Investigation of antibody to severe fever with thrombocytopenia syndrome virus (SFTSV) in blood samples donated in a SFTS-endemic area in Japan.

The risk of transfusion-transmitted infection (TTI) for severe fever with thrombocytopenia syndrome virus (SFTSV) is a concern because person-to-person transmission resulting from contact with SFTSV-contaminated blood has been reported. To obtain information regarding the risk of TTI-SFTSV, antibody testing was performed for blood samples donated in an severe fever with thrombocytopenia syndrome-endemic area in Japan. No antibody-positive samples were detected among 3990 samples. This finding suggested that there were few cases of SFTSV infection among donors and that the risk of TTI-SFTSV was also estimated low in Japan.

Efficacy of the Mirasol pathogen reduction technology system against severe fever with thrombocytopenia syndrome virus (SFTSV).

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tickborne virus in the Bunyaviridae family. This virus has recently been found in China, Japan and Korea. The risk of transfusion-transmitted SFTSV infection (TTI-SFTSV) is a concern because person-to-person transmission resulting from contact with SFTSV-contaminated blood has been reported. Therefore, we investigated the efficacy of the Mirasol pathogen reduction technology (PRT) system for inactivating SFTSV in vitro. The Mirasol PRT system achieved a > 4.11 log10 reduction value (LRV) for SFTSV. In conclusion, we showed that the Mirasol PRT system could potentially be used to reduce the risk of TTI-SFTSV.

Prevalence of Smqnr and plasmid-mediated quinolone resistance determinants in clinical isolates of Stenotrophomonas maltophilia from Japan: novel variants of Smqnr.

Stenotrophomonas maltophilia is an important pathogen in healthcare-associated infections. S. maltophilia may contain Smqnr, a quinolone resistance gene encoding the pentapeptide repeat protein, which confers low-level quinolone resistance upon expression in a heterologous host. We investigated the prevalence of Smqnr and plasmid-mediated quinolone resistance (PMQR) determinants in S. maltophilia isolates from Japan. A total of 181 consecutive and nonduplicate clinical isolates of S. maltophilia were collected from four areas of Japan. The antimicrobial susceptibility profiles for these strains were determined. PCR was conducted for Smqnr and PMQR genes, including qnrA, qnrB, qnrC, qnrS, aac(6')-Ib and qepA. PCR products for Smqnr and aac(6')-Ib were sequenced. For the S. maltophilia isolates containing Smqnr, pulsed-field gel electrophoresis (PFGE) was performed using XbaI. Resistance rates to ceftazidime, levofloxacin, trimethoprim-sulfamethoxazole, chloramphenicol and minocycline were 67.4%, 6.1%, 17.7%, 8.8% and 0%, respectively. The minimum inhibitory concentration required to inhibit the growth of 50% and 90% of organisms were 0.5 and 2 mg/L for moxifloxacin but 1 and 4 mg/L for levofloxacin, respectively. Smqnr was detected in 104 of the 181 S. maltophilia isolates (57.5%), and the most frequent was Smqnr6, followed by Smqnr8 and Smqnr11. Eleven novel variants from Smqnr48 to Smqnr58 were detected. The 24 Smqnr-containing S. maltophilia isolates were typed by PFGE and divided into 21 unique types. Nine S. maltophilia isolates (5.0%) carried aac(6')-Ib-cr. No qnr or qepA genes were detected. This study describes a high prevalence of Smqnr and novel variants of Smqnr among S. maltophilia from Japan. Continuous antimicrobial surveillance and further molecular epidemiological studies on quinolone resistance in S. maltophilia are needed.

Comparison of the BDProbeTec ET system with the Cobas Amplicor PCR for direct detection of Mycobacterium tuberculosis in respiratory samples.

In the study presented here, the performance of the BDProbeTec ET system (Becton Dickinson, USA) was compared with the Roche Cobas Amplicor-PCR (Roche, Switzerland) to detect Mycobacterium tuberculosis complex (MTB) in clinical respiratory samples. The Bactec MGIT 960 liquid culture system (Becton Dickinson) was used as a reference method. A total of 411 samples were tested. Of the 93 culture-positive samples, both the BDProbeTec ET system and the Cobas Amplicor-PCR detected 87 (sensitivity, 93.5%). When only smear-negative samples were considered, the BDProbeTec ET exhibited a sensitivity of 50% and the Cobas Amplicor-PCR 60%. Specificity was 99.7% for the BDProbeTec ET system and 100% for the Cobas Amplicor-PCR. Percent agreement between the two nucleic amplification methods was 98.7%. Inhibition occurred in three (0.7%) samples in the BDProbeTec ET system. The high sensitivity and specificity of the BDProbeTec ET system suggest it is a useful method for the rapid and direct detection of MTB in smear-positive respiratory samples.

