Gene expression profiles associated with early relapse during first remission induction in canine multicentric high-grade B-cell lymphoma.

Although chemotherapy using CHOP-based protocol induces remission in most cases of canine multicentric high-grade B-cell lymphoma (mhBCL), some cases develop early relapse during the first induction protocol. In this study, we examined the gene expression profiles of canine mhBCL before chemotherapy and investigated their associations with early relapse during the first whole CHOP-based protocol. Twenty-five cases of mhBCL treated with CHOP-based protocol as first induction chemotherapy were included in this study. Sixteen cases completed the first whole CHOP-based protocol without relapse (S-group), and nine developed relapse during the chemotherapy (R-group). RNA-seq was performed on samples from neoplastic lymph nodes. Differentially expressed genes (DEGs) were extracted by the comparison of gene expression profiles between S- and R-groups, and the differences in the expression levels of these genes were validated by RT-qPCR. Extracted 179 DEGs included the genes related to chemokine CC motif ligand, T-cell receptor signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway. We focused on chemokine CC motif ligand, and CCL4 was confirmed to be significantly downregulated in the R-group (P=0.039). We also focused on the genes related to T-cell signaling pathway, and CD3E (P=0.039), ITK (P=0.023), and LAT (P=0.023) genes were confirmed to be significantly upregulated in the R-group. The current results suggest that both changes in tumor cells and the interactions between tumor cells and immune cells are associated with the efficacy of the chemotherapy for first remission induction.

Abnormal movement of the pulmonary valve in dogs diagnosed with patent ductus arteriosus.

In the typical left-to-right patent ductus arteriosus (PDA), the shunt flows from the ductus arteriosus towards the pulmonary valve. Although hemodynamic changes have been carefully studied in dogs with PDA, there is very little information on the outcomes of the pulmonary valve after surgical correction of PDA. This study aimed to visualize the pulmonary valve by transthoracic echocardiography in dogs with PDA before and after surgical ligation. Prior to surgery, the movement of the anterior semilunar cusp of the pulmonary valve was obstructed by the shunted blood flow during systole in all nine dogs with PDA in this study. M-mode echocardiography revealed a continuous trajectory of the cusp, because the cusp was pushed towards the right ventricle during the whole cardiac cycle by the shunted flow. Epicardial echocardiography performed in one dog during surgical ligation of the ductus arteriosus revealed that the movement of the anterior semilunar cusp normalized immediately after ligation. B- and M-mode echocardiography may be used to support the diagnosis of PDA through observation of the pulmonary valve when color Doppler echography is not available. The findings in this study may be of importance in distinguishing PDA from PDA-mimicking diseases worth considering before the treatment process (e.g. aorticopulmonary fistulas or aberrant arteriovenous shunts).

Evaluation of prognostic factors and establishment of a prognostic scoring system for canine primary immune-mediated hemolytic anemia.

Clinical courses of primary immune-mediated hemolytic anemia (pIMHA) in dogs are highly variable, however, limited information is available to predict their accurate prognoses. To evaluate the prognostic significance of clinical factors and to propose a scoring system to predict prognoses, the medical records of seventy-one dogs with pIMHA were reviewed. Overall mortality rate of dogs with pIMHA was 39% and most of the dogs died within 3 months from diagnosis. Sex, body weight, seasonality, packed corpuscular volume (PCV), platelet count (PLT), total plasma protein (TP), blood urea nitrogen, albumin, total bilirubin, sodium ion, prothrombin time, and fibrin/fibrinogen degradation products before immunosuppressive treatment can influence on survival time in dogs with pIMHA. A prognostic scoring system using a combination of sex, seasonality, PCV, PLT and TP can be statistically significant for raising the accuracy of prognostic prediction. Using the scoring system for prognostication in dogs with pIMHA may enable veterinarians to predict a prognosis easily and accurately.

An epidemiological survey of feline hemoplasma infection in Japan.

