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.

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.

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.

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.

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.

A measurement of galvanic current and electrical potential in extracted human teeth.

When opposing teeth with amalgam and gold restoration are in contact, current flows in the mouth at the instant the dissimilar metals touch. In this study, this condition was simulated by use of resistors and extracted human teeth with amalgam and MOD gold inlay restorations. When both teeth were in contact in a physiological saline solution, we measured current and electrical potential generated in each pulp chamber. Galvanic current generated in the tooth with amalgam was always larger (as much as 18.2 times at the instant of contact) than that in the tooth with gold. Electrical potential generated in the tooth with amalgam was always larger (as much as 9.7 times at the instant of contact) than that in the tooth with gold. It should be emphasized that the larger current generated in the tooth with amalgam was caused mainly by its larger electrical potential. These results correspond well with the clinical phenomenon of galvanic pain, which occurs in the tooth with amalgam rather than in the tooth with gold.

Gradual increases in marginal leakage of resin composite restorations with thermal stress.

The effects of thermal stress on the marginal leakage of resin composite restorations in bovine teeth were investigated by a method that preserved the specimens. The changes in marginal leakage of specimens with increasing numbers of thermal cycles were measured by an electrical conductivity method. Four brands of posterior resin composites were used to fill cylindrical cavities (2.0 mm in diameter and 1.5 mm in depth) on the labial surfaces of bovine incisors, according to the manufacturer's instructions. Thermal cycling stress was applied to the specimens for up to about seven weeks (9000 cycles). During this time, the electrical conductance between the pulp and a drop of physiological saline solution covering the resin restoration was measured periodically by application of an electrical potential (60 Hz, 10 Vp-p). Thermal stress increased the marginal leakage gradually, rather than step-wise. Even before application of any thermal stress, wide variations of marginal leakage were found among different specimens restored with the same brand of resin. Specimens with less initial leakage showed less increase in leakage, and vice versa, throughout the experimental period.

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.

Establishment and characterization of a canine T-lymphoblastoid cell line derived from malignant lymphoma.

A canine lymphoma cell line (CL-1) was established in culture from tumor cells found in the pleural fluid of a 7-year old female Japanese terrier with thymic form lymphoma. The CL-1 cells were positive for CD45 and MHC class II and negative for CD4, CD5, CD8, Thy-1 and B-cell specific antigen and surface immunoglobulin. The CL-1 cells had a rearranged T-cell receptor beta-chain gene and a germ-line form immunoglobulin gene, indicating that the CL-1 cells represented a monoclonally expanded population of canine alpha beta T-cell lineage.

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.

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.

Molecular characteristics of malignant lymphomas in cats naturally infected with feline immunodeficiency virus.

Neoplastic disease, especially malignant lymphomas, are often observed in cats infected with feline immunodeficiency virus (FIV). In order to clarify the characteristics of lymphoma cells and to investigate the pathogenesis in FIV-infected cats, we examined the lymphoma tissues developed in five cats naturally infected with FIV by Southern blot analyses using feline immunoglobulin (Ig), T-cell receptors (TCR) and FIV probes. All of the five cases were serologically positive for anti-FIV antibody and negative for feline leukemia virus antigen. Of these five lymphoma samples, two displayed rearrangement of the Ig heavy chain gene and deletion of the Ig light (kappa) chain gene, indicating that the tumor cells were committed to B-cell development. One tumor sample was identified as a T-cell lymphoma because of the presence of a rearranged TCR beta-chain gene. The other two cases were considered to be non-T non-B cell lymphoma because they did not show any rearrangement of the Ig and TCR genes. Therefore, no consistent tumor type was found in lymphoma cases infected with FIV. Clonal integration of FIV provirus was not detected in any of the five lymphoma samples obtained from FIV-infected cats using Southern blot analysis, although FIV proviral genome was detected in the genomic DNA of all the lymphoma samples by using a polymerase chain reaction (PCR). These results indicated that FIV might not play a direct role in tumorigenesis of lymphoma in cats.

Gammopathy with two M-components in a dog with IgA-type multiple myeloma.