Accumulation of plant galactolipid affects cell morphology of Escherichia coli.

Monogalactosyldiacylglycerol (MGDG) is a major constituent of thylakoid membrane in chloroplasts. Therefore, it is considered to have an important role in the maintenance of the complicated structure of the thylakoid membrane. We have succeeded in cloning the enzyme for MGDG synthesis and overexpressed it in Escherichia coli. In this study we analyzed the morphology of the E. coli harboring the gene. The fatty acid composition of its membrane lipids did not differ between the wild type and transformant, except for the appearance of MGDG. However, transformant cells appeared to be elongated. DAPI staining revealed the entire intracellular region of filamentous cells to be stained; therefore, the elongation of the cells is probably due to a defect in cell division. Atomic force microscopy revealed that the transformant had a smooth but scratched surface. It was concluded that the excessive accumulation of a non-bilayer lipid, MGDG, interfered with the translocation of proteins across the plasma membrane, including those for cell division.

Molecular cloning of the cDNAs encoding the feline B-lymphocyte activation antigen B7-1 (CD80) and B7-2 (CD86) homologues which interact with human CTLA4-Ig.

We cloned the cDNAs encoding the feline homologues of B-lymphocyte activation antigens B7-1 (CD80) and B7-2 (CD86). We expressed recombinant feline CD80 and CD86 molecules by the baculovirus expression system, and demonstrated their binding ability to human CTLA4-murine immunoglobulin fusion protein.

Telomerase activity of needle-biopsied liver samples: its usefulness for diagnosis and judgement of efficacy of treatment of small hepatocellular carcinoma.

BACKGROUND/AIMS: High values for telomerase activity in malignant tumors have been reported. The clinical usefulness of measurements of telomerase activity as a diagnostic tool and to evaluate treatment efficacy in small hepatocellular carcinoma was investigated. METHODS: We investigated 22 patients (26 nodules) with intrahepatic abnormal nodules < or =20 mm in size determined by abdominal ultrasound. All underwent needle biopsies of tumorous nodules and extranodular regions of the liver by ultrasound guidance for histopathological diagnosis and measurement of telomerase activity by the fluorescence-based telomeric repeat amplification protocol. Re-biopsy of the same nodule was performed 1 week after percutaneous ethanol injection therapy to measure telomerase activity in 10 patients (10 nodules) found to have hepatocellular carcinoma. Liver-biopsied samples from 30 patients with chronic hepatitis C were used as a control. RESULTS: Telomerase activity increased with statistical significance stepwise: chronic hepatitis (n=30, mean: 0.00 U) extranodular regions (pre-cirrhosis or cirrhosis, n=22, mean: 1.80 U), atypical hyperplasia (borderline or premalignant lesions, n= 15, mean: 7.02 U) and low-grade malignant hepatocellular carcinoma (n=11, mean: 31.96 U) (p<0.0001 by the Kruskal-Wallis test). Percutaneous ethanol injection therapy resulted in loss (0.00 U) of telomerase activity in 9 nodules and persistence in 1 nodule. CONCLUSIONS: Measurement of telomerase activity appeared useful for diagnosis of intrahepatic abnormal nodules and assessment of the efficacy of percutaneous ethanol injection therapy and may be used as an alternative diagnostic method, especially when pathohistological discrimination between atypical hyperplasia and well-differentiated hepatocellular carcinoma is difficult.

Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG.

