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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/m

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.

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.

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.

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.

Background Little is known about the epidemic status of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cats living in Japan, and about the influence of cat lifestyles on the SARS-CoV-2 infection epidemic in cats. Results We developed protein A/G-based ELISA, which was standardized using positive rabbit antibodies. The measured values of this ELISA were consistent with those of conventional anti-feline IgG-based ELISA. We then collected blood samples from 1,969 cats that visited veterinary clinics in Japan from June to July 2020 and examined for the presence of anti-SARS-CoV-2 antibodies. Using protein A/G-based ELISA, nine cats were found to have SARS-CoV-2 S1-specific IgG, among which four had RBD-specific IgG. Among those nine samples, one showed neutralizing activity. Based on these, we estimated that the prevalence of SARS-CoV-2 neutralizing antibodies in cats living in Japan was 0.05% (1/1,969 samples). This prevalence did not differ much from the prevalence of neutralizing antibodies for SARS-CoV-2 in humans conducted in Japan at that time. Furthermore, we searched for factors associated with the prevalence of neutralizing antibodies in cats using our data and information from five countries (China, Croatia, France, Germany, and Italy). The prevalence of SARS-CoV-2 in cats was correlated with the rate of keeping indoor-only. Conclusions Protein A/G-based ELISA has the potential to be a standardized method to measure anti-SARS-CoV-2 antibodies in cats. The infection status of SARS-CoV-2 in cats in Japan is linked to that in humans, and the epidemic of SARS-CoV-2 infection in cats may be controlled by their living environment.

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.

Severe adverse reactions in cats after vaccination were examined from 316 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 130 (41%) showed anaphylaxis, and 99 (76%) of the 130 cases of anaphylaxis resulted in death. Veterinarians should be well prepared to deal with vaccine-associated anaphylaxis in cats. Bovine serum albumin (BSA) as indicator of purification was detected at high levels in commercially available feline vaccines. BSA might derive from fetal calf serum in culture media. This study provides useful information about anaphylaxis including critical details of the potential clinical signs associated with adverse events to feline vaccination.

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, Graphical abstract Image 1, Highlights • cHep spheroids was co-cultured using canine hepatocytes, mesenchymal stem cells, and human umbilical vein endothelial cells. • The ratio of spheroid formation was improved and expression of liver related genes and protein was maintained. • The three-mix culture could promote large spheroids without impairing liver function in canine hepatocyte.

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.

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 vaccine-associated anaphylaxis.

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.

With longevity, the prevalence of osteoporosis, which occurs when the activity of osteoclast surpasses that of osteoblasts, has increased in dogs. However, limited information is available on canine osteoclastogenesis. We herein described culture conditions to induce osteoclasts from canine bone marrow cells, and identified factors affecting canine osteoclastogenesis. Tartrate-resistant acid phosphatase-positive multinucleated cells were efficiently formed in a culture of bone marrow mononuclear cells with macrophage colony-stimulating factor (M-CSF 25 ng/mL) for 3 days and a subsequent culture in the presence of M-CSF (25 ng/mL) and soluble receptor activator of NF-κB ligand (RANKL 50 ng/mL) for 4 days. We previously reported in a murine cell system that gene induction of the E isoform of microphthalmia-associated transcription factor (Mitf-E) was required and sufficient for osteoclastogenesis, while transforming growth factor-β (TGF-β) enhanced RANKL-induced Mitf-E expression and osteoclastogenesis. Mitf-E expression also increased during RANKL-induced osteoclastogenesis in canine cells; however, TGF-β down-regulated Mitf-E expression and osteoclastogenesis, indicating a species-dependent response. The results of the present study show that, consistent with murine cells, M-CSF and soluble RANKL enable canine bone marrow cells to differentiate into osteoclasts, and Mitf-E expression is induced during osteoclastogenesis. However, the role of TGF-β in osteoclast formation is distinct between murine and canine cells, suggesting the necessity of analyses using canine cells to examine the factors affecting canine osteoclastogenesis.

