CD204⁺ tumor-associated macrophages are associated with clinical outcome in canine pulmonary adenocarcinoma and transitional cell carcinoma.

Tumor-associated macrophages are abundant infiltrating cells in the tumor microenvironment (TME). Macrophages can be classified into several types of subsets based on their immune responses. Among those subsets, M2 macrophages contribute to anti-inflammatory responses and create an immunosuppressive environment that promotes tumor cell proliferation. In a previous study, human cancer patients with high M2 macrophages showed a worse prognosis for many types of tumors. However, studies examining the relationship between M2 macrophages and clinical outcomes in canine tumors are limited. In the previous human and canine studies, CD204 has been used as the marker for detecting M2 macrophages. Then we evaluated CD204+ and total macrophages infiltration and its association with clinical outcomes in canine solid tumors. In this study, we examined dogs with oral malignant melanoma (OMM), pulmonary adenocarcinoma (PA), hepatocellular carcinoma (HCC), and transitional cell carcinoma (TCC). Compared to healthy tissues, CD204+ and total macrophages were increased in OMM, PA, and TCC, but not in HCC. High CD204+ macrophage levels were significantly associated with lung metastasis in TCC (P = 0.030). Kaplan-Meier analysis revealed that high CD204+ macrophage levels were associated with shorter overall survival (OS) in canine patients with PA (P = 0.012) and TCC (P = 0.0053). These results suggest that CD204+ macrophages contribute to tumor progression and could be a prognostic factor in dogs with PA and TCC.

Aberrant expression of microRNAs and the miR-1/MET pathway in canine hepatocellular carcinoma.

Canine hepatocellular carcinoma (HCC) is the most common primary hepatic tumour in dogs. MicroRNA (miRNA) dysregulation has been reported in human HCC and shown to have diagnostic and prognostic value; however, there are no data on miRNA expression in canine HCC. The aim of the present study was to investigate differentially expressed miRNAs in canine HCC. Analysis of miRNA expression in canine HCC tissues and cell lines by quantitative reverse transcription PCR showed that miR-1, miR-122, let-7a, and let-7g were downregulated, whereas miR-10b and miR-21 were upregulated in canine HCC. MET is one of the target genes of miR-1. MET was upregulated in canine HCC at the gene and protein levels, and a significant correlation between the concomitant downregulation of miR-1 and upregulation of MET was observed. Fast/intermediate-proliferating canine HCC cell lines had higher MET gene and protein expression levels than the slow-proliferating cell line. These findings suggest that miRNAs are differentially expressed in canine HCC, and that the miR-1/MET pathway may be associated with canine HCC cell proliferation.

Thoracic duct lymphography by subcutaneous contrast agent injection in a dog with chylothorax.

A 4-year-old male Japanese Shiba Inu presented with recurrent chylothorax. The thoracic duct was successfully imaged using computed tomography after the injection of an iodine contrast agent into the subcutaneous tissue surrounding the anus. The thoracic duct was successfully ligated and pericardectomy performed via an open thoracotomy. Pleural effusion improved but relapsed a week after the surgery. A second lymphography revealed a collateral thoracic duct that was not detected during the first lymphography. The collateral duct was ligated and chylothorax was resolved after the second surgery. The lymphography applied in this study was minimally-invasive and easily provided images of the thoracic duct in a dog with chylothorax.

Abrogation of protein phosphatase 6 promotes skin carcinogenesis induced by DMBA.

Somatic mutations in the gene encoding the catalytic subunit of protein phosphatase 6 (Ppp6c) have been identified in malignant melanoma and are thought to function as a driver in B-raf- or N-ras-driven tumorigenesis. To assess the role of Ppp6c in carcinogenesis, we generated skin keratinocyte-specific Ppp6c conditional knockout mice and performed two-stage skin carcinogenesis analysis. Ppp6c deficiency induced papilloma formation with 7,12-dimethylbenz (a) anthracene (DMBA) only, and development of those papillomas was significantly accelerated compared with that seen following DMBA/TPA (12-O-tetradecanoylphorbol 13-acetate) treatment of wild-type mice. NF-κB activation either by tumor necrosis factor (TNF)-α or interleukin (IL)-1ß was enhanced in Ppp6c-deficient keratinocytes. Overall, we conclude that Ppp6c deficiency predisposes mice to skin carcinogenesis initiated by DMBA. This is the first report showing that such deficiency promotes tumor formation in mice.