MGDG synthase, the enzyme that catalyzes the synthesis of the major chloroplast membrane lipid monogalactosyldiacylglycerol (MGDG), is encoded by a multigenic family. We have analyzed the biochemical properties, subcellular localization and membrane topology of a spinach chloroplast MGDG synthase, a representative member of the type A family from Spinacia oleracea (soMGD A), using a recombinant protein that was functionally overexpressed in Escherichia coli and specific polyclonal antibodies. We demonstrated that soMGD A could catalyze the synthesis of both 'prokaryotic' and 'eukaryotic' MGDG molecular species in vitro, with a selectivity for diacylglycerol similar to that of purified chloroplast envelope MGDG synthase activity. Furthermore, soMGD A was shown to be sensitive to chemical reagents (dithiothreitol, N-ethylmaleimide and o-phenanthroline) known to affect MGDG synthesis by the partially purified enzyme, as well as in isolated chloroplast envelope membranes. In spinach chloroplasts, soMGD A was localized by Western blot analysis in the inner envelope membrane. Topological studies demonstrated that soMGD A is a monotopic enzyme, embedded within one leaflet of the inner envelope membrane from spinach chloroplasts, a structure which may involve amphipathic alpha helices. We further demonstrated that in vitro, soMGD A precursor is imported and processed to its correct mature form in intact chloroplasts. These results show that soMGD A corresponds to a mature polypeptide of approximately 45 kDa. In addition, inactivation kinetics after gamma-ray irradiation strongly suggest that both native chloroplast envelope MGDG synthase and recombinant soMGD A have a functional molecular mass of 95-100 kDa, indicating that they are probably active as homodimers made of two 45-kDa subunits. This study suggests that, in spite of the growing evidence that MGDG synthesis is catalyzed by a multigenic family of enzymes, in spinach leaves both prokaryotic and eukaryotic MGDG syntheses could be attributable to a unique dimeric enzyme, provided that diacylglycerol is transported from the outer membrane to the inner membrane of the chloroplast envelope.

[Multicenter evaluation of broth microdilution test, BrothMIC MTB, to determine minimum inhibitory concentrations (MICs) of antimicrobial agents for Mycobacterium tuberculosis--evaluation of interlaboratory precision and interpretive compatibility with agar proportion method].

A newly developed microdilution antimycobacterial susceptibility test, BrothMIC MTB (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) to determine minimum inhibitory concentrations (MICs) was evaluated at multisites. The test method utilizes air-dried microplates containing serially diluted antimicrobial agents and the modified Middlebrook 7H9 broth. The eight antimycobacterial agents tested were rifampicin, isoniazid, ethambutol, streptomycin, kanamycin, levofloxacin, sparfloxacin and ciprofloxacin. The test plates were reconstituted by inoculation of 0.2 ml of cell suspensions (6 x 10(5) cells/ml) and were incubated at 36 degrees C in 5% to 10% CO2. The growth endpoints were visually read after 7-day and 10-day incubations. The reproducibility was evaluated with the four reference strains of Mycobacterium tuberculosis, and were compared with the agar proportion method described in the National Committee for Clinical Laboratory Standards (NCCLS) M24-T. Of the 1,022 testings of the reference strains, 1,020 (99.8%) of the MICs read after 7-day incubation fell within 3 log2 dilutions. The growth endpoints read after 7-day and 10-day incubations gave equal MIC ranges for the respective agents. The results obtained by the BrothMIC MTB for 93 clinical isolates of M. tuberculosis compared well with those determined by the NCCLS method with 98% to 99% agreements, except for ethambutol. According to the comparative analysis with the agar proportion method, the interpretive MIC breakpoints to discriminate between the isolates susceptible and resistant against the respective agents were proposed. In conclusion, this newly developed microdilution test for M. tuberculosis is a practical, rapid, quantitative, nonradiometric alternative for the determination of MICs in clinical mycobacteriology laboratories.

[Evaluation of a newly developed broth microdilution test method to determine minimum inhibitory concentrations (MICs) of antimicrobial agents for mycobacteria].

We developed a new broth microdilution antimycobacterial susceptibility test for determination of minimum inhibitory concentration (MICs) using an air-dried microplate containing serially diluted antimicrobial agents and the modified Middlebrook 7H9 broth. The eight agents included were streptomycin (SM), isoniazid (INH), rifampicin (RFP), ethambutol (EB), kanamycin (KM), levofloxacin (LVFX), sparfloxacin (SPFX) and clarithromycin (CAM). Serial dilutions of the agents (128 micrograms/ml to 0.125 micrograms/ml) were prepared in microplates, and were reconstituted by inoculation of 0.2ml of cell suspensions (approximately 3 x 10(5) cells/ml). The test plates were incubated at 36 degrees C in 5% CO2, and the growth endpoints were read visually after 5-day, 7-day and 10-day incubations. Four ATCC reference strains, Mycobacterium tuberculosis, M. avium, M. kansasii and M. intracellulare, were repeatedly tested at three sites. Of 480 determination against eight agents, 455 (94.8%), 470 (97.9%) and 455 (94.8%) of the MICs read after 5-day, 7-day and 10-day incubations fell within 3log2 dilutions, respectively. The MICs gradually elevated during the incubation, however those of 7-day incubation were highly precise and easily determined. A total of 160 clinical isolates of M. tuberculosis and 114 of nontuberculous mycobacteria were tested against eight agents. As for the primary drugs (SM, INH, RFP and EB), most isolates of M. tuberculosis were highly susceptible with MIC90, < or = micrograms/ml. Both LVFX and SPFX were also active. The MICs against nontuberculous mycobacteria distributed in a wide range, and the activities of RFP, LVFX, SPFX and CAM were more potent. These results demonstrate this newly developed test method to be a practical, rapid, quantitative and nonradiometric alternative for the determination of MICs in clinical mycobacteriology laboratories.