Feline hepatic lipidosis (HL), the most common hepatobiliary disease in cats, is characterized by the accumulation of excessive triglycerides (TGs) in more than 80% of hepatocytes. Forced oral feeding is recommended as the only therapy for this disease but the prognosis is often poor. As human placenta extract (Laennec) has been used to improve hepatic metabolism, we investigated the efficacy of this drug for the treatment of cats with HL. Ten cats diagnosed with HL in this study were treated with Laennec as a therapeutic drug. An immediate improvement in clinical symptoms was observed in all 10 cases. A statistical analysis indicated that the blood level of ALT (P = 0.029), AST (P = 0.029), ALP (P = 0.005), and T-Bil (P = 0.012) decreased significantly after treatment. All 7 hospitalized cases were eventually discharged from the hospital with a mean hospitalization duration of 4.8 days. The results of this study suggest that Laennec preparation has potential as an effective medicine for feline HL.

A 3-year-old, spayed female, Domestic Shorthair cat presented with anorexia, lethargy, vomiting, probable hemoabdomen, and multiple masses on the right lateral liver lobe. Clinicopathologic and imaging abnormalities included anemia, azotemia, icterus, and hepatomegaly with hypoechoic masses. On cytologic evaluation of a fine-needle aspiration of a liver mass there was abundant extracellular pink- to purple-colored material between hepatocytes. The amorphous material was stained with direct fast scarlet (DFS), and green birefringent areas were observed under polarized light, confirming the presence of amyloid. A unique finding on the cytologic smear were macrophages containing amorphous and fibrillar amyloid-like protein. Histopathologic examination using H&E and Congo red staining confirmed amyloid deposits within the space of Disse, along the sinusoids, portal tracts, blood vessel walls, and within the cytoplasm of macrophages. Immunohistochemical staining with anti-AA amyloid antibodies further confirmed the presence of AA amyloid. To the author's knowledge, this is the first report of the cytologic finding of AA amyloid protein within macrophages and DFS stain detection of amyloid on a cytologic smear.

Oncogene-containing retroviruses are generated by recombination events between viral and cellular sequences, a phenomenon called "oncogene capture". The captured cellular genes, referred to as "v-onc" genes, then acquire new oncogenic properties. We report a novel feline leukemia virus (FeLV), designated "FeLV-AKT", that has captured feline c-AKT1 in feline lymphoma. FeLV-AKT contains a gag-AKT fusion gene that encodes the myristoylated Gag matrix protein and the kinase domain of feline c-AKT1, but not its pleckstrin homology domain. Therefore, it differs structurally from the v-Akt gene of murine retrovirus AKT8. AKT may be involved in the mechanisms underlying malignant diseases in cats.

Based on the findings of the present study, we conclude that cats can be infected with human influenza viruses as well as avian influenza viruses. Actually, the recent study has shown that both human-type(α2, 6-linked sialic acid) and avian-type(α2, 3-linked sialic acid)influenza virus receptors were extensively detected in the respiratory organs such as trachea, bronchus, and lung of the domestic cats(Wang et al., 2013). Therefore, cats may act as a vector for human influenza virus transmission within households, posing a potential public health concern. Furthermore, we detected both H5N1 and human virus-seropositive cats in neighboring areas at similar sampling times, suggesting that cats can be simultaneously infected with both avian and human viruses in H5N1 virus-endemic areas. Thus, cats, like pigs, may act as an intermediate host for the emergence of new, potentially p and emic viruses.

We established a mass spectrometry-based quantitative method of assaying CD3ε, a component of the T-cell receptor complex. It revealed a CD3ε level of 1 mol per cell in a newly derived canine T-cell lymphoma cell line. Our results suggest that this method has sufficient sensitivity to quantify CD3ε levels in canine lymphoma cells reliably.

Feline leukemia virus (FeLV) induces neoplastic and nonneoplastic diseases in cats. The transduction of cellular genes by FeLV is sometimes observed and associated with neoplastic diseases including lymphoma and sarcoma. Here, we report the first natural case of feline Notch2 transduction by FeLV in an infected cat with multicentric lymphoma and hypercalcemia. We cloned recombinant FeLVs harboring Notch2 in the env gene. Notch2 was able to activate expression of a reporter gene, similar to what was previously reported in cats with experimental FeLV-induced thymic lymphoma. Our findings suggest that the transduction of Notch2 strongly correlates with FeLV-induced lymphoma.

