Establishment and characterization of urothelial carcinoma cell lines with and without BRAF mutation (V595E) in dogs.

Each 5 urothelial carcinoma (UC) cell lines with and without the v-Raf murine sarcoma virus oncogene homolog B (BRAF) gene mutation (V595E) were established and examined V595E-related tumorigenic characteristics in dogs. No typical morphological features were observed in cloned cells with and without V595E. The cell proliferation of both cloned cells showed logarithmic growth curve and those doubling time were 24.9 ± 4.1 h in V595E ( +) and 29.3 ± 11.3 h in V595E ( -). On the growth curve of xenotransplanted tumor in severe combined immunodeficiency mice, 3 out of 5 V595E ( +) and 2 out of 5 V595E ( -) cloned cells revealed gradually and remarkably increasing curve, indicating clearly tumorigenicity. The xenotransplanted tumors with V595E ( +) showed typical features of UC, such as solid proliferation of pleomorphic tumor cells, formation of papillary structure, and glandular structure. Additionally, various vascular formation was observed, probably indicating an advanced growth phase of UC. In mitogen-activated protein kinase (MAPK) signaling pathway, cytoplasmic phosphorylated-BRAF (pBRAF) and cytoplasmic and nuclear phosphorylated-ERK1/2 (pERK1/2) were detected in all 4 tumors with V595E ( +), whereas only cytoplasmic and nuclear pERK1/2 was detected in tumors with V595E ( -). Since V595E can directly activate MAPK signaling pathway, coincidence of V595E with pBRAF (phosphor Thr598/Ser601) indicates acquired resistance to BRAF inhibitors. These established UC cell lines, especially V595E ( +) cell lines, are useful tool for understanding pathophysiological states and controlling therapeutic manners of UC in dogs.

Coincidence of v-raf murine sarcoma viral oncogene homolog B mutation (V595E) with phosphorylated v-raf murine sarcoma viral oncogene homolog B in urothelial carcinoma in dogs.

Expression of phosphorylated v-raf murine sarcoma viral oncogene homolog B (pBRAF) and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) were investigated in urothelial carcinoma (UC) in dogs with or without the BRAF gene mutation (V595E). Among the 10 cases of UC with V595E (-), cytoplasmic immunoreactivity against pBRAF of neoplastic cells was reported in 8, with 7 displaying moderate reactivity and 1 displaying intense reactivity. Nuclear immunoreactivity against pBRAF was detected in 5 cases; however, these reactivities were non-specific, due to pBRAF being limited in the cytoplasm. In addition, positive cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells was detected in 7 cases and nuclear immunoreactivity against ERK1/2 was detected in 6 cases. Among the 13 cases of UC with V595E (+), cytoplasmic immunoreactivity against pBRAF of neoplastic cells was detected in all 13 cases and nuclear immunoreactivity against pBRAF was detected in 10 cases; however, the nuclear immunoreactivity was non-specific. Cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells was detected in all 13 cases and nuclear immunoreactivity against pERK1/2 was also detected in all cases. As nuclear pERK1/2 indicates a progressive signaling process in the mitogen-activated protein kinase pathway, V595E (+) UC might be in its growing stage. Probable phosphorylated sites of pBRAF at Thr598/Ser601, detected in this study, are major and essential sites of the upstream rat sarcoma viral oncogene homolog (RAS) signaling pathway. In human cancers, the BRAF mutation never coincides with oncogenic RAS. To our knowledge, this is the first report on the simultaneous occurrence of the BRAF mutation (V595E) and pBRAF expression (at Thr598/Ser601) in dogs with UC with V595E (+).

