Effect of suberoylanilide hydroxamic acid on peripheral blood mononuclear cell cytotoxicity towards tumor cells in canines.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor (HDACi) that suppresses the growth of tumor cells in humans and canines. SAHA reportedly enhances the antitumor activity of human peripheral blood mononuclear cell (PBMC). However, it is unclear whether a similar effect is exerted in canines. The present study focused on the effect of SAHA on the cytotoxicity of IL-2 activated PBMC in three tumor cell lines (CTAC, CIPm, and MCM-N1). The mRNA expression of a ligand for the NKG2D receptor was upregulated in SAHA-treated cell lines. Moreover, the SAHA-treated cell lines, except MCM-N1 demonstrated a significantly higher PBMC cytotoxicity compared to the untreated cell lines. Therefore, the NKG2DL upregulation likely enhanced the interaction of NKG2D-NKG2DL, leading to enhanced cytotoxicity of PBMC. It was also revealed that activated PBMC treated with SAHA significantly attenuated their cytotoxicity toward all the cell lines. Although the NKG2D, NKp46, NKp44, and NKp30 receptors, involved in PBMC cytotoxicity, were presumed to be downregulated, there was no significant reduction in the mRNA expression of these receptors. This study revealed that SAHA not only sensitizes the canine tumor cells to cytotoxicity due to PBMC activation, but also suppresses the cytotoxicity of PBMC themselves. Therefore, our results highlight the necessity of avoiding this inhibitory action to enhance the antitumor effect of SAHA in canines.

Cytotoxicity effect of trastuzumab on canine peripheral blood mononuclear cells.

BACKGROUND: Trastuzumab is an antibody drug used to treat human epidermal growth factor receptor 2 (HER2) overexpressing human metastatic breast cancer. Antibody-dependent cellular cytotoxicity (ADCC) is considered to be the major mechanism of cytotoxicity of the drug. However, its ability to induce an ADCC response in canine peripheral blood mononuclear cells (PBMCs) is not well established. AIMS: We aimed to evaluate the ability of trastuzumab in enhancing the cytotoxicity of PBMCs against canine tumor cells. METHODS: We used canine tumor cell lines isolated from metastatic mammary gland tumors (CHMm and CIPm) and thyroid adenocarcinoma (CTAC). The binding of trastuzumab to the cells was confirmed using flow cytometry analysis. Peripheral blood mononuclear cells obtained from healthy beagles and lymphokine-activated killer (LAK) cells, generated by interleukin-2 (IL-2) stimulation of PBMCs, were used as effector cells. Standard lactate dehydrogenase (LDH) release assay was used to measure the cytotoxicity of the LAK cells against tumor cell lines in the presence of trastuzumab. RESULTS: Trastuzumab enhanced the cytotoxicity of PBMCs against CHMm. Moreover, LAK cells killed CHMm synergistically in the presence of trastuzumab. However, the presence of trastuzumab did not produce such a synergistic effect when LAK cells acted against CIPm and CTAC. CONCLUSION: We confirmed the ability of trastuzumab to induce an ADCC response in canine PBMCs and determined its synergistic effect with LAK cells. Although the in vitro system in the present study did not show the induction of trastuzumab-mediated ADCC response against all canine tumor cell lines, the results of this study indicate the potential antitumor activity of trastuzumab in canines.

Cutaneous toxoplasmosis in a female Japanese cat.

A 16-year-old female Japanese cat was presented with a single mammary-gland nodule approximately 3 cm in diameter. Histologically, the nodule consisted of necrotizing granulomatous panniculitis, vasculitis, and mastitis, and contained free and clustered protozoal organisms. The organism was present in the cytoplasm of macrophages, fibroblasts, endothelial cells, and mammary-gland epithelia. The organism was positive for anti- Toxoplasma gondii and anti- Neospora caninum antibodies. Electron microscopy showed single and grouped tachyzoites, with morphologic features similar to those of T. gondii. Polymerase chain reaction and deoxyribonucleic acid sequence analysis was consistent with T. gondii infection. This is the first report of cutaneous toxoplasmosis in a Japanese cat.

Endotoxin-induced injury of the central, autonomic and enteric nervous systems and intestinal muscularis in Thoroughbred horses.

