evidence presented here supports the favorable preclinical evaluation that Evo may be effective for GIL in dogs.

Hypoxia-targeting therapy for intestinal T-cell lymphoma in dogs: Preclinical study using 3D in vitro models.

The transcription factor hypoxia-inducible factor 1α (HIF-1α) is activated in response to oxygen deficiency, and is expressed in several cancers under intratumoral hypoxic stress that arises during pathogenic processes. Hypoxic stimulation enhanced the growth potential of canine lymphoma cells by activating the HIF-1α signalling pathway in a previously reported study. The aim of this study was to establish a molecular design strategy for a novel hypoxia-targeting therapy for intestinal T-cell lymphoma (ITL) in dogs. We assessed the relationship between immunohistochemistry-based HIF-1α expression and clinical information, including signalment, tumour area, clinical signs, systemic diseases, treatment protocol, follow-up information, chemotherapy response and overall survivals (OS), using 48 tissue samples from dogs with ITL. We investigated the effects of hypoxic stimulation on the biological behaviour of cell lines from three different types of canine ITL. We assessed the effects of evofosfamide (Evo; hypoxia-activated prodrug) on cell lines cultured under hypoxic conditions. Our data showed that treatment response and overall survival might be significantly decreased in dogs with higher HIF-1α expression than in those with lower HIF-1α expression. Hypoxic culture (1% O2 , 72 h) enhanced the invasiveness of cell lines and decreased their sensitivity to CCNU, resulting in hypoxia-dependent aggressive behaviour. Sensitivity to Evo significantly increased in cell lines cultured under hypoxia compared with those cultured under normoxia, which exhibited hypoxia-dependent apoptosis. Additionally, Evo downregulated HIF-1α expression in cell lines cultured under hypoxia, suggesting that Evo might inhibit cell growth by inactivating HIF-1α-dependent cell signalling. Our results revealed the preclinical antitumor activity of Evo and provide a rationale for treatment strategies for dogs with ITL.

Genetic and phenotypic analyses of mcr-harboring extended-spectrum ß-lactamase-producing Escherichia coli isolates from companion dogs and cats in Japan.

The emergence of mcr plasmid-mediated colistin-resistant extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales among companion dogs and cats poses a risk of the animals acting as reservoirs for cross-species transmission. However, current knowledge of mcr-harboring ESBL-producing Enterobacterales in companion dogs and cats is still limited; thus, the genetic and phenotypic characteristics of the bacterial isolates and plasmids, in companion dogs and cats, remain to be elucidated. Here, we identified mcr gene-harboring ESBL-producing Escherichia coli isolates during whole-genome sequencing of ESBL-producing E. coli isolates from a dog and a cat in Osaka, Japan. Colistin-resistant MY732 isolate from a dog carried two plasmids: mcr-1.1-harboring IncI2 plasmid and blaCTX-M-14-harboring IncFIB plasmid. Conjugation assays revealed that both plasmids can be co-transferred even though the IncFIB plasmid lacked a conjugal transfer gene cassette. The other isolate MY504 from a cat harbored two bla genes and mcr-9 on the identical IncHI2 plasmid. This isolate was not resistant to colistin, which is likely to be due to deletion of the regulatory two-component QseBC system associated with the mcr-9 expression. To the best of our knowledge, this is the first report of a colistin-resistant ESBL-producing E. coli isolate harboring mcr-1 from a companion dog in Japan. Given that the mcr gene-harboring IncI2 and IncHI2 plasmids in this study shared high homology with plasmids from human or animal-derived Enterobacterales, companion dogs and cats may act as important reservoirs for cross-species transmission of the mcr gene in the community, in Japan.

Atypical hypoadrenocorticism with intact zona glomerulosa of the adrenal cortex after long-term observation: a case report of a dog.

An 8-year-old intact male pointer presented with lethargy and hypoalbuminemia. On abdominal ultrasonography, both adrenal glands were reduced in thickness. Based on the ACTH stimulation test results and the absence of electrolyte abnormalities, the dog was diagnosed with atypical hypoadrenocorticism. After treatment with low-dose prednisolone, his general condition improved, and blood tests normalized. The dog died 818 days later, and a complete autopsy was performed. Histologically, the architecture of the zonae fasciculata and reticularis was disrupted in both adrenal glands; however, the zona glomerulosa remained relatively normal. In summary, in this study, we detailed the pathological presentation of atypical hypoadrenocorticism without electrolyte abnormalities.

