Clinicopathologic findings of splenic marginal zone lymphoma with gallbladder involvement that progressed to diffuse large B-cell lymphoma in a dog.

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.

A canine case of Ehrlichia canis infection without a history of being in an endemic area in Japan.

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.

Usefulness of combining computed tomography and air sac fluid examination to rule out aspergillosis: case study in two gentoo penguins (Pygoscelis papua) with respiratory clinical signs.

Pulmonary aspergillosis occurring in captive penguins living in zoos and aquariums is a fatal disease owing to its high mortality rate. On conducting computed tomography, two gentoo penguins (Pygoscelis papua) with respiratory clinical signs that were housed at an aquarium in Hokkaido, Japan were found to have a certain amount of fluid in their air sacs. This fluid was collected and cultured for bacteria and fungi, the results of which both came back negative. These results enabled us to rule out bacterial infection or aspergillosis and supported our decision not to administer antibacterial and antifungal drugs. Overall, the combination of computed tomography and air sac fluid examination was useful for ruling out bacterial infection or aspergillosis in penguins with respiratory clinical signs.

Serum amyloid A (SAA) concentration in cats with gastrointestinal lymphoma.

The incidence of feline gastrointestinal (GI) 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 GI lymphoma. This study aimed to evaluate the clinical utility of SAA and other factors in feline GI lymphoma to assess the outcomes with potential differences. The study included 39 client-owned cats diagnosed with GI 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 GI 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 GI lymphoma.

Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis.

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.

Hepatosplenic lymphoma and visceral mast cell tumor in the liver of a dog with synchronous and multiple primary tumors.

An 11-year-old spayed female American Cocker Spaniel was presented with a 4-week history of anorexia and a 1-week history of abdominal distension. Clinicopathologic and imaging abnormalities included intra-abdominal hemorrhage, granular lymphocytes (GLs) in abdominal fluid smears, a splenic mass, and hepatomegaly with diffuse multiple hypoechogenic nodules. Based on the cytologic, histologic, and immunohistochemical evaluation of the spleen and liver, the diagnosis was hepatosplenic T-cell lymphoma (HSTCL) of GLs. Postoperatively, the dog was maintained in good condition with chemotherapy (ACNU [nimustine], L-asparaginase, and prednisolone). However, on day 85, ultrasound-guided fine-needle aspiration of the liver revealed a proliferation in neoplastic mast cells not associated with the GLs. The dog was diagnosed with a visceral mast cell tumor (MCT) originating from the liver. The chemotherapy was switched to vinblastine and toceranib. The dog remained in good condition until day 141 but died due to the progression of MCT on day 158. Liver cytology on day 155 showed no GLs, although HSTCL is thought to be resistant to chemotherapy. After the definitive diagnosis of HSTCL, we monitored this patient's response to chemotherapy with blood tests, including complete blood counts, ultrasound imaging, and cytologic aspirates of liver. Although canine HSTCL has a poor prognosis, the possibility of a new neoplasm, including visceral MCT, should be considered. Periodic liver cytology might be worthwhile in dogs receiving chemotherapy for HSTCL.

Screening of bacterial DNA in bile sampled from healthy dogs and dogs suffering from liver- or gallbladder-associated disease.

Although the biliary system is generally aseptic, gallbladder microbiota has been reported in humans and some animals apart from dogs. We screened and analyzed the bacterial deoxyribonucleic acid in canine gallbladders using bile sampled from 7 healthy dogs and 52 dogs with liver- or gallbladder-associated disease. PCR screening detected bacteria in 17.3% of diseased dogs (9/52) and none in healthy dogs. Microbiota analysis of PCR-positive samples showed that the microbial diversity differed between liver- and gallbladder-associated disease groups. Thus, a specific bacterial community appears to occur at a certain frequency in the bile of diseased dogs.

Establishment of large canine hepatocyte spheroids by mixing vascular endothelial cells and canine adipose-derived mesenchymal stem cells.

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 < 0.001). Histological evaluation revealed that the three-mix spheroids formed large canine hepatocyte spheroids (LcHS), and hepatocytes were distributed in the center of the spheroids. Quantitative gene expression analysis of LcHS showed that liver-related genes expression were maintained the same levels with that of a culture of cHep alone from days 4-7. CONCLUSION: These results revealed that the three-mix culture method using cHep, HUVECs, and cASCs was capable of promoting LcHS without impairing liver function in cHep, suggesting that LcHS could be used for the application of high-volume culture techniques in dogs.

A cat with myelodysplastic syndrome by administration of the methylation inhibitor Azacytidine.

