Tokuma Yanai

Expression of Vascular Endothelial Growth Factor, Basic Fibroblast Growth Factor, and Their Receptors (Flt-1, Flk-1, and Flg-1) in Canine Vascular Tumors

Expression of vascular endothelial growth factor (VEGF), its receptors (flt-1 and flk-1), and basic fibroblast growth factor (bFGF) in canine hemangiosarcoma (HSA) and hemangiomas was investigated by immunohistochemical analysis. In addition, expression of the mRNAs of VEGF, flt-1, flk-1, and flg-1 (a receptor for bFGF), was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) with cRNA probes. VEGF, bFGF, flt-1, and flk-1 were immunohistochemically detected in the neoplastic cells in HSAs; the staining intensity was stronger in HSAs than in hemangiomas. On the other hand, the neoplastic cells in hemangiomas exhibited very weak or no expression of VEGF, although they showed moderate expression of flt-1 and flk-1. The mRNAs of VEGF, flt-1, flk-1, and flg-1 were detected in the neoplastic cells in HSAs by ISH and RT-PCR. However, VEGF mRNA was not detectable in the neoplastic cells in hemangiomas by ISH, although it was detected in the inflammatory cells in the tumors by RT-PCR. Moreover, the HSAs that showed intense staining for flk-1 had a high proliferative activity, which was reflected as a high Ki-67 positive index. These results suggest that the expression of the growth factors and their receptors, especially flk-1, might be associated with the malignant proliferation of HSAs.

Site-Specific In Vivo Mutagenicity in the Kidney of gpt Delta Rats Given a Carcinogenic Dose of Ochratoxin A

Ochratoxin A (OTA) can induce renal tumors that originate from the S3 segment of the proximal tubules in rodents, but the results of conventional mutagenicity tests have caused controversy regarding the role of genotoxic mechanisms in the carcinogenesis. Human exposure to OTA from various foods is unavoidable. Therefore, an understanding of OTA-induced renal carcinogenesis is necessary for accurate estimates of the human risk hazard. In the present study, a 13-week exposure of gpt delta rats to OTA at a carcinogenic dose induced karyomegaly and apoptosis at the outer stripe of the outer medulla (OM) of the kidney but failed to affect the reporter gene mutations in DNA extracted from whole kidneys. This site specificity resulting from the kinetics of specific transporters might be responsible for the negative outcome of in vivo mutagenicity. The kidney was then macroscopically divided, based on anatomical characteristics, into the cortex, the OM, and the inner medulla, each of which was histopathologically confirmed. Spi⁻ mutant frequencies (MFs) but not gpt MFs in the OM after a 4-week exposure to OTA were significantly higher than in controls despite the absence of cortical changes. There were also no changes in 8-hydroxydeoxyguanosine levels in kidney DNA. These results strongly suggest the involvement of a genotoxic mechanism, with the exception of oxidative DNA damage in OTA-induced renal carcinogenesis. In addition, the reporter gene mutation assay using DNA from target sites could be a more powerful tool to investigate in vivo genotoxicities.

In Vitro Attenuation of Highly Virulent Infectious Bursal Disease Virus: Some Characteristics of Attenuated Strains

Some strains of highly virulent infectious bursal disease virus (HV-IBDV) were adapted through serial passage in embryonated eggs. The embryonated egg-adapted HV-IBDV was successfully adapted to grow in chicken embryo fibroblast (CEF) cell cultures showing a cytopathic effect by preparing the CEF cells from the virus-infected embryos. The embryonated egg- and cell culture-adapted strains significantly reduced their pathogenicity to, and did not kill any, young chickens in experimental infection. The bursal lesions of the adapted strain-infected chickens were similar to those observed in classical strain-infected chickens. Cross-virus neutralization analysis showed antigenic diversity between the cell culture-adapted HV-IBDV strains and classical strains. In immunization tests, the adapted strain-immunized chickens showed good protection against the fatal infection of HV-IBDV. Especially, in case of challenge at 3 days postimmunization, the adapted strains showed effective immunogenicity. The adapted strains appear to provide a new and effective live vaccine against HV-IBDV infection.

