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Introduction: Exploring techniques for differentiating and culturing canine hepatocytes serves as a means to establish systems for liver transplantation and drug metabolism testing. However, establishing consistent methods for culturing stable hepatocytes remains a challenge. Recently, several investigations have shown the reprogramming of mature hepatocytes into hepatic progenitor cells by applying specific small molecule compounds, including Y-27632, (a ROCK inhibitor), A-83-01 (a TGFβ inhibitor), and CHIR99021 (a GSK3 inhibitor) (termed YAC) in rat, mouse, and humans, respectively. However, reports or evidence of successful reprogramming using these small-molecule compounds in dogs are absent. This study aimed to induce the differentiation of mature canine hepatocytes into progenitor cells. Methods: Cryopreserved canine hepatocytes (cHep) were cultured for 14 d in a YAC-supplemented hepatocyte growth medium. Subsequently, an assessment was conducted involving morphological observations, quantitative real-time polymerase chain reaction (qRT-PCR), and immunocytochemistry. Results: Notably, cryopreserved cHep cells emerged and exhibited ongoing proliferation and concurrently developed colonies within the YAC-enriched culture. These observations indicated that the mature hepatocytes reprogrammed into hepatic progenitor cells. Moreover, qRT-PCR analysis revealed a notable enhancement in gene expression levels. Specifically, the genes encoding α-fetoprotein (AFP), epithelial cell adhesion molecule (EpCAM), Cytokeratin 19 (CK19) and SRY-box9 (Sox9) displayed approximately 12-, 2.2-, 517- and 2.9- increases in hepatic progenitor cells, respectively, on day 14 as compared to their state before induction of differentiation. Hepatocyte-related protein expression of AFP, EPCAM, SOX9 and CK19 was confirmed via immunocytochemistry on day 21. In contrast, ALB and MRP2, which are highly expressed in mature hepatocytes, were decreased compared to those before YAC addition, which is consistent with the characteristics of undifferentiated hepatocytes. Conclusions: Herein, we effectively promoted the reprogramming of cryopreserved cHep cells into hepatic progenitor cells using three small-molecule compounds. The mRNA and protein expression analyses demonstrated increased levels of hepatic progenitor cells-specific markers, whereas markers related to mature hepatocytes decreased, suggesting that reprogramming cryopreserved cHep cells to hepatic progenitor cells was achieved using YAC. Therefore, cultivating liver progenitor cells holds the potential to offer valuable insights into the development of artificial livers for drug discovery research and transplantation therapy aimed at addressing liver diseases in dogs.

Toceranib phosphate (toceranib) is approved for canine mast cell tumor treatment. However, no long-term response to toceranib in canine HSTCL has been reported. Here, we describe a case of a 10-year-old castrated mixed-breed dog that presented with a 3-month history of weight loss, polydipsia, and polyuria. The clinicopathological and imaging abnormalities included icterus, biliary obstruction, and splenomegaly with multiple diffuse splenic hypoechoic nodules. On day 21, a cholecystectomy was performed to remove the obstruction, followed by a liver biopsy and splenectomy. Cytology of the spleen and liver showed many small lymphocytes with intracytoplasmic granules (sGLs). Splenic and hepatic infiltration of neoplastic CD3/granzyme B-positive small cells and lymphocytic cholecystitis with granzyme B-negative small cells were noted. T-cell receptor gene clonal rearrangements were observed in the liver tissues. The dog was diagnosed with a hepatosplenic T-cell lymphoma (HSTCL) of sGLs concurrent with lymphocytic cholecystitis. The icterus resolved after surgery, but there was progressive elevation of liver enzyme levels. Toceranib was administered from day 39, resulting in decreased liver enzyme levels, and the dog remained in good condition. The dog stayed in remission after toceranib administration and survived for 460 days. Toceranib should be considered an effective treatment option for canine HSTCL.