A 5-year-old female cat with nonregenerative anemia and thrombocytopenia was diagnosed with myelodysplastic syndromes (MDS), since peripheral blood and bone marrow (BM) examination revealed various dysplasias and a blast ratio of 19%. Chemotherapy with azacytidine (AZA; 70-35 mg/m2, 3-5 days, three cycles) and treatment with prednisolone, antibiotics, and vitamin K2, and blood transfusion were performed. On day 106, blast cells and dysplasia had decreased in the BM, and the cat remained alive for at least 1,474 days. This report is the first on feline MDS treated with AZA, suggesting appropriate drug dosage, interval and effective combination should be investigated and the pharmacological and cell biological mechanisms needs to be elucidated in the future.

Presumptive hemophagocytic syndrome associated with immune-mediated anemia in two Miniature Dachshunds.

This report describes the cases of two Miniature Dachshunds who were suspected to have immune-mediated hemolytic anemia (IMHA) and were treated with immunosuppressive therapy. However, progression of anemia, increases in C-reactive protein (CRP) and total-bilirubin (T-Bil) levels, splenomegaly, transition to nonregenerative anemia, and thrombocytopenia occurred after the treatment. Splenectomy and bone-marrow aspirations were performed subsequently. Both dogs were diagnosed with hemophagocytic syndrome (HPS) associated with IMHA. Unfortunately, they died 9 and 6 days later. These findings indicate that some cases of refractory IMHA have the pathogenicity of HPS. HPS should be included as a differential diagnosis of refractory IMHA concurrent with thrombocytopenia. Continuously elevated CRP and T-Bil levels may be helpful indicators in the detection of HPS associated with IMHA.

Precursor-targeted immune-mediated anemia in a dog with a stage IV mast cell tumor and bone marrow infiltration.

A 12-year-old spayed female Shiba Inu dog was referred to our hospital for a suspected mast cell tumor (MCT) of the bone marrow (BM). Laboratory abnormalities included severe nonregenerative anemia (packed cell volume or PCV: 12.5%; reference interval (RI): 37.3-61.7%; reticulocytes: 35.1 × 103 /µL; RI: 10-110 × 103 /µL), and a few mast cells were visualized in the blood smear examination. The BM was hypercellular with hematopoietic cells, a decreased myeloid:erythroid (M:E) ratio (0.77; RI, 0.9-1.8), and no dysplastic hematopoietic cells. Mast cells accounted for 11.5% of the total nucleated BM cells. Neoplastic mast cells and histiocytes phagocytizing erythroid progenitor cells were occasionally noted. The dog was diagnosed with precursor-targeted immune-mediated anemia (PIMA) concurrent and a stage IV MCT infiltrating the BM. Multimodal treatment included toceranib, imatinib, vinblastine, lomustine (CCNU), prednisolone, cyclosporine, mycophenolate mofetil, and a blood transfusion. The dog died due to MCT progression lasting 139 days after the initial BM examination. To the best of our knowledge, this is the first report of a dog presenting with PIMA and a stage IV MCT infiltrating the BM.

Human placental hydrolysate promotes the long-term culture of hepatocyte-like cells derived from canine bone marrow.

Long-term culture of canine artificial hepatocytes has not been established. We hypothesized that human placental hydrolysate (hPH) may support the long-term culture of differentiated hepatocyte-like cells. Canine bone marrow cells were cultured using modified hepatocyte growth medium supplemented with hPH. Quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical analysis for albumin, qualitative RT-PCR for cytochrome P450 1A1 (CYP1A1), hepatocyte growth factor (HGF), Cytokeratin 7 (CK7), CD90, CD44, and CD34, and functional analyses of CYP450 activity and low-density lipoprotein (LDL) uptake were performed. Cultured hepatocyte-like cells were able to maintain hepatocyte characteristics, including morphology, albumin synthesis, CYP450 activity, and LDL uptake for 80 days. Thus, hPH may be a potential facilitator for the long-term culture of hepatocyte-like cells. Clinicopathologically, this culture protocol of artificial hepatocytes will contribute to liver function evaluation.

Successful treatment and long-term follow up of idiopathic immune-mediated polyarthritis with mycophenolate mofetil in a cat.

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.

Primary duodenal plasmacytoma with associated primary (amyloid light-chain) amyloidosis in a cat.

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.

Presumptive precursor-targeted immune-mediated anemia concurrent with gastrointestinal lymphoma in a cat.

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.

Characterization of Suicidal Erythrocyte Death (Eryptosis) in Dogs.