The characteristics of surface antigens in canine hepatocellular carcinoma (cHCC) have not been clarified. The objective of this study was to investigate surface antigens, which are considered as stem/progenitor or cancer cell markers, in cHCC cell lines. Expression of various antigens including CD29, CD34, CD44, CD90, CD133 and Dlk-1 was assessed in four cHCC cell lines by flow cytometry. CD44, CD133 and Dlk-1 expression was detectable in all cell lines, and three cell lines expressed CD29. These results indicate that CD29, CD44, CD133 and Dlk-1 have potential as suitable markers in cHCC identification, suggesting that these findings will contribute to the establishment of an early diagnostic tool for the identification of hepatocellular maturation processes.

Bone marrow cell infusion (BMI) has recently been suggested as an effective therapy for refractory liver disease; however, the efficiency of BMI using canine bone marrow cells (cBMCs) has not been reported. We evaluated the accumulation potential of cBMCs in a mouse model of acute liver failure. Acute hepatitis was induced by carbon tetrachloride (CCl4) treatment in NOD/SCID/γc(null)(NOG) mice and wild-type (WT) C57BL mice, and the characteristics of liver dysfunction and the degree of hepatic injury and regeneration were compared between the two mouse models. Next, female CCl4-treated NOG mice were xenotransplanted with male PKH26-labeled cBMCs, and the potential of cBMCs to accumulate in injured liver tissue compartments was examined. Fluorescence microscopy was performed to histologically detect the infused cBMCs, and DNA polymerase chain reaction was performed for detection of the male Y chromosome (SRY gene) in the recipient female NOG mice. The number of PKH26-positive cBMCs transplanted in the liver tissue gradually increased in the NOG mice. The infused cBMCs were located in the necrotic area of the liver at an early stage after transplantation, and most had accumulated a week after transplantation. However, the therapeutic efficacy of the xenotransplantation remained unclear, because no significant differences were observed concerning the extent liver injury and regeneration between the cBMC-transplanted and saline control mice. These results suggest that cBMCs will specifically accumulate in injured liver tissue and that BMC transplantation may have the potential to repair liver deficiency.

Cutaneous epitheliotropic T-cell lymphoma (CETL) is characterized by neoplastic T-cell infiltration of the epidermis, adnexal structures, and oral mucosa. The objective of this report was to describe the pathological findings of a canine case of terminal-stage CETL. A 10-year-old, mixed-breed, neutered male dog was presented with erosion of the oral mucosa and mucocutaneous junction. The dog was diagnosed with CETL with no evidence of metastasis. Despite chemotherapy, the dog was re-presented with oral pain, vomiting, and diarrhea, and died 17 months after the first visit to the hospital. A complete autopsy was performed. Histologic examination of the primary lesion and systemic organs was performed. Gross examination revealed an advanced-stage oral lesion. Distinct tumor formation was not observed in the primary sites and systemic organs. Histologically, the primary oral lesion was characterized by massive intraepithelial infiltration of a large number of neoplastic lymphocytes. The neoplastic cells in the metastatic sites also showed exclusive epitheliotropic proliferation in organs, including the trachea, tonsils, esophagus, stomach, small intestine, colon, anal mucosa, liver, pancreas, kidneys, urinary bladder, prostate gland, ear canals, and auricular and ventral skin. Immunohistochemically, the neoplastic cells were positive for CD3 and negative for CD20 as well as CD79α, supporting a diagnosis of CETL with systemic dissemination. In canine CETL with systemic signs, systemic metastasis should be considered even without evident mass formation. Neoplastic lymphocytes of CETL showed distinct epitheliotropism even in the systemic metastatic sites.

