Kazuhiko Suzuki

Dissemination of extracellular and intracellular Toxoplasma gondii tachyzoites in the blood flow.

Toxoplasma gondii is an intracellular parasite. It has been thought that T. gondii can disseminate throughout the body by circulation of tachyzoite-infected leukocytes (intracellular parasite) in the blood flow. However, a small number of parasites exist as free extracellular tachyzoites in the blood flow (extracellular parasite). It is still controversial whether the extracellular parasites in the blood flow disseminate into the peripheral tissues. In this study, we evaluated the dissemination efficiency of the extracellular and intracellular parasites in the blood flow using GFP-expressing transgenic parasite (PLK/GFP) and DsRed Express-expressing transgenic parasite (PLK/RED). When PLK/GFP and PLK/RED tachyzoites were injected, as intracellular and extracellular forms respectively, at the same time into the tail vein of a mouse, many disseminated green fluorescent PLK/GFP tachyzoites were observed in the lung, the spleen, the liver and the brain. However, only a few red fluorescent PLK/RED tachyzoites were detected in these organs. When PLK/GFP and PLK/RED tachyzoites were injected in the opposite manner, that is, as extracellular and intracellular forms respectively, the majority of tachyzoites in these tissues were PLK/RED tachyzoites. Collectively, these results indicate that intracellular tachyzoites mainly disseminate throughout the body and that extracellular tachyzoites hardly contribute to parasite dissemination.

Immunohistochemical Characterization of Canine Neuroepithelial Tumors

The expression of cell differentiation and proliferation markers of canine neuroepithelial tumors was examined immunohistochemically to identify the histogenesis of these tumors. Astrocytomas (n = 4) consisted of cells positive for glial fibrillary acidic protein (GFAP) and nestin and a few cells positive for doublecortin (DCX). Immunoreactive cells for receptor tyrosine kinases (epidermal growth factor receptor and c-erbB2) and their downstream molecules (phospho-extracellular signal-regulated kinase 1/2 and phospho-Akt) were often detected in astrocytomas, especially in medium- and high-grade tumors. Gliomatosis cerebri (n = 3) consisted of cells positive for ionized calcium–binding adaptor molecule 1 and GFAP, including a minor population of cells positive for nestin, DCX, and beta III tubulin, suggesting their glial differentiation. In choroid plexus tumors (n = 4), most tumor cells were positive for cytokeratins AE1/AE3 and 18, and few were positive for GFAP. The majority of cells of oligodendrogliomas (n = 5) were DCX positive, but the tumors also contained minor populations of cells positive for GFAP, nestin, or beta III tubulin. Primitive neuroectodermal tumors (PNETs; n = 2) consisted of heterogeneous cell populations, and the tumor cells were positive for nestin, beta III tubulin, and DCX, suggesting glial and neuronal differentiation. The major population of neuroblastoma cells (n = 3) were positive for beta III tubulin and DCX, suggesting single neuronal differentiation. As for antiapoptotic cell death molecules, most tumor cells in the choroid plexus tumors, PNETs, and neuroblastomas were intensely positive for Bcl-2 and Bcl-xL, whereas those in gliomatosis cerebri were almost negative. In astrocytomas, Bcl-xL-positive cells predominated over Bcl-2-positive cells, but the opposite was observed in oligodendrogliomas. The immunohistochemical results were analyzed by hierarchical clustering, and the constructed dendrogram clearly indicated a novel position of oligodendrogliomas: the primitive glial and neuronal differentiation.