Characterization of anti-feline CD8 monoclonal antibodies.

We generated three monoclonal antibodies (mAbs) (2D7, 10C7 and 12A3) reactive to the alpha-chain of feline CD8 (fCD8) molecule. Further we showed that reference anti-fCD8 mAbs, FT2, 3.357 and vpg9 recognize the beta-chain, alpha-chain and alphabeta-complex epitope, respectively. Flow cytometric analysis using these mAbs suggested that fCD8alpha(+)beta(-) cells were present in lymphocytes of spleen, but not significantly in those of thymus, lymph nodes and peripheral blood of normal kittens.

Eight-year observation and comparative study of specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) subtypes A and B: terminal acquired immunodeficiency syndrome in a cat infected with FIV petaluma strain.

Three specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) strains Petaluma, TM1 and TM2, respectively were observed for over 8 years. Without showing any significant clinical signs of immunodeficiency syndrome (AIDS) for 8 years and 4 months of asymptomatic phase, the Petaluma-infected cat exhibited severe stomatitis/gingivitis, anorexia, emaciation, hematological and immunological disorders such as severe anemia, lymphopenia, thrombocytopenia, and decrease of CD4/CD8 ratio to 0.075, and finally died with hemoperitoneum at 8 years and 8 months post-infection. Histopathological studies revealed that the cat had systemic lymphoid atrophy and bone marrow disorders indicating acute myelocytic leukemia (aleukemic type). Plasma viral titer of the cat at AIDS phase was considerably high and anti-FIV antibody titer was slightly low as compared with the other FIV-infected cats. In addition, immunoblotting analysis using serially collected serum/plasma samples of these cats revealed that antibodies against FIV proteins were induced in all the infected cats, however in the Petaluma-infected cat anti-Gag antibodies disappeared during the asymptomatic period. These results suggested that plasma viral load and anti-FIV Gag antibody response correlated with disease progression, and supported FIV-infected cats as a suitable animal model of human AIDS.

Expansion of CD8alpha+beta- cells in cats infected with feline immunodeficiency virus.

CD8+ lymphocytes have been subdivided into CD8alphabeta and CD8alpha alpha populations in the peripheral blood lymphocytes (PBL) of humans and in several animal species but have not yet been investigated in cats. Feline immunodeficiency virus (FIV) causes progressive immunological disorders similar to human AIDS. In this study, we analysed CD8+ cells in PBL of FIV-infected or uninfected cats by two-colour flow cytometric analysis. In specific pathogen-free adult cats, feline CD8alpha+beta(high) cells were observed but CD8alpha+beta- cells were not found in significant numbers. On the other hand, not only CD8alpha+beta(high) but also CD8alpha+beta- and CD8alpha+beta(low) cell populations were observed in cats chronically infected with FIV. The expansion of the CD8beta(low) or CD8beta- subpopulations resulted in the apparent differences in CD4/CD8 ratios depending on the anti-CD8 MAb used. These findings suggest a need to reconsider the CD4/CD8 ratio in studies of FIV infection. Furthermore, we found that the CD8alpha+beta- cell population expressed CD5 at a low level.

Appearance of fosfomycin resistant Rahnella aquatilis clinically isolated in Japan.

Among recent clinical isolates in Japan, strain CU264 was discovered which formed unusual colonies. This strain was identified as Rahnella aquatilis which is usually found in water. The antibiotic susceptibilities against tetracycline, carbenicillin, chloramphenicol, streptomycin, kanamycin, gentamicin, sulphonamide, neomycin, fosfomycin, rifampicin, norfloxacin and nalidixic acid, were investigated. The result demonstrated that the strain was highly resistant to fosfomycin only. It was further shown that this resistance was transmissible with low frequency to Serratia marcescens whereas it was not transmissible to Escherichia coli.