L'expression de l'homologue B de l'oncogène viral du sarcome murin phosphorylé v raf (pBRAF) et de la kinase1/2 régulée par le signal extracellulaire phosphorylé (pERK1/2) ont été étudiées dans le carcinome urothélial (CU) chez des chiens avec ou sans la mutation du gène BRAF (V595E). Parmi les 10 cas de CU avec V595E (−), une immunoréactivité cytoplasmique contre pBRAF de cellules néoplasiques a été rapportée chez huit, sept présentant une réactivité modérée et un présentant une réactivité intense. L'immunoréactivité nucléaire contre pBRAF a été détectée dans cinq cas; cependant, ces réactivités n'étaient pas spécifiques, car pBRAF était limité dans le cytoplasme. De plus, une immunoréactivité cytoplasmique positive contre pERK1/2 des cellules néoplasiques a été détectée dans sept cas et une immunoréactivité nucléaire contre ERK1/2 a été détectée dans six cas. Parmi les 13 cas de CU avec V595E (+), une immunoréactivité cytoplasmique contre pBRAF de cellules néoplasiques a été détectée dans les 13 cas et une immunoréactivité nucléaire contre pBRAF a été détectée dans 10 cas; cependant, l'immunoréactivité nucléaire était non spécifique. L'immunoréactivité cytoplasmique contre pERK1/2 des cellules néoplasiques a été détectée dans les 13 cas et l'immunoréactivité nucléaire contre pERK1/2 a également été détectée dans tous les cas. Comme le pERK1/2 nucléaire indique un processus de signalisation progressif dans la voie de la protéine kinase activée par les mitogènes, V595E (+) UC pourrait être dans sa phase de croissance. Les sites phosphorylés probables de pBRAF à Thr598/Ser601, détectés dans cette étude, sont des sites majeurs et essentiels de la voie de signalisation de l'oncogène viral (RAS) du sarcome de rat en amont. Dans les cancers humains, la mutation BRAF ne coïncide jamais avec le RAS oncogène. À notre connaissance, il s'agit du premier rapport sur la survenue simultanée de la mutation BRAF (V595E) et de l'expression de pBRAF (à Thr598/Ser601) chez des chiens atteints de CU avec V595E (+).(Traduit par Docteur Serge Messier).

Establishment and characterization of a novel cell line of medullary thyroid carcinoma from a dog.

A novel cell line of canine medullary thyroid carcinoma (MTC) was established from the neck mass, diagnosed histopathologically and immunohistochemically as ectopic MTC. The neoplastic cells arranging trabecular structures were characterized as pleomorphic cells with eosinophilic cytoplasm and nucleus, containing often clear nucleolus. These tumor cells were immuno-positive for calcitonin gene-related protein (CGRP), somatostatin, and chromogranin A. In addition, 8th passaged cultured cells were also immuno-positive for CGRP, somatostatin, and chromogranin A. The cloned tumor cells showed logarithmic cell growth with a doubling time of 33.3 h. From the results of DNA sequencing of rearranged during transfection (RET) proto-oncogene, the cloned tumor cells had four single base substitution, including exon 5 codon 82, exon 16 codon 750, exon 17 codon 777, and exon 24 codon 1085, all of which were single nucleotide polymorphism reported in RET gene of dogs. After the xenotransplantation into severe combined immunodeficiency (SCID) mice, the cloned cells showed tumorigenicity potentials. The morphological and immunohistochemical features of the xenotransplanted tumor were almost in conformity with those of the original tumor, including positive immunoreactivity for calcitonin, CGRP, and chromogranin A. To our knowledge, this is the first report of canine MTC cell line, which provides useful in vitro tool for understanding oncogenic mechanism and pathophysiological state of MTC in dogs.

Hypertrophic neuritis causing tetraparesis in a cat.

An 8-year-old castrated male cat presented with acute ataxia and paresis in all four limbs. The cat also exhibited signs of autonomic nervous system impairment. Magnetic resonance imaging revealed swelling of the brachial plexuses bilaterally. Despite treatment, the cat died after 10 days of treatment. A postmortem examination revealed swollen radial nerves and cervical nerve roots in which infiltration of inflammatory cells was histologically confirmed. Additionally, lymphocytic infiltration was found around the blood vessels of the sciatic nerve bundle and the vagus nerve. Histological features were comparable to previously reported brachial plexus hypertrophic neuritis in a cat. Our case was unique in that the autonomic nerves were also involved in addition to the somatic nerves in all four limbs.

Development of an ELISA for serological detection of feline morbillivirus infection.