Clinical guidelines for treating caries in adults following a minimal intervention policy--evidence and consensus based report.

OBJECTIVES: In 2002, FDI (World Dental Federation) published a policy advocating that caries be treated by minimal intervention (MI). This MI policy has been accepted worldwide and is taught in universities. But acceptance in general dental practice has been slower, especially in Japan where healthcare payment and practice favour drilling and filling. To help disseminate this MI policy into general practice, the Japanese Society of Conservative Dentistry developed an evidence-based clinical guideline for restoring carious permanent teeth in adult patients. METHODS: The guideline was developed by a committee of nine university clinicians and a librarian. The committee selected the most frequent clinical questions in treating caries and used electronic databases to search and assess the best scientific evidence for each. Members then added their clinical experience and discussed to reach consensus on each question on treating caries with MI policy. Graded recommendations and guidance were made for each clinical question. The provisional guideline was strengthened after review and discussion with university researchers and general practitioners. RESULTS: The guideline addresses the 16 most frequent clinical questions in treating adult caries, including restorative methods and how to tackle root caries. Recommendations for treatment using MI policy were developed using the best scientific evidence and consensus of experienced clinicians. CLINICAL SIGNIFICANCE: The guideline offers a practical expert view of treating caries with the MI policy that incorporates the best scientific evidence, the latest techniques, the most preferable materials and the general consensus of expert clinicians.

Quantitative determination of radio-opacity: equivalence of digital and film X-ray systems.

OBJECTIVES: To evaluate the equivalence of a digital X-ray system (DenOptix) to conventional X-ray film in terms of the measured radio-opacity of known filled-resin materials and the suitability of attenuation coefficient for radio-opacity determination. METHODS: Discs of five thicknesses (0.5-2.5mm) and step-wedges of each of three composite materials of nominal aluminum-equivalence of 50%, 200% and 450% were used. X-ray images of a set of discs (or step-wedge), an aluminum step-wedge, and a lead block were taken at 65 kV and 10 mA at a focus-film distance of 400 mm for 0.15s and 1.6s using an X-ray film or imaging plate. Radio-opacity was determined as equivalent aluminum thickness and attenuation coefficient. The logarithm of the individual optical density or gray scale value, corrected for background, was plotted against thickness, and the attenuation coefficient determined from the slope. The method of ISO 4049 was used for equivalent aluminum thickness. RESULTS: The equivalent aluminum thickness method is not suitable for materials of low radio-opacity, while the attenuation coefficient method could be used for all without difficulty. The digital system gave attenuation coefficients of greater precision than did film, but the use of automatic gain control (AGC) distorted the outcome unusably. CONCLUSION: Attenuation coefficient is a more precise and generally applicable approach to the determination of radio-opacity. The digital system was equivalent to film but with less noise. The use of AGC is inappropriate for such determinations.

Development of the needle insertion robot for percutaneous vertebroplasty.

Percutaneous Vertebroplasty (PVP) is an effective and less invasive medical treatment for vertebral osteoporotic compression fractures. However, this operative procedure is quite difficult because an arcus vertebra, which is narrow, is needled with accuracy, and an operator's hand is exposed to X-ray continuously. We have developed a needle insertion robot for Percutaneous Vertebroplasty. Its experimental evaluation on the basic performance of the system and needle insertion accuracy are presented. A needle insertion robot is developed for PVP. This robot can puncture with accuracy and an operator does not need to be exposed to X-ray. The mechanism of the robot is compact in size (350 mm x D 400 mm x H270 mm, weight: 15 kg) so that the robot system can be inserted in the space between C-arm and the patient on the operating table. The robot system is controlled by the surgical navigation system where the appropriate needle trajectory is planned based on pre-operative three-dimensional CT images. The needle holding part of the robot is X-ray lucent so that the needle insertion process can be monitored by fluoroscopy. The position of the needle during insertion process can be continuously monitored. In vitro evaluation of the system showed that average position and orientation errors were less than 1.0 mm and 1.0 degree respectively. Experimental results showed that the safety mechanism called mechanical fuse released the needle holding disk properly when excessive force was applied to the needle. These experimental results demonstrated that the developed system has the satisfactory basic performance as needle insertion robot for PVP.