Safety of autologous bone marrow stromal cell transplantation in dogs with acute spinal cord injury.

OBJECTIVE: To assess the feasibility and safety of transplantation of autologous bone marrow stromal cell (BMSC) in dogs with acute spinal cord injury (SCI). STUDY DESIGN: An open-label single-arm trial. ANIMALS: Dogs (n = 7) with severe SCI from T6 to L5, caused by vertebral fracture and luxation. METHODS: Decompressive and stabilization surgery was performed on dogs with severe SCI caused by vertebral fracture and luxation. Autologous BMSCs were obtained from each dog's femur, cultured, and then injected into the lesion in the acute stage. Adverse events and motor and sensory function were observed for >1 year after SCI. RESULTS: Follow-up was 29-62 months after SCI. No complications (eg, infection, neuropathic pain, worsening of neurologic function) were observed. Two dogs walked without support, but none of the 7 dogs had any change in sensory function. CONCLUSIONS: Autologous BMSC transplantation is feasible and safe in dogs with acute SCI. Further studies are needed to determine the efficacy of this therapy.

Canine induced pluripotent stem cells efficiently differentiate into definitive endoderm in 3D cell culture conditions using high-dose activin A.

Introduction: Endoderm-derived organs support indispensable functions in the body. Pluripotent stem cells can generate endoderm-derived cells or tissues and have excellent therapeutic potential to replace the functions of endodermal tissues. However, there is no viable method to induce endodermal precursor cells, definitive endoderm (DE), from canine induced pluripotent stem cells (ciPSCs). Methods: A ciPSC line was used in this study. In order to induce DE, ciPSCs were cultured with high dose activin A and fetal bovine serum. We considered the optimal differentiation period and starting cell density. Next, to reduce the remaining undifferentiated cells and improve the DE induction efficiency, DE was induced from 3D cell aggregates with knockout serum replacement instead of fetal bovine serum. Finally, hepatic and pancreatic induction were performed to investigate whether DE could differentiate into downstream lineages. Results: After differentiation, some cells expressed the DE markers FOXA2 and SOX17. DE induction period and starting cell density were found to be important for efficient DE induction. However, some cells remained undifferentiated even after optimization of cell density and culture period. Cell differentiation under 3D culture conditions reduced undifferentiated cells and the replacement of fetal bovine serum with knockout serum replacement improved the DE induction efficiency. After hepatic and pancreatic induction, cells expressed some early hepatic and pancreatic markers. Conclusions: A ciPSC line was successfully differentiated to DE efficiently using a high dose of activin A with knockout serum replacement under 3D cell culture conditions. We believe that this study will be fundamental to achieving the generation of canine endodermal tissues from ciPSCs.

Mesenteric lymph node abscesses due to Escherichia coli in a cat.

A 3-year-old, castrated male mixed-breed cat presented with an almost 2-year history of chronic loose stools. On radiography and ultrasound examination, there were two masses in the centre of the abdomen. Contrast-enhanced computed tomography revealed that the masses were enlarged mesenteric lymph nodes with fluid accumulation. Percutaneous lesion drainage yielded pus-like fluid. Fluid cytology revealed numerous neutrophils and Gram-negative rods. Pus culture identified Escherichia coli as the causative organism. Consequently, mesenteric lymph node abscesses were definitively diagnosed. Since computed tomography showed that the abscesses adhered to the surrounding tissues, it was difficult to remove them surgically. With drainage and antimicrobial therapy, the mesenteric lymph nodes gradually decreased in size. However, loose stools persisted. The cat's diet was changed to a hydrolysed diet, and the clinical symptoms improved, suggesting food-responsive enteropathy. This may be an underlying disease of lymph node abscesses. Lymph node abscesses limited to the mesenteric lymph nodes rarely occur in veterinary medicine, and this is the first report in cats.