Hemoplasma (hemotropic mycoplasma) often causes hemolytic anemia in infected cats, especially those with immune suppression. An updated nationwide epidemiological survey of feline hemoplasmosis was conducted in Japan. Blood samples were collected from 1,770 outdoor-accessing cats from March to October 2008. The infections were molecularly detected by PCR analyses, which are able to distinguish Mycoplasma haemofelis (Mhf), `Candidatus M. haemominutum' (CMhm), and `Candidatus M. turicensis' (CMt) infections. Of the 1,770 cats, 468 cases (26.4%) revealed a single- or co-infection of feline hemoplasmas [Mhf alone, 42 cases (2.4%); CMhm alone, 280 cases (15.8%); CMt alone, 48 cases (2.7%); Mhf+CMhm, 28 cases (1.6%); Mhf+CMt, 6 cases (0.3%); CMhm+CMt, 50 cases (2.8%); Mhf+CMhm+CMt, 14 cases (0.8%)]. In addition, male gender, middle to old age, history of fight wounds, and feline immunodeficiency virus infection were shown to be risk factors for hemoplasma infection. Close attention must be paid to the acute onset of disease in feline practice because a prevalence of hemoplasma infection was detected even in clinically healthy cats.

An updated nation-wide epidemiological survey of feline immunodeficiency virus (FIV) infection in Japan.

An updated nation-wide epidemiological survey of feline immunodeficiency virus (FIV) infection was conducted in Japan. Blood samples were collected from 1,770 outdoor accessing cats from March to October 2008. Serologically, 410 cats (23.2%) were positive for anti-FIV antibody. Proviral DNA of the FIV env V3-V5 region isolated from 348 cases could be phylogenetically analyzed. The present study disclosed a geographic distribution of four subtypes (A, B, C and D) of FIV in Japan. Even though an FIV vaccine was introduced in Japan, we do not currently know whether this vaccine is effective against all strains of FIV in Japan or not. Therefore, close attention still has to be paid to epidemic and genotypic trends of FIV.

Antiviral activity of membrane fusion inhibitors that target gp40 of the feline immunodeficiency virus envelope protein.

For the entry of lentivirus into target cells, fusion between its viral membrane and cellular membrane is essential. The present study was conducted to examine the inhibitory effect of modified peptides corresponding to heptad repeats (HR) 1 and 2 of feline immunodeficiency virus (FIV) envelope gp40 on the fusion between the viral and cellular membranes. FIV-N36 and FIV-C35 were synthesized as authentic peptides of the N-terminal HR1 domain and C-terminal HR2 domain of FIV gp40, respectively. FIV-C35EK1, FIV-C35EK2, and FIV-C35EK3 were peptides synthesized by modifying FIV-C35 as the X-EE-XX-KK concept to increase their solubility in water and the stability of their alpha-helicity. FIV-C35 and FIV-C35EK1 inhibited the cell membrane fusion mediated by FIV-infected cells and the replication of FIV. FIV-N36, FIV-C35EK2, and FIV-C35EK3 did not show any apparent inhibitory effect. These results indicated that the newly developed membrane fusion inhibitors could facilitate the development of novel anti-lentiviral chemotherapies.

Cutaneous nocardiosis in a cat.

A 4-year-old spayed female domestic shorthair cat was presented to us for swelling of all foot pads. The skin lesions were histopathologically diagnosed as suppurative pyogranulomatous panniculitis. The lesions did not respond to cephalexin, prednisolone or itraconazole. However, complete resolution of the skin lesions was obtained with doxycycline. A littermate living in the same household developed similar skin lesions that were also successfully treated with doxycycline. Polymerase chain reaction analysis detected the 16S ribosomal RNA gene of Nocardia spp. in DNA extracted from lesion pus, and direct nucleotide sequencing analysis revealed 100% homology with Nocardia elegans. We diagnosed this case as nocardiosis.

A case report: a dog with acute onset of Hepatozoon canis infection.