Canine coronavirus induces apoptosis in cultured cells

Abstract Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups. In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown. We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in order to characterize the apoptotic effect in homologous cell system. Following CCoV infection A-72 cell line, which is permissive to CCoV, showed reduced growth rate, as detected by MTT assay, a standard colorimetric assay for measuring cellular proliferation, and underwent to apoptotic death. Starting from 24h after CCoV infection, cells morphology appeared dramatically changed, with cells rounding and detachment from culture surface. Morphologic and biochemical features of apoptosis, such as blebbing of the plasma membrane, translocation of phosphatidilserine to cell surface and annexin V positive staining, nuclear fragmentation, apoptotic bodies formation and DNA laddering, were detected in CCoV-infected cells. Propidium iodide staining of infected culture indicated the appearance of hypodiploid DNA peak corresponding to apoptotic cell population. Commonly to other animal coronavirus infection caspase-3 is likely to contribute to the execution phase of apoptosis induced by CCoV in A-72 cells since we found activation of enzymatic activity as well as procaspase-3 activating cleavage. Apoptotic death of infected cells is detrimental as it causes cell and tissue destruction as well as inflammatory responses. Therefore in the case of CCoV associated gastroenteritis, apoptosis of epithelial mucosa cells may be responsible for pathology induced by CCoV infection.

Kinetics of cytokines mRNAs expression in the dorsal skin of WBN/ILA-Ht rats following topical application of T-2 toxin

The kinetics of cytokines mRNAs expression was examined in the dorsal skin of Wistar-derived hypotrichotic WBN/ILA-Ht rats topically applied with T-2 toxin. After the application of 10 microl (0.5 microg/microl) of T-2 toxin solution, the total mRNA was obtained from skin biopsies at 3, 6, 12 and 24 hours after treatment (HAT). Reverse transcription-polymerase chain reaction (RT-PCR) was carried out with pairs of oligonucleotide primers corresponding to the cDNA sequences of rat TNF-alpha, IL-1 alpha, IL-1beta, IL-6 and IL-10 cytokines. The level of TNF-alpha mRNA showed marked elevation at 3HAT and decreased toward 24HAT, but it remained significantly higher level even at 24HAT. In addition, the level of IL- 1beta mRNA expression showed a sligth but significant elevation at 3 and 24HAT. On the other hand, no significant differences were observed in other cytokines mRNAs expression between T-2 toxin-treated and control groups througth the observation period. Together with our previous report describing the sequence of epidermal cell apoptosis (Albarenque et al. 1999), the present results suggest that the elevation of TNF-alpha mRNA expression may play an important role in T-2 toxin-induced epidermal cell apoptosis.

Vaginal rhabdomyosarcoma in a dog.

A 10-year-old, female, mongrel showed hemorrhage from vulva. By magnetic resonance image (MRI) and endoscopic examination, a multipapillary mass with a grape-like appearance was found around the urethral opening. Histologically, the mass consisted of variable-sized round-, spindle-to-polygonal-shaped tumor cells including many multinuclear cells. Mitotic figures were also frequently observed. In some areas, that tumor cells were loosely arranged, with intercellular myxoid components. immunohistochemically, these tumor cells were strongly positive for vimentin and focally positive for desmin but negative for myoglobin. Thus, the case was diagnosed as a relatively poorly differentiated botryoid rhabdomyosarcoma by the macroscopic, histopathologic, and immunohistochemical identification. This is the first report of botryoid rhabdomyosarcoma developing in the vagina of a dog.

Expression of Cell Adhesion Molecules and Doublecortin in Canine Anaplastic Meningiomas

Tumor cell invasion into the surrounding nervous tissue is one of the histologic hallmarks of anaplastic meningiomas. To identify other possible markers for aggression in canine meningiomas, the relationship between histologic features and the expression of molecules involved in cell adhesion, cell proliferation, and invasion was examined. Immunohistochemistry for epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), β-catenin, doublecortin (DCX), and Ki-67 was performed for 55 cases of canine meningioma. DCX was preferentially expressed in tumor cells invading the brain parenchyma (12 of 14 cases), suggesting its involvement in the invasion process. Regardless of the histologic type, E-cadherin and N-cadherin expression was observed in 31 of 55 and 44 of 55 cases, respectively. There was a significant positive correlation between DCX and N-cadherin expression and a significant negative correlation between E-cadherin and N-cadherin expression, suggesting that decreased E-cadherin and increased N-cadherin expression induce DCX expression. Typical membranous β-catenin expression was observed in 10 of 55 cases, whereas nuclear translocation was observed in 33 cases. Nuclear β-catenin expression was frequently found in anaplastic meningiomas (12 of 14 cases). The Ki-67 labeling indices were significantly higher in anaplastic meningiomas than in other types. These findings indicate that the expression of N-cadherin and DCX and the nuclear translocation of β-catenin are closely associated with the presence of invasion and anaplasia in canine meningiomas. Notably, granular cell meningiomas were negative for almost all the molecules examined, suggesting that they have a different tumor biology than other meningiomas.