To evaluate the effects of endotoxin on the morphology of the equine central, autonomic and enteric nervous system and intestinal muscularis, six Thoroughbred horses with experimentally induced endotoxaemia were examined. The lesions in the central nervous system consisted of perivascular oedema around arterioles, suggesting brain oedema, and ring haemorrhages around veins, similar to those in human patients with septic shock. In the cranial mesenteric ganglia, neuronal cell bodies became pink or red, with shrinkage of cytoplasm indicative of ischaemic changes; intramural and perivascular infiltration by erythrocytes and neutrophils occurred around arterioles in the epineurium (acute focal interstitial inflammation). In addition, transmission electron microscopy revealed oedema of the endoneurium and mesoaxon in the nerve fascicles running inside or outside the ganglia. Myenteric neurons showed shrinkage of the cytoplasm with multiple cytoplasmic vacuoles, suggesting ischaemic changes. Oedematous degeneration and coagulation necrosis of smooth muscle cells, with dissociation of the cells, were prominent in the tunica muscularis. It is suggested that arterionecrosis elicited by endotoxin and frequently observed in the autonomic and enteric nervous system and intestinal muscularis, was the result of vasoconstriction or vasospasm.

Molecular characterization of a putative protein disulfide isomerase from Babesia caballi.

We produced a mAb against the Babesia caballi extracellular merozoite termed mAb 2H2 and used it to screen a cDNA expression library prepared from B. caballi merozoite mRNA for highly expressed proteins. The complete nucleotide sequence of the cloned gene had 1547 nucleotides and contained a 36-nucleotide intron. The 1398 nucleotide open reading frame predicts a 51 kDa protein showing similarity to protein disulfide isomerase (PDI) from other species. The PDI gene had a predicted N-terminal signal sequence of 19 amino acids and a C-terminal tetrapeptide sequence (His-Thr-Glu-Leu; HTEL) for retention in lumen of the endoplasmic reticulum (ER). The recombinant protein expressed in baculovirus showed an apparent mass of 51 kDa, identical to that the native B. caballi protein. Moreover, the ER retention signal site (HTEL) of the recombinant protein retained its function in ER of insect cells. This 51 kDa protein was strongly expressed by extracelluar B. caballi merozoites in indirect immunofluorescence antibody tests, and was not expressed in the early phase of trophozoite development. Interestingly, detailed observation showed that the reaction of anti-P51 antibody and mAb 2H2 against pear-shaped forms was very erratic, some displaying one or two brightly fluorescent patterns.

Effects of orientation, intermittent rest and vehicle cleaning during transport on development of transport-related respiratory disease in horses.

The effects of various factors on the inflammatory and stress response in horses during transportation were examined in Experiments 1 and 2, carried out in April and August, respectively. In Experiment 1, three groups (G1-G3) of four Thoroughbreds were used, and in Experiment 2, two groups (G4, G5). G1 animals were loaded into lorries with their heads facing forwards (FF) and given periods of short rest (SR) (30 min for every 4 h driven). G2 horses were loaded facing rearwards (FR) and given SR. G3 horses were FF and given periods of long rest (LR) (2 h rest for every 4 h driven). G4 horses were transported FF with hay suspended in front of them and no cleaning of faeces or urine during rest stops. G5 horses were FF and given pellets instead of hay, and the lorry was cleaned and washed at each rest stop. G4 and G5 horses were rested for 1 h after every 5 h of driving. All groups were driven a distance of 1500 km, and the total journey time was 37 h for G1 and G2, 49 h for G3, and 40 h for G4 and G5. In Experiment 1, indicators of inflammatory and stress responses tended to be highest in G1, intermediate in G2 and lowest in G3. In Experiment 2 they tended to be higher in G4 than in G5. The results suggested that increasing the rest time and cleaning the interior of the vehicle during rest stops reduced transportation stress and respiratory insults, factors that may lead to respiratory disease.

Well-differentiated biphasic synovial sarcoma in the atlanto-occipital joint of a Holstein cow.

A 7-year-old Holstein cow developed a large cystic mass in the region between the atlantoaxis and larynx. The mass extended to the synovium in the atlanto-occipital joint. Many villous projections were present on the inner surface of the tumor tissue, and irregular clefts were formed in the inside. Two cell types, epithelioid-like synovioblasts and spindle cells, were present. Immunohistochemical analysis showed that the cells stained positively for cytokeratin (AE1/AE3) and vimentin. Both cells had similar fine structures ultra-structurally. Vacuoles present in the cytoplasm were full of an acid mucous substance. The tumor was diagnosed as a well-differentiated biphasic synovial sarcoma. This is the first report of a rare case of synovial sarcoma, from the viewpoint of its origin.