We present a clinical overview of a dog with acute onset of Hepatozoon canis infection. A stray female beagle dog of unknown age was referred to Kagoshima University showing anemia. Blood tests revealed the presence of anemia, thrombocytopenia, hyperproteinemia, polyclonal gammopathy, hypoalbuminemia, and elevated creatine kinase and alkaline phosphatase activities. In addition, capsule-like organisms were detected in the cytoplasm of approximately 50% of neutrophils in blood smears. H. canis infection was confirmed by polymerase chain reaction and DNA sequencing analyses. Amplified DNA fragments revealed 100% identity to the 18S ribosomal RNA gene of H. canis. The clinical symptoms improved after the administration of antibiotics. Hepatozoonosis in dogs is rare, but veterinarians should be alert to its possible acute onset.

C-reactive protein concentration in dogs with various diseases.

To investigate the clinical utility of C-reactive protein (CRP) determination in dogs, its plasma concentration was measured by a laser nephelometric method in 928 dogs with various diseases, and was compared with other inflammatory parameters. CRP concentration was elevated in various inflammatory diseases, this was most frequently observed in cases with neoplastic and immune-mediated diseases. All cases of pyometra, panniculitis, acute pancreatitis, polyarthritis, and hemangiosarcoma showed significantly elevated CRP concentrations. On the other hand, the CRP concentration was elevated only in few cases of neurological diseases such as epilepsy, meningoencephalitis, and hydrocephalus and endocrine diseases such as hypothyroidism, hyperadrenocorticism, and diabetes mellitus. Only a weak correlation was observed between the CRP concentration and white blood cell (WBC) counts (r=0.44) but no correlation with band neutrophil counts. There was no correlation between the CRP and albumin concentrations, but a weak negative correlation (r=-0.40) when excluding chronic intestinal diseases and nephrotic syndrome, which can cause protein loss. Thus, CRP can be useful to detect inflammations that cannot be detected by WBC and, or band neutrophil counts, suggesting that the examination of CRP concentration is essential as routine diagnostic test.

Measurement of intestinal mucosal permeability in dogs with lymphocytic-plasmacytic enteritis.

Lymphocytic-plasmacytic enteritis (LPE) is a type of canine inflammatory bowel disease (IBD). One of its most probable causes is a defect in the mucosal permeability barrier. In the present study, intestinal permeability in LPE dogs was examinated to evaluate its clinical value. Twenty-nine dogs with LPE diagnosed by clinical and histological examinations were included in this study. Intestinal permeability was evaluated by measuring the ratio of the concentrations of two sugars (lactulose (L) and rhamnose (R)) with different molecular weights in urine samples after oral administration of a solution containing them. Biopsy specimens of duodenum were evaluated according to histological criteria. The urinary L:R ratio in the 29 LPE dogs (1.68 +/- 1.17, mean +/- SD) was significantly higher than that in the 10 healthy control dogs (0.75 +/- 0.38, P<0.01). In the LPE dogs, a weak correlation was observed between the histopathological grading score of the duodenum and the urinary L:R ratio (r=0.408, P<0.05). The urinary L:R ratio in the 20 dogs showing hypoalbuminemia (< 2.5 g/dl) was significantly higher than that in the 9 dogs with normal serum albumin levels > 2.5 g/dl (P<0.01). In conclusion, permeability of the intestinal mucosa as determined by the urinary L:R ratio could be a useful laboratory parameter for evaluating intestinal damage in LPE dogs.

Rapid immunocytochemistry for the detection of cytokeratin and vimentin: assessment of its diagnostic value in neoplastic diseases of dogs.