A 12-year neutered male mixed-breed dog was referred to hospital for evaluation of chronic diarrhea. Cellulose acetate electrophoresis of its serum revealed two monoclonal peaks in the gamma-globulin fraction. On immunoelectrophoretic analysis, the two monoclonal peaks in the gamma-globulin region were strongly precipitated with anti-dog IgA serum. On sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis, the fractions corresponding to these two peaks were shown to be dimer and trimer or tetramer of immunoglobulin consisting of heavy and light chains. These results indicated that the studied dog had gammopathy with two M-components with dimer and trimer or tetramer of IgA. Accumulations of large amounts of these immunoglobulins with very high molecular weight in the serum were concluded to induce the hyperviscosity syndrome in this dog in the terminal stage.

Fluoride release from light-activated glass ionomer restorative cements.

Fluoride release from four light-activated glass ionomer cements, including newly developed restorative cements, was evaluated and compared with four conventional acid-base glass ionomer cements. There was no significant difference between the group of light-activated cements and the group of conventional cements, and light-activated cements were found to have a potential for releasing fluoride equivalent to that of conventional cements. The amount and rate of release varied among cements. It may be affected not only by the formation of complex fluoride compounds and their interaction with polyacrylic acid but also by the type and amount of resin used for the photochemical polymerization reaction.

In vitro toothbrush-dentifrice abrasion of resin-modified glass ionomers.

OBJECTIVES: This study was conducted to compare the rate of abrasive wear and change in surface roughness of resin-modified and conventional acid-base glass ionomers when subjected to toothbrush-dentifrice abrasion. METHODS: Two resin-modified and two conventional glass ionomers were used. Samples of a high-copper amalgam and a hybrid resin composite were used as reference materials. Specimens of each material were prepared and subjected to toothbrush-dentifrice abrasion using 20,000 strokes of brushing. The amount of vertical loss of material was determined by profilometry (Surfcom-4A, Tokyo-Seimitsu, Tokyo, Japan). The surface characteristics after abrasion were evaluated on secondary electron images by SEM, and the surface hardness (KHN) was also measured for all materials. Data were analyzed by one-way ANOVA (p < 0.05), followed by a multiple comparisons test using LSD (Least-significant difference) at a level of p < 0.05. RESULTS: The abrasion resistance of resin-modified glass-ionomers was statistically lower for the amalgam and the resin composite reference materials. Statistically lower abrasion resistance and surface hardness were observed for the resin-modified glass ionomers than for their conventional acid-base counterparts when two forms of products from the same manufacturer were compared. SEM observations made after abrasion testing showed a significantly rougher surface for all glass ionomer materials than for the amalgam and the resin composite. SIGNIFICANCE: When comparisons were made between products from the same manufacturers, it was found that in vitro resistance to toothbrush-dentifrice abrasion of the resin-modified glass ionomers is inferior to that of the conventional acid-base glass ionomers. The lower abrasion resistance found in the resin-modified products appears to be related to their lower surface hardness.

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.

Plasma thymidine kinase activity in dogs with lymphoma and leukemia.

Plasma thymidine kinase (TK) activity was evaluated as a plasma marker for canine lymphoma and leukemia. A tentative "cut-off" value was set at 6.0 U/l as the upper level of plasma TK based on the mean + 2SD of plasma TK activity in 13 clinically healthy dogs. The levels of plasma TK activity in all of the 20 dogs with lymphoma and leukemia were higher than the cut-off value, whereas those in dogs with lymphoma decreased in parallel with the reduction of the tumor mass after chemotherapy. These findings suggested that estimation of plasma TK activity can be used as a plasma marker for lymphoma and leukemia in the dog.

Establishment and characterization of a new canine B-cell leukemia cell line.

A new cell line derived from a spontaneous canine leukemia was established and designated GL-1. The cells have been cultured in a floating fashion and passaged for over two years. They were round with rich cytoplasm containing many rough endoplasmic reticula and mitochondria. Peroxidase staining was negative. The nuclei of many cells were round, but segmented nuclei were seen frequently. The doubling time of the cells was 27.3 hr and they had 78 chromosomes. Surface marker analysis using monoclonal antibodies (MABs) and flowcytometry revealed that GL-1 possessed CD45 and surface IgG. However, the cells did not react with MABs detecting T-cell markers. These results indicate that GL-1 has a lymphocytic lineage and is derived from a B-cell leukemia.