Detection of circulating autoantibodies using living keratinocyte staining on MCA-B1 method in dogs with pemphigus foliaceus.

In this study, we compared the sensitivity and specificity of three immunofluorescence techniques used to detect circulating autoantibodies in dogs with pemphigus foliaceus (PF); living keratinocyte staining on a canine keratinocyte cell line, MCA-B1, indirect immunofluorescence (IIF) on canine lip and IIF on bovine esophagus. Sera from canine PF cases were positive in four out of 27 dogs (14.8%) using living keratinocyte staining on MCA-B1 cells method, and five (18.5%) and eight sera (29.6%) using IIF on canine lip and bovine esophagus methods, respectively. By contrast, none of the 31 sera from dogs with non-pemphigus dermatoses reacted with MCA-B1 cells, whereas two (6.5%) as well as five sera (16.1%) obtained from those dogs showed positive reactivity with IIF on canine lip and bovine esophagus, respectively. Our results suggest that, although it exhibits the least sensitivity, the positive reactivity obtained by living keratinocyte staining on MCA-B1 cells can support the diagnosis of canine PF.

Quantitative analysis of Fas and Fas ligand mRNAs in a feline T-lymphoid cell line after infection with feline immunodeficiency virus and primary peripheral blood mononuclear cells obtained from cats infected with the virus.

Apoptosis is frequently observed in feline lymphocytes in association with feline immunodeficiency virus (FIV) infection. In this study, to investigate the mechanism of FIV-induced apoptosis, levels of Fas and Fas ligand mRNAs were measured by real-time reverse transcription-PCR. In a feline T-lymphoid cell line the amounts of Fas ligand mRNA increased along with the induction of apoptosis after in vitro infection with FIV. In PBMC collected from 10 cats naturally infected with FIV, Fas ligand mRNA levels were significantly higher than those in PBMC from five uninfected cats. These results indicate that the increased expression of Fas ligand may be involved in the induction of apoptosis of lymphocytes in FIV infection.

A severe hepatic disorder with myelodysplastic syndrome, treated with cytarabine ocfosfate, in a dog.

An 8-year-old female Shih Tzu was presented with weight loss and vomiting. Alanine aminotransferase was high and abdominal radiographs revealed hepato- and splenomegaly. Mild anaemia, neutrophilia with left shift, eosinophilia, a thrombocytosis with dysplastic features of eosinophils and platelets, were detected. The animal was initially considered to have hepatitis and was treated accordingly, but clinical signs persisted. Histological examination of liver biopsy samples showed disruption of the hepatic lobule, with extensive infiltration by haemopoietic cells. Further investigation of the bone marrow suggested a diagnosis of myelodysplastic syndrome. The animal was treated with cytarabine ocfosfate, a prodrug of cytosine arabinoside, and appeared to recover.

Increase in plasma lipid peroxide in cats fed a fish diet.

Plasma lipid peroxide levels were examined in cats. Plasma lipid peroxide levels in 3 of 4 clinical cases which had been fed raw fish were higher than those in normal cats. When healthy cats were put on a raw fish diet in controlled conditions, a remarkable increase in plasma lipid peroxide was observed. This increase occurred within 1 to 3 weeks in cats without obvious clinical disorders. We also showed that a continuous raw fish diet is necessary to cause the increase, but the increase was transient and restored in spite of a continuous raw fish diet, indicating the development of an unidentified antioxidant process. Our results clearly indicate that intake of foods high in polyunsaturated fatty acid can induce oxidative stress in cats.

Breed differences in allele frequency of the dopamine receptor D4 gene in dogs.