BACKGROUND: Immunocytochemistry (ICC) is an advanced diagnostic technique used in the field of veterinary cytology. We recently developed a rapid ICC method for the detection of cytokeratin and vimentin in dogs, which helps to determine whether tumor cells are of epithelial or nonepithelial origin. However, the diagnostic value of this rapid ICC method in neoplastic diseases of dogs has not been assessed yet. OBJECTIVES: The aim of the present study was to assess the diagnostic accuracy of rapid ICC compared to standard immunohistochemistry (IHC). METHODS: Air-dried smear samples and formalin-fixed paraffin sections were prepared from tumors excised from dogs (n = 30). Immunosignals for cytokeratin and vimentin were detected in smear samples by rapid ICC, and in paraffin sections by standard IHC. Signals in smear samples detected by rapid ICC were compared with positive staining in paraffin sections detected by standard IHC and analyzed for statistical significance (kappa statistic). RESULTS: Rapid ICC detected specific immunosignals in 25/30 cases (83.3%), and nonspecific signals were detected in 5/30 cases. Statistical analysis revealed fair agreement in epithelial tumors (n = 16) with cytokeratin (κ = 0.236) and vimentin (κ = 0.294). In nonepithelial tumors (n = 14), almost perfect agreement was demonstrated with cytokeratin (κ = 0.857) and vimentin (κ = 0.857). CONCLUSIONS: The rapid ICC method can be a useful tool for the diagnostic cytology of neoplastic tissues in dogs.

Hypoxia-activated prodrug TH-302 decreased survival rate of canine lymphoma cells under hypoxic condition.

We tested the hypotheses that hypoxic stimulation enhances growth potentials of canine lymphoma cells by activating hypoxia-inducible factor 1α (HIF-1α), and that the hypoxia-activated prodrug (TH-302) inhibits growth potentials in the cells. We investigated how hypoxic culture affects the growth rate, chemoresistance, and invasiveness of canine lymphoma cells and doxorubicin (DOX)-resistant lymphoma cells, and influences of TH-302 on survival rate of the cells under hypoxic conditions. Our results demonstrated that hypoxic culture upregulated the expression of HIF-1α and its target genes, including ATP-binding cassette transporter B1 (ABCB1), ATP-binding cassette transporter G2 (ABCG2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and survivin, and enhanced the growth rate, DOX resistance, and invasiveness of the cells. Additionally, TH-302 decreased the survival rate of the cells under hypoxic condition. Our studies suggest that hypoxic stimulation may advance the tumorigenicity of canine lymphoma cells, favoring malignant transformation. Therefore, the data presented may contribute to the development of TH-302-based hypoxia-targeting therapies for canine lymphoma.

Effects of toceranib phosphate (Palladia) monotherapy on multidrug resistant lymphoma in dogs.

We examined whether multidrug resistant (MDR) canine lymphoma increases gene expression for platelet-derived growth factor receptor α (PDGFRα), vascular endothelial growth factor receptor 2 (VEGFR2), and c-KIT, and whether toceranib phosphate (TOC) has potential as a treatment for MDR canine lymphoma. The clinical data showed that PDGFRα expression was higher in canine subjects with MDR T-cell lymphoma than in those with untreated T-cell lymphoma, and that c-KIT expression was greater in subjects with T-cell lymphoma than in those with B-cell lymphoma. TOC monotherapy was well tolerated without serious adverse effects, and two of the five subjects that received TOC exhibited partial responses. The data presented here could contribute to the assessment of TOC-based therapy for dogs with MDR or T-cell lymphoma.

C-reactive protein concentration in canine idiopathic polyarthritis.

To investigate the clinical utility of C-reactive protein (CRP) in idiopathic polyarthritis (IPA), its concentration was measured in dogs with IPA. The CRP concentration was markedly increased in all the IPA dogs at the time of diagnosis and decreased significantly in response to the initial corticosteroid treatment; this indicated that CRP can be used as an index for therapeutic response in IPA cases. Furthermore, at 6 months after the diagnosis, a significant association was observed between the CRP concentration at follow-up (6-13 days after the treatment was started) and the frequency of medication ("no or seldom-medicated (NSM)" groups or "continuing medication (CM)" groups). These results suggest that the initial response of CRP to corticosteroid treatment may be a prognostic factor of canine IPA.

Prognostic factors associated with survival in dogs with lymphocytic-plasmacytic enteritis.