Visualization of Toxoplasma gondii stage conversion by expression of stage-specific dual fluorescent proteins.

To recognize the stage conversion of Toxoplasma gondii between tachyzoite and bradyzoite in live host cells, a transgenic T. gondii line, which expressed stage-specific red and green fluorescence, was constructed. T. gondii PLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7.0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.

Renal Collecting Duct Carcinoma in a Dog

An 8-year-old, male, mongrel dog developed severe cough and anorexia and died within 3 months. Autopsy revealed an invasive grayish-white mass in the right kidney and multiple nodules in the lungs, thoracic wall, and spleen. Histologically, the renal mass and the other nodules were mainly composed of papillotubular structures lined by oval-to-polygonal pleomorphic cells. The cells were reactive with DBA, PNA, and UEA-1 lectins and positive for vimentin but negative for CD10 and high molecular weight cytokeratin. Because of its histological, histochemical, and immunohistochemical similarities with human collecting duct carcinoma (CDC), a diagnosis of renal collecting duct carcinoma with pulmonary, thoracic, and splenic metastases was established. To our knowledge, this is the first case report of CDC in animals.

Development of loop-mediated isothermal amplification method for diagnosis of bovine leukemia virus infection

A rapid, sensitive loop-mediated isothermal amplification (LAMP) assay was established for diagnosis of bovine leukemia virus (BLV) infection. The LAMP assay for targeting the BLV-LTR region can detect at least 2 copies of proviral DNA in a 2microl sample and its sensitivity is equivalent to or greater than the conventional single PCR. In addition, amplification is obtained in less than 1h by incubating a single tube in a water bath. The data obtained by the LAMP assay applied to field samples were compared with PCR and serological tests for BLV. The results showed a high level of agreement with these serological methods, but there was one animal positive only by the LAMP assay. When the blood was collected from the cow after 6 months, the BLV antibody was detected. This suggested that the LAMP assay could help the detection of the cattle in the early stage of BLV infection. The LAMP assay is a rapid, sensitive and simple method for the diagnosis of BLV infection as reported for other pathogens, and is available for use in local laboratories without any special equipment.

MAXI observations of gamma-ray bursts

Monitor of all-sky image (MAXI) Gas Slit Camera (GSC) detects gamma-ray bursts (GRBs) including the bursts with soft spectra, such as X-ray flashes (XRFs). MAXI/GSC is sensitive to the energy range from 2 to 30 keV. This energy range is lower than other currently operating instruments which is capable of detecting GRBs. Since the beginning of the MAXI operation on August 15, 2009, GSC observed 35 GRBs up to the middle of 2013. One third of them are also observed by other satellites. The rest of them show a trend to have soft spectra and low fluxes. Because of the contribution of those XRFs, the MAXI GRB rate is about three times higher than those expected from the BATSE log N – log P distribution. When we compare it to the observational results of the Wide-field X-ray Monitor on the High Energy Transient Explorer 2, which covers the the same energy range to that of MAXI/GSC, we find a possibility that many of MAXI bursts are XRFs with Epeak lower than 20 keV. We discuss the source of soft GRBs observed only by MAXI. The MAXI log N – log S distribution suggests that the MAXI XRFs distribute in closer distance than hard GRBs. Since the distributions of the hardness of galactic stellar flares and X-ray bursts overlap with those of MAXI GRBs, we discuss a possibility of a confusion of those galactic transients with the MAXI GRB samples.