We previously reported that the dog dopamine receptor D4 (DRD4) gene is polymorphic as observed in humans, and four alleles were identified based on the number and/or order of the 12 and 39 bp sequences located in the homologous region of human DRD4. To assess the diversity of the DRD4 gene in dogs we examined the allelic variations in four breeds (beagle, golden retriever, Shetland sheepdog, and shiba) employing the polymerase chain reaction (PCR). As a result, we found three novel alleles and determined the DNA sequences of these alleles. The beagle shared four alleles, including 396, 435, 447a, and 447b, with the 435 (52.6%) and 447a (39.5%) alleles being common. The golden retriever had the 435 and 447a alleles, and the 435 allele was frequent (73.3%). In the Shetland sheepdog, the 435, 447a, and 498 alleles were observed, of which the 447a allele was most frequent (82.5%). The shiba had five alleles-447a, 447b, 486, 498, and 549-and the 447b allele was most common (55.4%). These findings suggest that the allele frequency varied among the four dog breeds, and analysis of the DRD4 polymorphism may therefore be useful for elucidating the relationships among dog breeds.

Quantification of viral ribonucleic acid in plasma of cats naturally infected with feline immunodeficiency virus.

OBJECTIVE: To assess plasma viral RNA concentration in cats naturally infected with feline immunodeficiency virus (FIV). ANIMALS: 28 FIV-infected cats. PROCEDURE: Cats were categorized into 1 of the 3 following stages on the basis of clinical signs: asymptomatic (nonclinical) carrier (AC; n = 11), acquired immunodeficiency syndrome-related complex (ARC; 9), or acquired immunodeficiency syndrome (AIDS; 8). Concentration of viral RNA in plasma (copies per ml) was determined by use of a quantitative competitive polymerase chain reaction (QC-PCR) assay. Total lymphocyte count, CD4+ cell and CD8+ cell counts, and the CD4+ cell count-to-CD8+ cell count ratio were determined by use of flow cytometry. RESULTS: Plasma viral RNA concentration was significantly higher in cats in the AIDS stage, compared with cats in AC and ARC stages. Most (5/7) cats in the AIDS stage had low total lymphocyte, CD4+ cell, and CD8+ cell counts. CONCLUSIONS AND CLINICAL RELEVANCE: Concentration of plasma viral RNA is a good indicator of disease progression in FIV-infected cats, particularly as cats progress from the ARC to the AIDS stage. Determination of CD4+ and CD8+ cell counts can be used as supportive indicators of disease progression.

Pertussis toxin enhances human immunodeficiency virus type 1 replication.

Pertussis toxin (PTX) has been used as a reagent to identify involvement of the G protein-mediated signal transduction pathway. In this study, we found that PTX enhanced HIV-1 replication in acute infection systems at a high dose (1-10 microg/ml) in vitro. PTX treatment enhanced the infectivity of HIV-1-based pseudovirus enveloped with HIV-1 or amphotropic murine leukemia virus (A-MuLV), but not with vesicular stomatitis virus (VSV). This high dose of PTX treatment did not affect HIV-1 gene expression. These data suggested that the effect was virus envelope dependent and that PTX acted on an early stage of viral infection. Treatment with B-oligomer, a nonenzymatic subunit of PTX, mimicked this enhancing effect of PTX. However, desialylation of viral and cellular surface glycoproteins, which are receptors for B-oligomer, did not affect the augmentation induced by PTX. These results indicate that the enhancement of HIV-1 replication is mediated through an unknown biological function of B-oligomer.

Genetic heterogeneity of env gene of feline immunodeficiency virus obtained from multiple districts in Japan.

Feline immunodeficiency virus (FIV) infection is widespread in many countries. FIV isolates have been classified into five distinct subtypes, A, B, C, D and E based on their env gene sequences. Several reports indicate that most of the FIVs isolated in Japan belong to subtype B which includes the first Japanese isolate, TM2 strain. To examine the distribution of FIV subtypes in Japan, proviral DNA sequences of the env gene were directly amplified by nested PCR from FIV-infected cats that had been kept in multiple districts throughout Japan. Phylogenetic analysis of the 11 strains showed that four FIV subtypes, A, B, C and D, were present in Japan. Among these subtypes, subtypes B and D were the two most common subtypes in Japan, and they were mainly distributed in the eastern and western parts of Japan, respectively. The present study provides information that is fundamental for development of a vaccine to protect against FIV infection in cats.