Prognostic factors associated with survival in dogs with lymphocytic-plasmacytic enteritis (LPE) were investigated through a retrospective study. Using case records, 48 dogs diagnosed with LPE were classified as survivors (n = 32) or non-survivors (n = 16), and the clinical and clinicopathological parameters were reviewed between the 2 groups by using univariate and multivariate prognostic analysis. Compared to the hospital population, non-survivors had an overrepresentation of the Shiba breed. Results of univariate analysis indicated that anorexia, severe weight loss, packed cell volume, and total protein were significantly associated with survival for 6 months after diagnosis. In multivariate analysis, anorexia and hypoproteinemia were significantly associated with survival. Furthermore, initial response to treatment was strongly associated with poor prognosis. Based on these clinical and laboratory parameters such as anorexia, hypoproteinemia and initial response to treatment, it may be possible to predict poor prognosis in canine LPE.

Minimal residual disease in canine lymphoma: An objective marker to assess tumour cell burden in remission.

Lymphoma is the most common haematopoietic malignancy in dogs. Since a high proportion of dogs with lymphoma achieve remission soon after initiation of chemotherapy, an objective marker assessing treatment efficacy is required. Following clinical remission, the residual population of tumour cells can be referred to as the minimal residual disease (MRD). MRD traditionally has been detected by cytology and flow cytometry; however, if the burden of malignant cells is low, these methods might not be sufficiently sensitive to detect MRD. As an extension of the development of PCR for antigen receptor gene rearrangements (PARR) in dogs, there has been recent progress in the application of real-time quantitative PCR (RT-qPCR) to canine lymphoma. With the RT-qPCR system, a very high sensitivity (1 cell per 10,000 cells) has been achieved by preparing allele-specific oligonucleotide primers and probes designed from neoplastic clones of each dog. A series of MRD diagnostics studies employing the RT-qPCR system has revealed its usefulness as a prognostic indicator, an objective marker of treatment efficacy and a predictor of relapse for dogs with lymphoma receiving chemotherapy. Introduction of the MRD monitoring system will provide an innovative scientific tool in the development of superior treatments and monitoring strategies for canine lymphoma.

Urinary transforming growth factor-beta1 in feline chronic renal failure.

Transforming growth factor-beta1 (TGF-beta1), an inflammatory cytokine, plays a role in tissue fibrosis, such as glomerular sclerosis and tubulointerstitial fibrosis of the kidneys. In the present study, the urinary TGF-beta1 level of cats diagnosed with chronic renal failure (CRF) was measured to investigate its relationship to the pathogenesis of feline CRF. Urinary TGF-beta1 levels (TGF-beta1/creatinine ratio) were significantly increased compared with healthy controls, whereas serum levels of TGF-beta1 were not. These results indicate that TGF-beta1 is expressed in the kidneys of CRF cats, and that it was reflected in the urinary TGF-beta1 level. Therefore, TGF-beta1 may play a role in feline CRF, and urinary TGF-beta1 could be used as a clinical marker for renal fibrosis.

Development and application of multiple immunofluorescence staining for diagnostic cytology of canine and feline lymphoma.

BACKGROUND: Immunophenotyping of canine and feline lymphoma to determine B-cell or T-cell origin is important for predicting prognosis and for development of treatment protocols. For advanced diagnostic cytology tests that can be performed on smears are required to predict the immunophenotype of lymphomas. OBJECTIVES: The aim of this study was to develop a multiple immunofluorescence (MIF) staining method for the determination of lymphocyte immunophenotype in cytologic specimens, and to evaluate its clinical utility. METHODS: B cells and T cells were detected using anti-CD79α and anti-CD3 antibodies, respectively, followed by specific fluorescence-labeled secondary antibodies. The MIF staining method was first developed using fresh-frozen sections of normal canine lymph nodes. The optimal fixative, the necessity of antigen retrieval (AR), and the optimal concentration of the antibodies were determined. The MIF method was then applied to smears of normal lymph nodes, and to clinical samples from dogs and cats with lymphoma. The MIF results were compared to genetic clonality results. RESULTS: B and T cells were detected based on specific fluorescence in frozen sections, using formalin fixation without AR. Specific fluorescence was also detected in smears from normal lymph nodes and lymphomas, and the immunophenotypes predicted from this MIF staining method completely corresponded to those from genetic clonality analysis. CONCLUSIONS: The MIF staining method that we developed in this study effectively distinguished lymphocyte immunophenotypes with high specificity and sensitivity using a single smear sample, and was useful as a diagnostic tool for canine and feline lymphoma.