Abstract Background Little is known about the epidemic status of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cats in Japan due to insufficiently reliable seroepidemiological analysis methods that are easy to use in cats. Results We developed a protein-A/G-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies against SARS-CoV-2 in cats. The assay was standardized using positive rabbit antibodies against SARS-CoV-2. The ELISA results were consistent with those of a conventional anti-feline-immunoglobulin-G (IgG)-based ELISA. To test the protein-A/G-based ELISA, we collected blood samples from 1,969 cats that had been taken to veterinary clinics in Japan from June to July 2020 and determined the presence of anti-SARS-CoV-2 antibodies. Nine cats were found to have SARS-CoV-2 S1-specific IgG, of which 4 had recombinant receptor-binding domain-specific IgG. Of those 9 samples, one showed neutralizing activity. Based on these findings, we estimated that the prevalence of SARS-CoV-2 neutralizing antibodies in cats in Japan was 0.05% (1/1,969 samples). This prevalence was consistent with the prevalence of neutralizing antibodies against SARS-CoV-2 in humans in Japan according to research conducted at that time. Conclusions Protein-A/G-based ELISA has the potential to be a standardized method for measuring anti-SARS-CoV-2 antibodies in cats. The infection status of SARS-CoV-2 in cats in Japan might be linked to that in humans.

Introduction: Differentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs). Methods: Spheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes (ALB, AFP, α1-AT, CDH1, CYP2E1, CYP3A12, and TAT) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes. Results: The ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P

Haematopoietic stem and progenitor cells (HSPCs) are used for transplantation to reconstruct the haematopoietic pathways in humans receiving severe chemotherapy. However, the characteristics of canine HSPCs, such as specific surface antigens and gene expression profiles, are still unclear. This study aimed to characterise the haematopoietic ability and gene expression profiles of canine bone marrow HSPCs in healthy dogs. In this study, the CD34 positive (CD34+) cells were defined as classical HSPCs, CD34+/CD45 diminished (CD45dim) cells as more enriched HSPCs, and whole viable cells as controls. Haematopoietic abilities and gene expression profiles were evaluated using a colony-forming unit assay and RNA-sequencing analysis. Canine CD34+/CD45dim cells exhibited a significantly higher haematopoietic colony formation ability and expressed more similarity in the gene expression profiles to human and mouse HSPCs than those of the other cell fractions. Furthermore, the canine CD34+/CD45dim cells expressed candidate cell surface antigens necessary to define the canine haematopoietic hierarchy roadmap. These results indicate that the canine CD34+/CD45dim cells express the HSPC characteristics more than the other cell fractions, thereby suggesting that these cells have the potential to be used for studying haematopoietic stem cells in dogs.

Introduction: Primary cultured hepatocytes are an important model for early safety evaluations of newly developed drugs. Many factors, however, hinder the wider applications of this technology, especially the difficulty to maintain these cells in long-term culture. To date, creating a stable supply of human or animal hepatocytes with proper hepatic function in vitro has not been achieved. Furthermore, frequently harvesting hepatocytes from living donors for use in culture is highly invasive and simply not feasible. We have previously reported that canine bone marrow-derived mesenchymal stem cells (cBMSCs) can be effectively converted into induced hepatocyte-like cells (iHep cells); however, these cells had reduced function in comparison to mature hepatocytes. In recent studies, spheroid formation-based three-dimensional (3D) culture has been noted to greatly increase hepatocyte function; nevertheless, no reports have described the use of this technology for culturing canine hepatocytes. Therefore, in this study, we aimed to establish a 3D spheroid culture using converted canine iHep cells to investigate their function as hepatocytes. Methods: The iHep cells were prepared by introducing two genes, namely, the Forkhead box A1 (Foxa1) and hepatocyte nuclear factor 4 homeobox alpha (Hnf4α), into cBMSCs seeded onto an ultra-low attachment microplate to induce spheroid formation. Thereafter, the hepatic functions of these spheroids were evaluated using immunocytochemistry, as well as qualitative and quantitative PCR. Results: Notably, albumin was observed in the iHep spheroids and the expression of hepatic genes, such as albumin and drug metabolism CYP genes, could also be detected. Another interesting finding was evident upon further comparing the quantified albumin gene and CYP2E1 gene expressions in the two-dimensional and three-dimensional culture systems; notably, a 100- to 200-fold increase in gene expression levels was observed in the three-dimensional spheroids when compared to those in conventional monolayers. Conclusions: Upon incorporating three-dimensional technology, we managed to achieve iHep spheroids that are closer in gene expression to living liver tissue compared to conventional monolayer cultures. Thus, we are one step closer to creating a sustainable in vitro hepatocyte model. Furthermore, we believe that this system is capable of maintaining the stable drug metabolizing capacity of canine hepatocytes in vitro, which might be useful in improving current drug assessment studies.