Non-functional C-cell adenoma in aged horses.

Thyroid tumours occur in older horses, and most such tumours have been considered to be of follicular epithelial origin. However, their immunohistochemical characterization has not been investigated. The purpose of this study was to confirm a suspicion that most of these tumours are in fact parafollicular cell (C cell)-derived adenomas, and to evaluate their pathogenesis and functional state. Thyroid glands from 38 horses aged 10-29 years were evaluated, all tissue samples being examined histologically, immunohistochemically and ultrastructurally. Nodular tumour masses were found in the thyroids of 12 of 38 horses older than 10 years (31.6%), and in nine of 12 horses older than 20 years (75.0%), regardless of sex or breed. Nodular lesions were composed of solid proliferations of polygonal cells with eosinophilic granular cytoplasm. Immunohistochemically, tumour cells were positive for calcitonin and neuron specific enolase, but negative for thyroglobulin. Ultrastructurally, few if any secretion granules were found in tumour cells. On the basis of these results it was concluded that the nodular lesions were C-cell adenomas, not follicular adenomas. It was suspected that the C-cell adenomas were non-functional and unlikely to lead to calcitonin hypersecretion-related diseases.

Malignant ameloblastic fibro-odontoma in a dog.

An 11-year-old male Collie was presented with a swelling of the face caused by tumor masses arising from the gingiva. Postmortem examination revealed metastases to the lymph nodes, lung, liver, and orbital cavity. Histologically, the tumor represented a combination of fibrosarcomatous proliferation, pulpal mesenchyme, and undifferentiated odontogenic epithelium, with a follicular or plexiform growth pattern. In addition, the follicular areas of the tumor showed a biphasic character, and there were numerous apoptotic cells in plexiform areas. Furthermore, acidophilic material resembling dysplastic dentine or enamel matrix was observed in the metastatic lesion in the lung. Based on the histological characters, the present case was diagnosed as malignant ameloblastic fibro-odontoma. This study is the first known description of a possible malignant ameloblastic fibro-odontoma in a dog with metastasis to distant organs.

Characterization of monoclonal antibodies against Gnathostoma nipponicum.

Monoclonal antibodies (mAbs) were produced against the proteins of advanced third-stage larvae (AdL3) of Gnathostoma nipponicum. Six mAbs (Gn2C3, Gn2H3, Gn4C3, Gn4E9, GnSH1, and Gn10B7) were obtained as determined by enzyme-linked immunosorbent assay (ELISA). Gn4E9 and GnSH1 seemed to be genus-specific, as they did not cross-react with Anisakis sp., Dirofilaria immitis, Gongylonema pulchrum, Toxocara canis, Trichinella sp., Trichuris vulpis, Metagonimus sp., or Spirometra erinaceieuropaei by ELISA. Immunohistochemistry showed that Gn2C3, Gn4E9, and Gn5H1 reacted strongly with the central esophagus; Gn2H3 reacted with cuticle,muscle, intestine, and the cervical sac; and Gn4C3 and Gn10B7 reacted with cuticle, muscle, esophagus, intestine, and the cervical sac of AdL3. In Western blotting analysis, Gn2C3, Gn4E9, and Gn5H1 reacted to 60-, 53-, 46-, and 41-kDa proteins; Gn4C3 reacted to the AdL3 protein of G. nipponicum (>42 kDa). Moreover, proteins purified using a mAb Gn4E9 immunoprecipitation method (sizes 60-, 53-, 46-, and 41-kDa) were used as antigens in ELISAs. A significant difference (P < 0.01) was shown between mouse sera infected with G. nipponicum and sera infected with Trichnella sp. or not infected. These results provide a rationale for evaluating esophageal proteins for the development of diagnostic methods for detecting G. nipponicum or Gnathostoma sp. infections.

Congenital hepatic fibrosis in a newborn calf.

Congenital hepatic fibrosis was observed in a newborn calf. Light microscopy revealed that periportal areas were linked via connective tissue to the central vein regions and to other periportal areas. Hyperplastic fibers were positive for type I collagen. A remarkable increase in the number of myofibroblasts that were positive for alpha-smooth muscle actin and vimentin was observed in the inner wall of the sinusoids, indicating the occurrence of various fibrogenesis. Ultrastractually, foci of cells resembling cholangiole epithelium cells were observed within the sinusoids, thereby suggesting either ductal plate dysplasia or a bile duct anomaly.

