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1.  Serosurveillance for Francisella tularensis Among Wild Animals in Japan Using a Newly Developed Competitive Enzyme-Linked Immunosorbent Assay 
Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis–seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild.
PMCID: PMC3993067  PMID: 24689989
Francisella tularensis; Tularemia; Serosurveillance; Wild animals; Japan
2.  Role of Pathogenicity Determinant Protein C (PdpC) in Determining the Virulence of the Francisella tularensis Subspecies tularensis SCHU 
PLoS ONE  2014;9(2):e89075.
Francisella tularensis subspecies tularensis, the etiological agent of tularemia, is highly pathogenic to humans and animals. However, the SCHU strain of F. tularensis SCHU P0 maintained by passaging in artificial media has been found to be attenuated. To better understand the molecular mechanisms behind the pathogenicity of F. tularensis SCHU, we attempted to isolate virulent bacteria by serial passages in mice. SCHU P5 obtained after 5th passages in mice remained avirulent, while SCHU P9 obtained after 9th passages was completely virulent in mice. Moreover, SCHU P9 grew more efficiently in J774.1 murine macrophages compared with that in the less pathogenic SCHU P0 and P5. Comparison of the nucleotide sequences of the whole genomes of SCHU P0, P5, and P9 revealed only 1 nucleotide difference among P0, P5 and P9 in 1 of the 2 copies of pathogenicity determinant protein C (pdpC) gene. An adenine residue deletion was observed in the pdpC1 gene of SCHU P0, P5, and P9 and in the pdpC2 gene of SCHU P0, and P5, while P9 was characterized by the wild type pdpC2 gene. Thus, SCHU P0 and P5 expressed only truncated forms of PdpC protein, while SCHU P9 expressed both wild type and truncated versions. To validate the pathogenicity of PdpC, both copies of the pdpC gene in SCHU P9 have been inactivated by Targetron mutagenesis. SCHU P9 mutants with inactivated pdpC gene showed low intracellular growth in J774.1 cells and did not induce severe disease in experimentally infected mice, while virulence of the mutants was restored by complementation with expression of the intact PdpC. These results demonstrate that PdpC is crucial in determining the virulence of F. tularensis SCHU.
PMCID: PMC3928404  PMID: 24558472
3.  Detection of Francisella tularensis-Specific Antibodies in Patients with Tularemia by a Novel Competitive Enzyme-Linked Immunosorbent Assay 
A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R2 = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.
PMCID: PMC3535769  PMID: 23114700
4.  Inhibition of Both Protease and Helicase Activities of Hepatitis C Virus NS3 by an Ethyl Acetate Extract of Marine Sponge Amphimedon sp 
PLoS ONE  2012;7(11):e48685.
Combination therapy with ribavirin, interferon, and viral protease inhibitors could be expected to elicit a high level of sustained virologic response in patients infected with hepatitis C virus (HCV). However, several severe side effects of this combination therapy have been encountered in clinical trials. In order to develop more effective and safer anti-HCV compounds, we employed the replicon systems derived from several strains of HCV to screen 84 extracts from 54 organisms that were gathered from the sea surrounding Okinawa Prefecture, Japan. The ethyl acetate-soluble extract that was prepared from marine sponge Amphimedon sp. showed the highest inhibitory effect on viral replication, with EC50 values of 1.5 and 24.9 µg/ml in sub-genomic replicon cell lines derived from genotypes 1b and 2a, respectively. But the extract had no effect on interferon-inducing signaling or cytotoxicity. Treatment with the extract inhibited virus production by 30% relative to the control in the JFH1-Huh7 cell culture system. The in vitro enzymological assays revealed that treatment with the extract suppressed both helicase and protease activities of NS3 with IC50 values of 18.9 and 10.9 µg/ml, respectively. Treatment with the extract of Amphimedon sp. inhibited RNA-binding ability but not ATPase activity. These results suggest that the novel compound(s) included in Amphimedon sp. can target the protease and helicase activities of HCV NS3.
PMCID: PMC3492463  PMID: 23144928
5.  Inhibition of Hepatitis C Virus Replication and Viral Helicase by Ethyl Acetate Extract of the Marine Feather Star Alloeocomatella polycladia 
Marine Drugs  2012;10(4):744-761.
Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC50 of 11.7 µg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC50 value of 23 to 44 µg/mL, which is similar to the IC50 value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.
PMCID: PMC3366673  PMID: 22690141
marine organism; Alloeocomatella polycladia; hepatitis C virus; NS3 helicase
6.  Renal cell carcinoma with a tumor thrombus in the ureter: a case report 
BMC Urology  2011;11:16.
Renal cell carcinoma (RCCs) is the most common malignancy of the kidney. When RCC progresses, it is known to form tumor thrombus in the renal vein and/or inferior vena cava. However, RCC does not normally form tumor thrombus in the ureter or renal pelvis.
Case presentation
A 43-year-old man presented to our department for the treatment of a renal tumor with asymptomatic gross hematuria. In a dynamic CT study, contrast enhancement revealed a tumor suspected to be RCC, but atypical finding as a tumor thrombus that filled the renal pelvis and the whole ureter was also observed. Nephroureterectomy was performed, and the tumor was diagnosed histopathologically as RCC.
We report here a very rare case of RCC with a tumor thrombus in the whole ureter.
PMCID: PMC3161958  PMID: 21806792
7.  Preparation of Monoclonal Antibodies for Detection and Identification of Francisella tularensis▿  
Monoclonal antibodies (MAbs) against Francisella tularensis were obtained. Three MAbs specifically reacted with F. tularensis, while four MAbs reacted with other members of the genus Francisella as well. Fluorescent isothiocyanate-conjugated MAbs unequivocally stained bacterial cells in specimens from experimentally infected mice. Two MAbs agglutinated F. tularensis antigen in the agglutination tests. These MAbs should improve methods for detection and identification of F. tularensis.
PMCID: PMC1797713  PMID: 17121981

Results 1-7 (7)