Molecular cloning of feline Fas antigen and Fas ligand cDNAs.

The Fas antigen (FasA) and Fas ligand (FasL) are key molecules which mediate apoptosis. For investigation of apoptosis in cats, we isolated molecular clones of feline FasA and FasL cDNAs by using the polymerase chain reaction (PCR) method to amplify cDNAs from feline lymphoma cell lines. These feline FasA and FasL clones contained complete open reading frames encoding 314 and 280 amino acids, respectively. These feline FasA and FasL cDNA clones had structures characteristic of the tumor necrosis factor (TNF) receptor family and TNF family, respectively. The deduced amino acid sequence of feline FasA and feline FasL, respectively showed 45.0%-60.0% and 75.0%-90.0% similarity with their human, mouse and bovine counterparts. These data will be helpful for investigating the role of the FasA and FasL system in apoptosis and for studying the various diseases associated with the deregulation of apoptosis in cats.

Biocompatibility of Clearfil Liner Bond 2 and Clearfil AP-X system on nonexposed and exposed primate teeth.

OBJECTIVE: Recent studies have demonstrated that acid etching of vital dentin and pulpal tissue does not retard pulpal healing, odontoblastoid cell differentiation, or dentinal bridge formation when the pulp is capped with adhesive resins. The purpose of this study was to evaluate the pulpal response in nonexposed and exposed monkey pulps to treatment with the Clearfil Liner Bond 2 and Clearfil AP-X system. METHOD AND MATERIALS: Class V and Class I cavities in nonexposed and exposed pulps were observed at 7 or 8, 27, and 97 days. RESULTS: There were no differences in pulpal inflammation between the Clearfil Liner Bond 2/Clearfil AP-X specimens and calcium hydroxide controls in either Class V or Class I cavities at the various time periods. CONCLUSION: Clearfil Liner Bond 2 and Clearfil AP-X system is not toxic to either nonexposed or exposed pulpal tissues when placed according to manufacturer's directions.

Clinicopathological and immunological characteristics of six cats with granular lymphocyte tumors.

Clinical and immunological characteristics were investigated in six cases of feline granular lymphocyte (GL) tumor. The ages of the affected cats were relatively old, ranging from 4 to 13 years of age. Gastrointestinal signs were commonly observed in these cases. Only one of the six GL tumor cases was positive for feline leukemia virus (FeLV) antigen. Phenotypic analysis revealed that the GL tumor cells from all of the six cases lacked the T- or B-cell markers. These GL tumor cells were examined by Southern blot analysis using feline immunoglobulin (Ig) and T-cell receptor (TCR) gene probes. GL tumor cells obtained from two cases were identified as cells of T-cell lineage by the presence of a rearranged TCR beta gene, whereas those from the other four cases were considered to be derived from non-T- non-B-cell lineage because of the absence of rearrangement of these genes. These findings indicated that feline GL tumors can be considered as a specific disease entity in feline lymphomas because the cases examined in this study showed onset at an older age, a low incidence of FeLV infection and frequent involvement of gastrointestinal lesions, which are not found in typical FeLV-associated lymphomas. Although no specific phenotypes was observed by phenotypic analysis, the feline GL tumor cells were divided into two consistent genotypes of T-cell or non-T- non-B-cell lineages.

Apoptosis enhanced by soluble factor produced in feline immunodeficiency virus infection.

Feline immunodeficiency virus (FIV)-infected cells have been shown to undergo apoptosis by treatment with tumor necrosis factor alpha (TNF-alpha). This study detected a soluble factor which enhanced the apoptosis induced by TNF-alpha treatment. The sensitivity to TNF-alpha in the induction of apoptosis in a feline fibroblastoid cell line (CRFK) cells was significantly enhanced when the culture supernatant of FIV-infected CRFK cells or plasma samples from FIV-infected cats was added to the culture. These findings suggested that FIV infection induces production of a soluble factor which enhances CRFK cells sensitivity to TNF-alpha induction of apoptosis both in vitro and in vivo. This factor may contribute to the loss of lymphocytes in cats infected with FIV.