Reactivity of autoantibodies of autoimmune hemolytic anemia with recombinant rhesus blood group antigens or anion transporter band3.

The specificity of autoantibodies in autoimmune hemolytic anemia (AIHA) has been studied using the serological procedure and immunoprecipitation technique with rare phenotype red cells. We attempted to analyze specificity using recombinant rhesus (Rh) blood group and band3 antigens expressed on erythroleukemic cell lines, KU812E. The autoantibody eluates were isolated by the acid elution procedure from the red cells of 20 AIHA patients. The recombinant Rh antigens, RhD, cE, ce, CE, and chimera antigens CE-D and D-CE, were obtained by retroviral cDNA transduction into KU812E cells, and the cell line expressing the antigens was cloned. Band3 cDNA was also obtained and introduced into KU812E and cloned KU812 expressing RhcE. The reactivities of AIHA eluates with recombinant Rh and band3 antigens were studied by flow cytometry. Fifteen eluates reacted with at least one of the RhcE, ce, or CE antigens, and four eluates reacted with RhD. Seven eluates with strong Rh specificity were studied further using chimera antigen. Five eluates showed reduced or lost reactivity, although two eluates reacted identically with the chimera antigens as wild type. These results indicated that conformational epitopes constituted by RhD or CE specific exofacial peptide loops are important for autoantibodies in most cases. Seven eluates reacted with band3, five exclusively. The coexpression study of RhcE and band3 did not enhance the expression of either antigen nor the reactivity with patient eluates, indicating that association of Rh and band3 was not involved in the appearance of autoantigen.

The first host record of trichinosis in a red fox, Vulpes vulpes japonica, from Aomori Prefecture, northern Honshu, Japan.

A case of wildlife trichinosis was found in a red fox (Vulpes vulpes japonica) captured at Rokkasho, Aomori Prefecture on November 27, 1998. Trichinella larvae were obtained from almost all of the muscle tissues except for the masseter. The highest number of larvae per gram of tissue was found in the muscles of the gluteal region and throat. The lowest number was found in the diaphragm and tongue. Trichina cysts within the muscle fibers had groups of fatty cells at the poles, and minimal tissue reaction was observed around the cyst. No calcification was found in the cyst. These morphological findings suggested that the considerable time had elapsed since the invasion. This is the first case of trichinosis in a red fox in Japan.

Direct-antiglobulin-test-negative immune haemolytic anaemia and thrombocytopenia in a patient with Hodgkin's disease.

A case of direct-antiglobulin-test (DAT)-negative auto-immune haemolytic anaemia (AIHA) and immune thrombocytopenia (ITP) associated with Hodgkin's disease (HD) is reported. A 52-year-old male was admitted with anaemia, thrombocytopenia, and lymphadenopathy. The patient was DAT negative, although he exhibited the clinical features of warm-type AIHA and elevated levels of red-blood-cell-associated IgG (RBC-IgG). The serum level of platelet-associated IgG (PA-IgG) was markedly increased. A biopsy specimen of the inguinal lymph nodes showed HD of mixed cellularity. Marked improvement of subjective symptoms, normalization of haematological values and a decrease in the level of both RBC- and PA-IgG were observed after the start of combination chemotherapy for HD. Although the association of HD, ITP, and/or AIHA has been infrequently reported, the measurement of RBC-IgG is recommended in cases of HD with anaemia even though DAT is negative, since HD is known to be associated with various protean immunological abnormalities.

Age-related changes in the testes of horses.

Atrophy of seminiferous tubules and interstitial fibrosis are frequently observed in aged horses. Samples from 8 male Thoroughbreds, age 4-24 years, were subjected to histological, electron microscopical and immunohistochemical examination and statistical analysis. There were statistically significant increases in collagen fibres in the lamina propria of seminiferous tubules and testicular interstitium in 3 horses age 23 and 24 years compared with 5 horses age 4-20 years (P<0.001). Lamina propria surrounding atrophic tubules was thickened by an increase in collagen type IV and elastic fibres and by proliferation of bizarre myoid cells. Basal lamina was also thickened but had decreased reactivity for collagen type IV. Some myoid cells changed morphologically to a swollen and irregular shape and contained abundant cytoplasmic organelles. Laser scanning microscopy revealed that cytoplasmic actin filaments were decreased; the remaining filaments were positive for alpha-smooth muscle actin and vimentin, and matrix metalloproteinase-2 was secreted. These myoid cells transformed into myofibroblasts. The changes are interpreted as evidence of injured structure and function of the lamina propria and basal lamina and may explain the functional decline of the blood-testis barrier. Myoid cells may play an important role in the progression of testicular fibrosis.