Background Platelet concentrates (PC) are prepared by centrifugation of platelet-rich plasma (PRP) that is prepared by centrifugation of whole blood. The resuspension of the platelet pellet during PC preparation from dogs is difficult because of platelet activation induced by centrifugation. Objectives To investigate the efficacy of adding prostaglandin E1 (PGE1) to prevent platelet activation during PC preparation from dogs. Animals Fifteen healthy Beagle dogs. Methods Prospective, experimental trial: PGE1 was added to PRP before the high-speed centrifugation during PC preparation. To estimate the effect of this addition, we assessed the platelet aggregability before transfusion, the survival of the platelets after transfusion, and the platelet reactivity after transfusion, which is estimated by the P-selectin expression of the platelets when stimulated by thrombin. Results The difficulty associated with platelet resuspension was resolved by PGE1.PGE1 strongly inhibited platelet aggregation induced by collagen and ADP; however, it recovered after the platelets were resuspended in plasma without PGE1 (mean aggregation ratio; collagen: 10.00–80.80%, ADP: 8.20–53.60%). Survival of the platelets after transfusion was not affected by PGE1 (mean 8.04 and 7.56 days, without and with PGE1), and thrombin-induced P-selectin expression after transfusion in PGE1-treated PC was also well maintained (mean positive ratio 53.7 and 47.9%, before and 24 hours after transfusion). Conclusions and Clinical Importance The addition of PGE1 in PRP before the centrifugation of PRP can improve the preparation efficiency of PC from dogs, while maintaining the therapeutic efficacy of the platelets.

Background: Intravenous administration of human immunoglobulin G (hIVIgG) has been suggested to potentiate thromboembolism in dogs, but supportive scientific reports are lacking. Objectives: To determine if hIVIgG therapy promotes hypercoagulability and inflammation in dogs. Animals: Twelve healthy Beagle dogs. Methods: Prospective, experimental trial. An hIVIgG/saline solution was infused IV at 1 g/kg BW over 8 hours to 6 dogs, and physiological saline was infused to the other 6 dogs. Blood samples were drawn before, during, and after infusion for serial measurement of indicators of coagulation and inflammation. Data were analyzed by 2-way repeated measures analysis of variance. Results: Dogs administered hIVIgG developed mildly decreased blood platelet concentrations without thrombocytopenia (median, 200 × 103/μL; range, 150–302 × 103/μL; P < .01), leukopenia (median, 3.5 × 103/μL; range, 20–62 × 103/μL; P < .001), and mildly increased plasma total protein concentrations (median, 6.3 g/dL; range, 5.6–6.7 g/dL; P < .001). Administration of hIVIgG was also associated with increases in fibrin/fibrinogen degradation products in all dogs (either 5 μg/mL or 10 μg/dL), thrombin-antithrombin III complexes (median, 7.2 ng/mL; range, 4.9–14.2 ng/mL; P < .001), and C-reactive protein concentrations (median, 2.5 mg/dL; range, 0.5–4.3 mg/dL; P < .01). Conclusion and Clinical Importance: Administration of hIVIgG to dogs promotes hypercoagulability and an inflammatory state. This should be further evaluated and considered when using hIVIgG in dogs with IMHA or other prothrombotic conditions.

Hepatocyte growth factor (HGF) can stimulate human and rat bone marrow (BM) cells to differentiate into hepatocytes. A human placental hydrolysate (hPH) stimulates proliferation of hepatocytes, but its role as a potential inducer of BM cells to form hepatocytes is unclear. To determine if canine BM cells stimulated with HGF or hPH differentiate into hepatocyte-like cells, BM cells were cultured with HGF or hPH. The cultured cells underwent morphological examination, expression of albumin and cytokeratin 18 (CK18), hepatic function tests including uptake of low-density lipoprotein (LDL) and cytochrome P (CYP) 450 activity. Albumin mRNA and protein expression of albumin and CK18 proteins were detected in cultures with HGF and hPH. Furthermore, these cells demonstrated LDL uptake and CYP450 activity. These results indicate that canine BM cells can differentiate into hepatocyte-like cells when stimulated by both HGF and that hPH may be an effective inducer of hepatic differentiation.