Characteristics of a Novel Type of Bovine Cryptosporidium andersoni

ABSTRACT We isolated oocysts that resemble Cryptosporidium andersoni from cattle grazing on a farm in Japan. The partial sequences of genes from the isolate were coincident with published sequences of genes of C. andersoni. Since the isolate was able to infect SCID mice, the isolate appears to be a novel type of C. andersoni.

A hamster model of equine herpesvirus 9 induced encephalitis

An acute and lethal infection of equine herpesvirus 9 (EHV-9), a new type of equine herpesvirus, was established in Syrian hamsters by intranasal inoculation. Clinical symptoms included the loss of body weight, nasal and ocular discharges and apparent neurological symptoms. Both LD50 and ID50 were equal at 33 plaque forming units. Histological and immunohistochemical examination demonstrated that the virus replicated in the olfactory mucosal cells and in the neurons of the olfactory bulbs, cerebrum and mesencephalon. The induction of encephalitis by intranasal but not by other routes of inoculation (i.v., i.p., i.m.) indicated that EHV-9 entered the brain via the olfactory nerve and then spread trans-synaptically to connecting neurons along the olfactory tract. This animal model should be useful for studying the pathogenesis and neurovirulence of this newly discovered neurotropic virus as well as other neurotropic herpesviruses.

Detection of canine pemphigus foliaceus autoantigen by immunoblotting

The antigens targeted by autoantibodies in sera from canine patients with pemphigus foliaceus (PF) were detected by indirect immunofluorescence and Western immunoblotting. The extracted proteins from canine keratinocytes cultured in high-calcium condition for 48 h after confluency and from bovine nose epidermis were used as antigens in Western blotting. Canine keratinocytes cultured in high-calcium condition showed fluorescent deposits in intercellular spaces by incubation with sera from both canine and human pemphigus patients. By Western blotting, eight out of 16 canine PF sera recognised 160 kDa protein. 85 kDa and 120 kDa proteins were also recognised by four to five canine PF sera, respectively. The 160 kDa band, recognised by eight canine PF sera, had an identical mobility to the protein identified by a human PF serum. These results suggested that the autoantibodies in sera from canine PF recognised the 160 kDa desmosomal proteins, which may correspond to the desmoglein 1.

Equine herpesvirus 9 induced lethal encephalomyelitis in experimentally infected goats

Summary. The pathogenicity of a new neurotropic equine herpesvirus 9 (EHV-9) formerly designated gazelle herpesvirus 1 was evaluated using the goat as a representative of domesticated ruminants. Two goats inoculated intranasally with EHV-9 showed salivation, teeth grinding and other neurological disorders on day 8 post inoculation. One goat died 30 min after the onset of clinical signs and the other was sacrificed 3 h after the sudden onset of teeth grinding and foamy salivation. EHV-9 was recovered from peripheral white blood cells, the olfactory bulbs and brain, nasal swabs, concha, and lungs. Neuropathological lesions were located in the olfactory bulbs, cerebrum, midbrain and medulla oblongata with degeneration and necrosis of neurons, rarefaction, perivascular infiltration of mononuclear cells, and nodal glial reaction. EHV-9 antigen was detected in neurons in the lesions. These findings indicated that EHV-9 is highly pathogenic with high neurotropism for goats.