Analysis of a growth-promoting factor for Babesia caballi cultivation.

Serum-free media were examined to culture Babesia caballi. Daigo's T (DT) basal medium supplemented with Daigo's GF21 (GF21) or GIT medium, which already contains GF21, supported the parasite propagation at 37 C in a humidified atmosphere under 5% CO2 in air. Growth of B. caballi was dependent of the suitable concentration (10-20%) of GF21. Therefore, GF21 was suggested as the growth-promoting factor for B. caballi. However, GIT medium did not support the growth of parasites from cryopreserved stabilates, and serum supplementation was essential for the retrieval of parasites.

Content analysis of group work sessions in the context of an educational program for inpatient diabetes patients.

This study investigated the changes in emotions and attitudes of diabetic patients as observed in group work sessions linked to an inpatient diabetic educational program. Using content analysis of transcripts from Sessions 1 and 3 of 8 selected groups (40 patients), in Session 1 negative emotions or attitudes such as anxiety, remorse, or self-blame, a lack of understanding of diabetes, along with a willingness to undertake self-care were frequently expressed. In Session 3, more positive emotions and attitudes towards self-care (willingness, specific plans, and resolutions), satisfaction, and relief or hope were frequently expressed. These results suggest that the patients participating in the group work sessions moved toward adaptation to diabetes, although the effects of the group work were not separated from those of the inpatient educational program.

Ultrastructural study of Langerhans cells in equine insect hypersensitivity "Kasen".

Ultrastructural features of Langerhans cells (LCs) of equine "Kasen" were studied. Electron microscopic observation revealed that LCs were dendritic and had irregular nuclear membranes. A number of Birbeck granules (Bgs) of various types were observed in the cytoplasm of LCs. In LCs in the upper stratum spinous, many Bgs were observed (Type 2 LC). LCs in the epidermo-dermal junction (EDJ) had a few Bgs, vesicles (multivesicular bodies) and highly electron-dense granular endosomes in the cytoplasm (Type 3 LC). Inactive LCs were also observed between the keratinocytes (Type 1 LC). Various types of LCs observed in the skin lesions of equine "Kasen" were interpreted as representing those that recognize, intake and process antigens.

A neural mechanism of hierarchical discrimination of odors in the olfactory cortex based on spatiotemporal encoding of odor information.

We propose a neural mechanism for discrimination of different complex odors in the olfactory cortex based on the dynamical encoding scheme. Both constituent molecules of the odor and their mixing ratios are encoded simultaneously into a spatiotemporal activity pattern (limit cycle attractor) in the olfactory bulb [Hoshino O, Kashimori Y, Kambara T (1998) Biol Cybern 79:109-120]. We present a functional model of the olfactory cortex consisting of some dynamical mapping modules. Each dynamical map is represented by itinerancy among the limit cycle attractors. When a temporal sequence of spatial activity patterns corresponding to a complex odor is injected from the bulb to the network of the olfactory cortex, the neural activity state of each mapping module is fixed to a relevant spatial pattern injected. Recognition of an odor is accomplished by a combination of firing patterns fixed in all the mapping modules. The stronger the response strength of the component, the earlier the component is recognized. The hierarchical discrimination of an odor is made by recognizing the components in order of decreasing response strengths.

The kinetics of Langerhans cells in equine insect hypersensitivity "Kasen".

An immunohistochemical study was carried out on the kinetics of Langerhans cells (LCs) at various pathological stages of "Kasen". Skin lesions of "Kasen" that were collected by biopsy from May to October were classified histopathologically into three stages: initial (Group I, 31 cases), developing (Group II, 50 cases) and regressing (Group III, 13 cases). LCs showed a positive reaction with anti-equine thymocytes (EqT6) monoclonal antibody (MoAb) and anti-major histocompatibility complex (MHC) class II MoAb by immunohistochemical staining. The anti-EqT6 MoAb was intensely positive along the cytoplasmic process. The number of LCs per unit area increased markedly with the passage of time from the initial to the developing stage of the disease, particularly in the epidermo-dermal junction (EDJ). However, the number of LCs tended to decrease in the epidermal layer. In conclusion, the LCs moving into the epidermal layer moved into the EDJ and dermis during the time course of lesion development, and the changes occurring in LCs possibly influenced the progression of the skin lesions of "Kasen".