Introduction: Hepatocytes, which account for the majority of liver tissue, are derived from the endoderm and become hepatocytes via differentiation of hepatic progenitor cells. Induced hepatocyte-like (iHep) cells and artificial liver tissues are expected to become useful, efficient therapies for severe and refractory liver diseases and to contribute to drug discovery research. The establishment of iHep cell lines are needed to carry out liver transplants and assess liver toxicity in the rising number of dogs affected by liver disease. Recently, direct conversion of non-hepatocyte cells into iHep cells was achieved by transfecting mouse adult fibroblasts with the Forkhead box protein A1 (Foxa1) and hepatocyte nuclear factor 4 homeobox alpha (Hnf4α) genes. Here, we applied this conversion process for the differentiation of canine bone marrow stem cells (cBMSCs) into hepatocyte-like cells. Methods: Bone marrow specimens were collected from four healthy Beagle dogs and used to culture cBMSCs in Dulbecco's Modified Eagle's Medium (DMEM). The cBMSCs displayed the following characteristic features: plastic adherence; differentiation into adipocytes, osteoblasts and chondrocytes; and a cell surface antigen profile of CD29 (+), CD44 (+), CD90 (+), CD45 (−), CD34 (−) and CD14 (−), or CD11b (−) and CD79a (−), or CD19 (−) and HLA class II(−). The cBMSCs were seeded in a collagen I-coated plate and cultured in DMEM with 10% fetal bovine serum and transfected with retroviruses expressing Foxa1 and Hnf4α the following day. Canine iHep cells were differentiated from cBMSCs in culture on day 10, and were analyzed for morphology, RNA expression, immunocytochemistry, urea production, and low-density lipoprotein (LDL) metabolism. Results: The cBMSCs expressed CD29 (98.06 ± 1.14%), CD44 (99.59 ± 0.27%) and CD90 (92.78 ± 4.89%), but did not express CD14 (0.47 ± 0.29%), CD19 (0.44 ± 0.39%), CD34 (0.33 ± 0.25%), CD45 (0.46 ± 0.34%) or MHC class II (0.54 ± 0.40%). The iHep cells exhibited morphology that included circular to equilateral circular shapes, and the formation of colonies that adhered to each other 10 days after Foxa1 and Hnf4α transfection. Quantitative RT-PCR analysis showed that the expression levels of the genes encoding albumin (ALB) and cadherin (CDH) in iHep cells on day 10 were increased approximately 100- and 10,000-fold, respectively, compared with cBMSCs. Corresponding protein expression of ALB and epithelial-CDH was confirmed by immunocytochemistry. Important hepatic functions, including LDL metabolic ability and urea production, were increased in iHep cells on day 10. Conclusion: We successfully induced cBMSCs to differentiate into functional iHep cells. To our knowledge, this is the first report of canine liver tissue differentiation using Foxa1 and Hnf4α gene transfection. Canine iHep cells are expected to provide insights for the construction of liver models for drug discovery research and may serve as potential therapeutics for canine liver disease.

Case summary A 7-year-old neutered male Norwegian Forest Cat was presented with decreased appetite and activity, weight loss, fever, neutrophilia and hyperglobulinaemia. A physical examination showed painful stifle joints and enlarged popliteal lymph nodes. Blood examination showed neutrophilia, hyperglobulinaemia and increased serum amyloid A. Urinalysis, thoracic and abdominal radiographs, and abdominal ultrasonography were unremarkable. Synovial fluid from the knee joints had diminished viscosity and revealed neutrophilic inflammation on the smear. There was no evidence of infection in a microbiological culture of the synovial fluid. A diagnosis of idiopathic immune-mediated polyarthritis (IMPA) was made. Prednisolone was initiated at 2 mg/kg q24h PO and tapered with additional immunosuppressants (leflunomide, ciclosporin A and methotrexate); however, prednisolone could not be discontinued. Informed consent was obtained from the owner and mycophenolate mofetil (MMF) at a dosage of 10 mg/kg q12h PO was initiated on day 798. There were no adverse effects of MMF and prednisolone was discontinued on day 1183. Clinical signs resolved and the cat’s general condition remained stable with MMF alone at a dosage of 10 mg/kg q48h PO on day 1600. Relevance and novel information There is limited information describing feline IMPA and its treatment options other than the use of prednisolone. This is the first report of the successful treatment and long-term follow-up of feline IMPA with MMF. MMF may be a safe and effective option as an additional immunosuppressant in feline IMPA.