Feline morbillivirus (FeMV), a member of the family Paramyxoviridae, is an emerging virus that was discovered in 2012. Despite the importance of FeMV infection in cats because of its postulated involvement in kidney diseases, no simple serological assay has been reported in its detection. Here, FeMV phosphoprotein (P protein) was expressed and purified as a glutathione-S-transferase (GST)-fusion protein and used for an enzyme-linked immunosorbent assay (ELISA) to detect FeMV-specific antibodies. With a cutoff value determined by immunoblotting, anti-FeMV P protein was detected with this assay in 22 (22%) of the 100 cat plasma samples collected from various regions of Japan. This ELISA is useful for epidemiological and immunological studies, as well as for diagnosis of FeMV infection.

Quantitative PCR detection of feline morbillivirus in cat urine samples.

Feline morbillivirus (FmoPV) is a new virus species and its detection is important, since correlation has been reported between FmoPV virus infection and tubulointerstitial nephritis in cats. Here, we report a real-time reverse transcription (RT)-PCR system that can detect the FmoPV L-gene sequence with more than 10-time higher sensitivity than a conventional PCR system, resulting in detection of less than 10 copies of the template DNA. The total FmoPV positive rate of urine samples from veterinary clinics and hospitals in Japan was 15.1% (25/166) using this system. This study demonstrates usefulness of the real-time RT-PCR system for detection of FmoPV for cat urine samples.

Molecular epidemiological study of feline coronavirus strains in Japan using RT-PCR targeting nsp14 gene.

BACKGROUND: Feline infectious peritonitis is a fatal disease of cats caused by infection with feline coronavirus (FCoV). For detecting or genotyping of FCoV, some RT-PCR plus nested PCR techniques have been reported previously. However, referring to the whole genome sequences (WGSs) registered at NCBI, there are no detection methods that can tolerate the genetic diversity among FCoV population. In addition, the quasispecies nature of FCoV, which consists of heterogeneous variants, has been also demonstrated; thus, a universal method for heteropopulations of FCoV variants in clinical specimens is desirable. RESULTS: To develop an RT-PCR method for detection and genotyping of FCoV, we performed comparative genome analysis using WGSs of 32 FCoV, 7 CCoV and 5 TGEV strains obtained from NCBI. As the PCR target, we focused on the nsp14 coding region, which is highly conserved and phylogenetically informative, and developed a PCR method targeting nsp14 partial sequences. Among 103 ascites, 45 pleural effusion and 214 blood specimens from clinically ill cats, we could detect FCoV in 55 (53.4%), 14 (31.1%) and 19 (8.9%) specimens using the present method. Direct sequencing of PCR products and phylogenetic analysis allowed discrimination between type I- and II-FCoV serotypes. Our nsp14 amino acid sequence typing (nsp14 aa ST) showed that the FCoV clone with sequence type (ST) 42, which was the most predominant genotype of WGS strains, was prevalent in domestic cats in Japan. CONCLUSIONS: Our nsp14 PCR scheme will contribute to virus detection, epidemiology and ecology of FCoV strains.

Existence of feline morbillivirus infection in Japanese cat populations.

Feline morbillivirus (FmoPV) is a member of a new virus species that has only been found in the Hong Kong cat population. For the first time, however, we have now detected nucleotide sequences similar to FmoPV in samples from Japanese cat populations. The positive rates for urine and blood samples from Japanese cats were 6.1 % (5/82) and 10 % (1/10), respectively. These sequences are similar to the previously reported FmoPV, with 92-94 % identity, and substantially different from all other morbilliviruses. Phylogenetic analysis of the identified Japanese FmoPVs and other morbilliviruses demonstrated a pattern similar to those previously published for the FmoPV viruses isolated in Hong Kong. FmoPV RNA was also detected from formalin-fixed paraffin-embedded (FFPE) kidney tissues of cats with nephritis, with a positive rate of 40 % (4/10). By using nested-set primers based on the FmoPV sequence and RNA from FFPE tissues, we demonstrated the existence of FmoPV infection in Japanese cats and established the method for detection of the FmoPV RNA from kidney tissues prepared for pathology examinations, which is useful for studies on the pathogenicity of the virus.