Molecular survey of arthropod-borne pathogens in ticks obtained from Japanese wildcats.

The Iriomote cat (IC), Prionailurus bengalensis iriomotensis, and the Tsushima leopard cat (TLC), Prionailurus bengalensis euptilurus, are endangered subspecies of leopard cats in Japan. In addition to habitat destruction and road kills, infectious diseases may threaten their populations, and infection with arthropod-borne pathogens has been reported in both subspecies. Infestations with ectoparasites, especially ticks, have frequently been observed in ICs and TLCs. In the present study, ticks collected from captured ICs and TLCs between November 2011 and January 2012 were morphologically identified and the prevalence of the pathogens Bartonella sp., Babesia sp., Ehrlichia sp., Anaplasma sp., Hepatozoon sp., and hemoplasmas in the ticks was molecularly evaluated. The ticks Haemaphysalis longicornis, H. hystricis, and Amblyomma testudinarium were obtained from ICs, and H. megaspinosa, Ixodes tanuki, H. campanulata, and A. testudinarium were collected from TLCs. The pathogens Hepatozoon felis, Babesia sp., and Anaplasma bovis were detected in ticks obtained from ICs, while H. felis. Babesia sp., Ehrlichia sp., E. muris, 'Candidatus Mycoplasma haemominutum', and Bartonella henselae were found in ticks from TLCs. To protect and conserve these endangered animals, continuous monitoring and additional surveys will be necessary to understand the role of ticks as disease vectors in Japanese wildcats.

Effect of an adenoviral vector that expresses the canine p53 gene on cell growth of canine osteosarcoma and mammary adenocarcinoma cell lines.

OBJECTIVE: To generate an adenoviral vector that expressed the canine p53 gene and investigate its growth-inhibiting effect on canine osteosarcoma and mammary adenocarcinoma cell lines. SAMPLE POPULATION: 2 canine osteosarcoma cell lines (HOS, OOS) and 3 canine mammary adenocarcinoma cell lines (CHMp, CIPm, and CNMm). PROCEDURE: An adenoviral vector that expressed the canine p53 gene (AxCA-cp53) was generated. p53 gene expression was examined by use of reverse transcription (RT)-polymerase chain reaction (PCR) assay and immunohistochemistry. Susceptibility of cell lines to the adenoviral vector was determined by infection with an adenoviral vector that expresses beta-galactosidase (AxCA-LacZ) and 3-indolyl-beta-D-galactopyranoside staining. Growth inhibitory effects were examined by monitoring the numbers of cells after infection with mock (PBS) solution, AxCA-LacZ, or AxCA-cp53. The DNA contents per cell were measured by flow cytometry analysis. Apoptotic DNA fragmentation was detected by use of a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. RESULTS: AxCA-cp53-derived p53 gene mRNA and P53 protein were detected by RT-PCR analysis and immunohistochemistry, respectively. Multiplicity of infection at which 50% of cells had positive 3-indolyl-beta-D-galactopyranoside staining results ranged from 10 to 50. AxCA-cp53 induced growth inhibition in a dose-dependent manner. Arrest of the G1-phase population and apoptotic DNA fragmentation were observed in cells infected with AxCA-cp53. CONCLUSIONS AND CLINICAL RELEVANCE: AxCA-cp53 inhibits cell growth via induction of cell cycle arrest and apoptosis in canine osteosarcoma and mammary adenocarcinoma cell lines that lack a functional p53 gene. AxCA-cp53 may be useful to target the p53 gene in the treatment of dogs with tumors.