Case summary A 14-year-old spayed female American Shorthair cat was presented with weight loss and a palpable abdominal mass. Abdominal ultrasound and CT revealed a duodenal mass with suspected perforation and an enlarged jejunal lymph node. Cytological evaluation from a fine-needle aspiration of the abdominal mass displayed many atypical round cells, some with a small amount of light pink material at the cellular edge. The duodenal mass was surgically removed, and was diagnosed as a plasma cell tumour immunohistochemically positive for CD79 alpha, IgA and lambda immunoglobulin light chains. In addition, amyloidosis was detected. PCR to assess the antigen receptor rearrangement of the tumour cells showed a monoclonal rearrangement of the immunoglobulin heavy chain gene. Postoperatively, the cat received chemotherapy with cyclophosphamide and prednisolone. Owing to progressive enlargement of the jejunal lymph node, different chemotherapy protocols were used sequentially, namely chlorambucil, lomustine and L-asparaginase. However, the cat died 96 days after the initial diagnosis. Post-mortem examination confirmed systemic dissemination of tumour cells. The cause of death was considered to be a result of a complication of the tumour itself and associated amyloidosis. Relevance and novel information This patient was diagnosed with a primary duodenal plasmacytoma, and primary (amyloid light-chain) amyloidosis. In cats, intestinal plasmacytoma is rarely reported and associated amyloidosis is an uncommon feature, when compared with humans. To our knowledge, this is the first clinical report of duodenal plasmacytoma in a cat. The present report shows that feline plasmacytomas should be included in the differential diagnosis of a duodenal mass.

Canine primary hypoplasia of the portal vein (PHPV) is a microscopic malformation of the hepatic vasculature. The prevalence, clinical signs, and clinicopathological findings of PHPV in dogs are unclear, because there are few reports concerning PHPV in the veterinary literature. This retrospective study reviewed clinical records and liver biopsy data from 48 dogs with hepatic disease that were examined at a private veterinary hospital in Japan between April 2011 and March 2014 to determine the prevalence of PHPV among dogs that underwent liver biopsy and to determine the clinical and clinicopathological findings of PHPV in dogs. Records for all 48 dogs that underwent liver biopsy were investigated. Collected data included signalment, clinical signs, physical examination findings, complete blood cell count, chemistry results, pre-and postprandial serum total bile acid concentrations, coagulation profiles (prothrombin time, activated partial thromboplastin time, fibrinogen, and antithrombin), and abdominal ultrasonography findings at the first medical examination. The diagnosis of PHPV was made on the basis of histological examination of hepatic biopsy specimens and portography or CT angiography. Among the 48 canine cases, 28 dogs (58.3%) were diagnosed with PHPV, which was the most common diagnosis. The most frequent clinical sign in dogs with PHPV was asymptomatic persistently increased liver enzymes (57.1%). Toy poodles were at a significantly higher risk of PHPV than other breeds among dogs that underwent liver biopsy (P

A 12-year-old spayed female Dalmatian presented with acute vomiting and anorexia. The clinicopathological and imaging abnormalities included icterus, biliary obstruction, and multiple diffuse splenic hypoechogenic nodules. Cholecystectomy was performed to remove the obstruction, followed by liver biopsy and splenectomy. Histopathological and immunohistology evaluation of the spleen, liver, and gallbladder revealed splenic marginal zone lymphoma (MZL) with gallbladder and hepatic infiltration of neoplastic CD20/CD79α-positive cells. Moreover, we observed clonal rearrangements of the immunoglobulin heavy-chain (IgH) gene in all three tissues. The dog was in good condition without chemotherapy. However, there was progressive elevation of liver enzymes, which could be attributed to neoplastic hepatic infiltration. Chlorambucil and prednisolone were administered until day 108, when the liver enzyme levels normalized. On day 156, the dog developed diffuse large B-cell lymphoma (DLBCL) of the peripheral lymph nodes. Sequence analysis of the clonally rearranged IgH gene revealed that all neoplastic cells in the spleen, gallbladder, and liver at initial presentation, as well as lymph nodes on day 156, possessed the same sequence identity of the amplified IgH fragments. This demonstrated that all neoplastic cells were derived from the same B-lymphocyte clone. The DLBCL was considered to have transformed from the splenic MZL, with gallbladder involvement. In cases of splenic MZL, it is important to consider gallbladder involvement and transformation to DLBCL. Moreover, gallbladder lymphoma should be included in the differential diagnosis of dogs with gallbladder abnormalities. Further studies are warranted to investigate the prognosis of splenic MZL. [ABSTRACT FROM AUTHOR]