Immunophenotyping and gene rearrangement analysis in dogs with lymphoproliferative disorders characterized by small-cell lymphocytosis.

Lymphocytosis caused by neoplastic proliferation of small lymphocytes is occasionally difficult to distinguish by morphological examination from nonneoplastic lymphocytosis. To examine the clinical utility of gene rearrangement analysis for demonstrating neoplastic proliferation of small lymphocytes, gene rearrangement analysis was performed in comparison with immunophenotyping using peripheral lymphocytes in dogs with small lymphocytosis. Thirty-one dogs with small-cell lymphocytosis (8,100-884,300/microl) were enrolled. By immunophenotyping, lymphocytosis of all dogs was suggested to be neoplastic in nature based on the detection of marked expansion of phenotypically homogeneous lymphocytes or the presence of an aberrant antigen-expressing population of lymphocytes. In contrast, gene rearrangement analysis represented clonality in 27 dogs (detection rate of 87%). From the present study, gene rearrangement analysis was considered to be worthwhile to strengthen the evidence of neoplastic proliferation of small lymphocytes when coupled with immunophenotyping and to be a suitable diagnostic substitute if immunophenotyping is not available in clinical practice.

Generation of monoclonal antibody against canine neural-cell adhesion molecule.

A monoclonal antibody, K9BYU, was generated using Escherichia coli recombinant extracellular domain of canine neural-cell adhesion molecule (N-CAM) as an antigen. Immunoreactivity of K9BYU to insect cell recombinant canine N-CAM was demonstrated by Western blotting using Sf9 insect cells transfected with the canine N-CAM gene. In Western blotting against canine brain tissue, K9BYU detected three isoforms of N-CAM that correspond to three major isoforms of human and mouse N-CAM (N-CAM-120, -140, and -180). From these results, K9BYU was considered to be a useful tool for research of canine N-CAM.

Changes in peripheral lymphocyte subsets in the type 1 diabetic dogs treated with insulin injections.

Plasma metabolites and peripheral lymphocyte subsets were measured in ten diabetic and ten control dogs to investigate their significances as indicators to evaluate immune states in the diabetic dogs. Diabetic dogs were treated with insulin injections, however their plasma glucose and fructosamine concentrations were significantly higher than those of the controls. There were no significant differences in counts of total white blood cells (WBC) and lymphocyte CD8(+) cells (cytotoxic T cells) between the control and the diabetic dogs. In the diabetic dogs, the counts of CD3(+) (T cells), CD4(+) (Helper T cells) and CD21(+) (B cells) cells and the peripheral lymphocytes CD4/CD8 ratio were significantly lower than those in the control dogs. We confirmed abnormality of lymphocyte subsets in insulin treated diabetic dogs and it may relate to depression of immunocompetence and high susceptibility to common infectious diseases.

Disassembly of actin filaments by botulinum C2 toxin and actin-filament-disrupting agents induces assembly of microtubules in human leukaemia cell lines.

BACKGROUND INFORMATION: C(2) toxin produced by Clostridium botulinum types C and D ADP-ribosylates actin monomers and inactivates their polymerization activities. The disassembly of actin filaments by C(2) toxin induces a polarization of cultured human leukaemia cell lines. RESULTS: The polarization induced by C(2) toxin was temperature dependent and was prevented by nocodazole, a microtubule-disrupting agent, whereas it was promoted by paclitaxel, a microtubule-stabilizing agent. The fluorescence staining of polarized cells indicated an increase in microtubule assembly accompanying disassembly of actin filaments. Furthermore, several actin-filament-disrupting agents, other than C(2) toxin, also induced microtubule assembly and cell polarization, irrespective of their different mechanisms of action. The effects induced by some of the agents, which have lower binding affinities for actin, were reversible in response to the re-assembly of actin filaments. CONCLUSIONS: Thus the disassembly of actin filaments by C(2) toxin and actin-filament-disrupting agents induces assembly of microtubules followed by polarization of human leukaemia cell lines, indicating that the assembly/disassembly equilibrium of actin filaments influences the dynamics of microtubules, which control cell morphology and, in turn, diverse cellular processes.