Feline leukemia virus (FeLV) clone33 was obtained from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV from other cats with AML, differs from the LTRs of other known FeLV in that it has 3 tandem direct 47-bp repeats in the upstream region of the enhancer (URE). Here, we injected cats with FeLV clone33 and found 41% developed myelodysplastic syndromes (MDS) characterized by peripheral blood cytopenias and dysplastic changes in the bone marrow. Some of the cats with MDS eventually developed AML. The bone marrow of the majority of cats with FeLV clone33 induced MDS produced fewer erythroid and myeloid colonies upon being cultured with erythropoietin or granulocyte-macrophage colony-stimulating factor (GM-SCF) than bone marrow from normal control cats. Furthermore, the bone marrow of some of the cats expressed high-levels of the apoptosis-related genes TNF-alpha and survivin. Analysis of the proviral sequences obtained from 13 cats with naturally occurring MDS reveal they also bear the characteristic URE repeats seen in the LTR of FeLV clone33 and other proviruses from cats with AML. Deletions and mutations within the enhancer elements are frequently observed in naturally occurring MDS as well as AML. These results suggest that FeLV variants that bear URE repeats in their LTR strongly associate with the induction of both MDS and AML in cats.

本研究では,犬の肝細胞癌株化細胞または正常肝細胞を用いHGFの細胞増殖に及ぼす影響について検討した。肝癌細胞株4種を用いて検討した結果,3様の反応が認められた。一定の濃度まで増殖が促進され,それ以上の濃度では増殖がプラトーに達する株(CHKS-rL),一定の濃度まで増殖が促進され,さらに濃度を増すと増殖が抑制される傾向を示す株(95-1044),HGFに反応が乏しく増殖促進,抑制の影響が明らかではない株(KO, 930-599A)に分類された。一方,正常犬肝細胞とHGFに増殖反応を示す肝癌細胞で,増殖を促進する濃度が類似しているため,HGFの利用は困難であり,HGFで増殖を示さない肝癌細胞(KO, 930-599A)で利用可能と考えられた。治療に際しHGFに対する反応を簡易に検出することが重要と考えられた。, In this study, we examined the effects of feline recombinant HGF (frHGF) on proliferation of four types of canine hepatocellular carcinoma cells and normal canine hepatic cells in culture. Three types of responses were observed in the tumor cells. One was increased growth correlated with increasing HGF up to a certain concentration when a growth ceiling was reached (CHKS-rL cells). The second was growth correlated with increasing concentration of HGF, after which growth decreased gradually in inverse proportion to HGF concentration (95-1044 cells). The remaining two cell lines (KO, 930-599A) did not show a consistent reaction to HGF. Normal canine hepatic cells also proliferated at an HGF concentration close to that of CHKS-rL and 95-1044 cells. Thus, HGF for treatment of patients bearing tumors of CHKS-rL or 95-1044 cells will be ineffective; however, it is a useful treatment for patients with tumors of KO or 930-599A cells.

Antisilencing function 1 (ASF1) is a conserved histone chaperone implicated in nucleosome assembly, transcriptional silencing, and the cellular response to DNA damage. Here, we report the identification of human ASF1B, but not ASF1A, as a direct transcriptional target of transcription factor E2F1. We demonstrated that overexpression of E2F1 by adenoviral-mediated gene transfer upregulated ASF1B mRNA expression in HeLa cells. Analysis of human ASF1B promoter constructs showed that an E2F-responsive sequence was necessary for E2F1-induced activation of the ASF1B gene transcription. Oligonucleotides including an E2F consensus sequence were specifically bound by E2F1 protein in vitro. Chromatin immunoprecipitation analysis demonstrated that E2F1 bound to an E2F-responsive sequence of the human ASF1B gene. Among the members of the E2F family, E2F1 to E2F5, but not E2F6, activated the ASF1B reporter construct. Sp1 and NFYA failed to induce the activity of the ASF1A and ASF1B promoter. ASF1A and ASF1B mRNA were upregulated by serum stimulation. Taken together, our results suggest that the expression of human ASF1A and ASF1B are upregulated followed by cell proliferation signal, but that of ASF1B is uniquely regulated by transcription factors E2F during cell cycle progression.