A mixed-breed, 8-year-old male dog developed neutropenia, thrombocytopenia, and hyperglobulinemia. Bone marrow hyperplasia and splenic plasmacytosis were cytologically observed. The dog had never been outside of Tokyo or Shizuoka Prefecture. Splenectomy was performed to confirm and remove the cause of splenic plasmacytosis. A histopathological diagnosis of splenic plasmacytoma was made; however, serum protein electrophoresis showed polyclonal gammopathy. Further screening was performed, and Ehrlichia canis infection was confirmed. The dog was treated with doxycycline for 5 weeks. After the antibiotic therapy, no relapse of neutropenia, thrombocytopenia, hyperglobulinemia, or positive polymerase chain reaction result of E. canis infection was observed for 3 years. Careful attention should be given to ehrlichiosis when exploring the cause of pancytopenia or hyperglobulinemia, regardless of the travel history. [ABSTRACT FROM AUTHOR]

The incidence of feline gastrointestinal (Gl) lymphoma has recently increased. Serum amyloid A (SAA) levels are elevated in feline lymphoma. However, no reports have evaluated SAA concentrations and outcomes in feline Gl lymphoma. This study aimed to evaluate the clinical utility of SAA and other factors in feline Gl lymphoma to assess the outcomes with potential differences. The study included 39 client-owned cats diagnosed with Gl lymphoma, which were divided into two groups: high- and low-grade lymphomas. Changes in SAA concentration, complete blood count (CBC), and biochemical profiles were analyzed at the time of initial presentation as well as on days 1, 28, and 56. Differences between the two groups were investigated. High-grade lymphoma was observed in 17 cats, whereas 22 cats showed low-grade lymphoma. SAA concentrations on the day of initial presentation were significantly higher in low-grade lymphoma than those in high-grade lymphoma (median, 12.4 µg/mL; range, 4.8-46.5 µg/mL vs. 3.8 µg/mL; 3.8-13.7 µg/mL; P=0.011). Elevated SAA concentration on day 56 in high-grade Gl lymphoma was a poor prognostic factor. (Hazard Ratio=1.012, per 1 µg/mL increase; 95% confidence interval; 1.004-1.020, P=0.002). The SAA concentration on the day of initial presentation did not serve as a suitable prognostic factor and did not depend on the grade or stage of the lymphoma. However, continuous SAA concentration measurement may be useful for predicting the outcome of feline Gl lymphoma. [ABSTRACT FROM AUTHOR]

Severe adverse reactions after rabies vaccination in dogs were examined from 317 cases reported to the Ministry of Agriculture, Forestry and Fisheries (MAFF) in Japan during 15-year period from April 2004 to March 2019. We found that 109 of the 317 dogs showed anaphylaxis (0.15/100,000 vaccinated dogs), and 71 of the 109 cases of anaphylaxis resulted in death (0.10/100,000 vaccinated dogs). We measured bovine serum albumin (BSA) in four commercially available rabies vaccines and found the levels ranged from 0.1 to 16.6 µg/dose. Our survey showed that the rate of anaphylaxis to rabies vaccines in dogs is rare, although some cases of anaphylaxis resulted in death. Veterinarians should be well prepared to deal with vaccineassociated anaphylaxis. [ABSTRACT FROM AUTHOR]

A 10-year-old spayed female mixed-breed cat presented with progressive nonregenerative anemia. Clinicopathological abnormalities included severe nonregenerative anemia (packed cell volume [PCV]: 7%, aggregate reticulocytes: 1.12 × 10³/µl) and a hypoechogenic mass well-localized in the stomach. Bone marrow (BM) smears revealed increased particle hematopoietic cellularity with decreased myeloid:erythroid (M:E) ratios, no dysplasia of any lineage, and presence of erythroid precursors phagocytized by macrophages. The cat was diagnosed with presumptive precursor-targeted immune-mediated anemia (PIMA). The stomach mass was consistent with CD 20 positive T-cell lymphoma. The lymphoma was completely resected via surgery, and the PIMA was cured by immunosuppressive therapy. On day 410, both diseases have not recurred without medications. This is the first report of feline PIMA and concurrent gastrointestinal lymphoma. [ABSTRACT FROM AUTHOR]