Neuropathological study of gazelle herpesvirus 1 (equine herpesvirus 9) infection in Thomson's gazelles (Gazella thomsoni)

Gazelle herpesvirus (GHV-1), correctly designated as equine herpesvirus 9, is a new type of equine herpesvirus immunologically related to equine herpesvirus 1 (EHV-1). As a sequel to a virological study, the neuropathology of encephalitis caused by GHV-1 in Thomsons gazelles (Gazella thomsoni) was examined. Seven gazelles died with or without neurological symptoms between early September and mid-October in 1993. No gross abnormalities were observed at necropsy, but all animals had non-suppurative encephalitis, characterized by necrosis and degeneration of neurons, glial reactions and perivascular cuffing in the cerebrum. Five cases showed intranuclear inclusion bodies, with the appearance of herpesvirus in the degenerating neurons. Immunohistochemically, all seven animals showed a positive reaction to EHV-1 antigen in neurons in the necrotic areas of the cortex. The clinical course and morphological features of GHV-1 encephalitis were distinct from those of EHV-1-induced encephalitis in the horse, which is characterized by vasculitis, thrombosis, ischaemia, and lack of intranuclear inclusions in neurons.

Experimental Infection of Equine Herpesvirus 9 in Dogs

Equine herpesvirus 9 (EHV-9), a new neurotropic equine herpesvirus, was inoculated intranasally at 107 plaque-forming units in five dogs to assess its pathogenicity. Dogs showed weight loss, pyrexia, anorexia, and neurologic signs on the fourth day. The EHV-9 virus was recovered from the examined brains. Histologically, dogs had a fulminant nonsuppurative encephalitis characterized by severe neuronal degeneration and loss, with intranuclear inclusions, slight glial reactions, perivascular cuffing, and multifocal hemorrhage. The olfactory bulb and the frontal and temporal lobes were predominantly affected. Immunohistochemistry revealed reactivity for EHV-9 antigen in neurons. All dogs had mild bronchopneumonia and various degrees of lymphoid necrosis. These findings indicate that dogs are fully susceptible to EHV-9 and that EHV-9 can cause fulminant encephalitis with high mortality in dogs, as in gazelles and goats.

Roles of cyclooxygenase‐2 and microsomal prostaglandin E synthase‐1 expression and β‐catenin activation in gastric carcinogenesis in N‐methyl‐N‐nitrosourea‐treated K19‐C2mE transgenic mice

K19‐C2mE transgenic (Tg) mice, simultaneously expressing cyclooxygenase‐2 (COX‐2) and microsomal prostaglandin E synthase‐1 (mPGES‐1) in the gastric mucosa under the cytokeratin 19 gene promoter, were here treated with N‐methyl‐N‐nitrosourea (MNU) and inoculated with Helicobacter pylori (H. pylori) to investigate gastric carcinogenesis. Wild‐type (WT) and Tg mice undergoing MNU treatment frequently developed tumors in the pyloric region (100% and 94.7%, respectively); multiplicity in Tg was higher than that in WT (P < 0.05) with H. pylori infection. Larger pyloric tumors were more frequently observed in Tg than in WT (P < 0.05). In addition, Tg developed fundic tumors, where WT did not. No gastric tumors were observed without MNU treatment. Transcripts of TNF‐α, iNOS, IL‐1β, and CXCL14 were up‐regulated with H. pylori infection in both genotypes and were also increased more in Tg than in WT within H. pylori‐inoculated animals. Immunohistochemical analysis demonstrated significantly greater β‐catenin accumulation in pyloric tumors, compared with those in the fundus (P < 0.01) with mutations of exon 3; 18.2% and 31.6% in MNU‐alone and MNU + H. pylori‐treated WT, whereas 21.4% and 62.5% was observed in the Tg, respectively; the latter significantly higher (P < 0.05), suggesting the role of H. pylori in Wnt activation. In conclusion, K19‐C2mE mice promoted gastric cancer in both fundic and pyloric regions. Furthermore β‐catenin activation may play the important role of pyloric carcinogenesis especially in H. pylori‐infected Tg. Induction of various inflammatory cytokines in addition to overexpression of COX‐2/mPGES‐1 could be risk factors of gastric carcinogenesis and may serve as a better gastric carcinogenesis model. (Cancer Sci 2008; 99: 2356–2364)