BACKGROUND/AIMS: Suicidal erythrocyte death (eryptosis) is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface following a Ca2+ entry in the cell. Eryptosis is stimulated by increased cytosolic Ca2+ ([Ca2+]i), oxidative stress, energy depletion, or high osmotic shock. Eryptosis signaling includes p38 mitogen-activated protein kinase (MAPK), caspases, casein kinase 1 (CK1), janus kinase 3 (JAK3), and protein kinase C (PKC). Dog and human erythrocytes have different characteristics, for example, dog erythrocytes lack Na+/K+- ATPase activity. Whether eryptosis occurs in dog erythrocytes in an analogous way as that in humans remains unclear. Eryptosis in dogs has not been investigated. This study aimed to explore which stimulator and signaling molecules are involved in eryptosis in healthy dog erythrocytes. METHODS: Erythrocytes were isolated from 10 dogs, and eryptosis was stimulated by oxidative stress with tert-butyl hydroperoxide (tBOOH), high osmotic shock with excessive sucrose condition, energy depletion with minus glucose condition, and high [Ca2+]i with ionomycin. Phosphatidylserine exposure was estimated using annexin V binding. Erythrocyte volume and [Ca2+]i were measured by forward scatter and Fluo3-fluorescence, respectively. In addition, the role of certain mediators was assessed using the following inhibitors to determine the detailed mechanisms of eryptosis in dog erythrocytes: p38MAPK, caspase family, CK1, JAK3, and PKC inhibitors. RESULTS: All eryptosis-inducing factors resulted in phosphatidylserine exposures, except for ionomycin. In addition, the erythrocyte volume increased with ionomycin and tBOOH but decreased with excessive sucrose and minus glucose condition. All treatments increased [Ca2+]i. Furthermore, WH1-P154 and chelerythrine significantly blunted the increase of annexin V binding erythrocytes following the tBOOH treatment. CONCLUSION: Eryptosis in dogs is triggered by oxidative stress, hyperosmotic shock, and energy depletion. It is suggested that JAK3 and PKC play an important role in eryptosis following an oxidative stress in dog erythrocytes.

Conversion of mesenchymal stem cells into a canine hepatocyte-like cells by Foxa1 and Hnf4a.

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.

Three-dimensional spheroid culture of canine hepatocyte-like cells derived from bone marrow mesenchymal stem cells.

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.

A case of hemophagocytic syndrome progressing into large granular lymphoma in a dog.

A 12-year-old castrated male mixed breed dog was presented with anorexia, lethargy, intermittent vomiting, diarrhea, and weight loss. Clinicopathologic and imaging abnormalities included pancytopenia, icterus, and splenomegaly with multiple minute hypoechogenic nodules. Bone marrow (BM) smears revealed 2.5% hemophagocytic macrophages. In addition, an increased number of small to intermediate lymphocytes (16.3%) and plasma cells (3.2%) were recognized in the BM smears. More than 80% of the lymphocytes contained multiple small intracytoplasmic magenta granules. Histopathologic findings of the spleen revealed hemophagocytosis. Large granular lymphocytes (LGLs) were not found on the liver cytology or splenic histopathology at this time. PCR for antigen receptor rearrangement (PARR) analysis showed a clonal reaction in the T-cell receptor ɤ (TCRɤ) gene in the BM sample. The dog was diagnosed with hemophagocytic syndrome (HPS). The dog was maintained in good condition with immunosuppressive therapy. However, the dog developed hepatic LGL lymphoma 7 months later. At this time, PARR analysis showed a clonal TCRɤ gene rearrangement in the hepatic LGL lymphoma samples. The BM and liver sample clonal rearrangements showed 100% homology, indicating that the small to intermediate granular lymphocytes in the BM at the HPS stage had progressed to hepatic LGL lymphoma. To our knowledge, this is the first report of canine secondary HPS caused by the occurrence of a BM LGL lymphoma clone that progressed to hepatic LGL lymphoma.

Akita dogs possess GLUT1 in erythrocytes, and Na,K-ATPase activity enables more efficient ascorbic acid recycling.

We investigated hematologic characteristics of healthy Akita dogs. All were found to contain glucose transporters, GLUT1 and GLUT4, in erythrocyte membrane, whereas Beagle and any other Western dogs have only GLUT4. Of 47 Akitas, ten showed high K and low Na concentrations with elevated glutathione (GSH) in erythrocytes due to Na,K-ATPase activity in the membrane (HK). Akitas showed increased capacity for recycling vitamin C or ascorbic acid (AA) from oxidized ascorbic acid (DHA) compared to Beagle dogs. Particularly, HK Akitas performed even greater AA recycling and ferricyanide reduction than normal Akitas which have normal GSH, low K and high Na concentrations (LK). All HK Akitas also had stomatin in erythrocyte membrane, while half of LK Akitas had it at lower levels than HK Akitas. Stomatin did not have any influence on AA recycling. GLUT1, Na,K-ATPase and stomatin in erythrocytes are characteristics of Akita dogs, and the high prevalence of these proteins suggests their positive roles in biological efficiency.