Diagnostic value of the strand displacement amplification method compared to those of Roche Amplicor PCR and culture for detecting mycobacteria in sputum samples.

We compared the ability of the semiautomated BDProbeTec-SDA system, which uses the strand displacement amplification (SDA) method, with that of the Roche Amplicor-PCR system and the Septi-Chek AFB culture system to directly detect Mycobacterium tuberculosis complex (MTB) and other mycobacteria in sputum samples. A total of 530 sputum samples from 299 patients were examined in this study. Of the 530 samples, 129 were culture positive for acid-fast bacilli with the Septi-Chek AFB system; 95 for MTB, 29 for M. avium-M. intracellulare complex (MAC), and 5 for other mycobacteria. The BDProbeTec-SDA system detected 90 of the 95 samples culture positive for MTB (sensitivity, 94.7%), and the Amplicor-PCR system detected 85 of the 95 samples culture positive for MTB (sensitivity, 89.5%). The specificity of each system, based on the clinical diagnosis, was 99.8% for SDA and 100% for PCR, respectively. Among the 29 samples culture positive for MAC, the BDProbeTec-SDA system detected MAC in 24 samples (sensitivity, 82.8%), whereas the Amplicor-PCR system detected MAC in 23 samples (sensitivity, 79.3%). The specificities of the systems were 98.3 and 100%, respectively. The high degrees of sensitivity and specificity of the BDProbeTec-SDA system suggest that it should be very useful in clinical laboratories for the rapid detection of mycobacteria in sputum samples.

Generation of monoclonal antibodies against a feline CD antigen (CD4) expressed by a recombinant baculovirus.

We attempted to establish a system to generate monoclonal antibodies (mAbs) recognizing CD antigens on lymphocytes of domestic animals using the expressed CD antigens by a baculovirus expression system. For this purpose, we selected feline CD4 (fCD4) antigen and expressed it in an insect cell line (Sf9 cells). To obtain mAbs, BALB/c mice were immunized with feline peripheral blood mononuclear cells (fPBMCs) or Sf9 cells expressing the fCD4 (Sf-fCD4 cells), and then hybridomas secreting antibodies were screened by indirect immunofluorescence assay against the opposite antigens of Sf-fCD4 cells or fPBMCs, respectively. Five mAbs recognizing the fCD4 were obtained in total. The system established here might be useful to obtain mAbs recognizing CD antigens of domestic animals.

Cloning of the gene for monogalactosyldiacylglycerol synthase and its evolutionary origin.

Monogalactosyldiacylglycerol (MGDG) synthase (UDPgalactose:1,2-diacylglycerol 3-beta-D-galactosyltransferase; EC 2.4.1.46) catalyzes formation of MGDG, a major structural lipid of chloroplast. We cloned a cDNA for the synthase from cucumber cDNA library. The full-length cDNA clone was 2142 bp, and it contains a 1575-bp open reading frame encoding 525 aa. The open reading frame consists of the regions for a mature protein (422 aa; Mr of 46,552) and transit peptide to chloroplast (103 aa). Although the molecular weight of mature protein region matched that purified from cucumber cotyledons, it was quite different from those purified from spinach (approximately 20 kDa) reported by other groups. The mature region of the protein was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The expression in E. coli showed that the protein catalyzed MGDG synthesis very efficiently. Therefore, we concluded that the cDNA encodes MGDG synthase in cucumber. In addition, the deduced amino acid sequence of the MGDG synthase cDNA showed homology with MurG of Bacillus subtilis and E. coli, which encode a glycosyltransferase catalyzing the last step of peptidoglycan synthesis in bacteria. This sequence homology implies that the machinery of chloroplast membrane biosynthesis is evolutionarily derived from that of cell wall biosynthesis in bacteria. This is consistent with the endosymbiotic hypothesis of chloroplast formation.

The effects of treatment with chemical agents or infection with feline viruses on protein-binding properties of the feline immunodeficiency virus long terminal repeat.

The effects of treatment with chemical agents or infection with feline viruses on protein-binding properties of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were examined by gel-mobility-shift assays using oligonucleotides designed to represent putative AP-1 or ATF motif from the FIV LTR. Infection with FIV led to less nuclear proteins binding to the AP-1 and ATF sites, suggesting that proteins binding to the sites were consumed or suppressed by FIV-replication in FIV-infected cells. Nuclear proteins that bind to the AP-1 or ATF site were examined by using extracts from Crandell feline kidney (CRFK) cells treated with TPA (a phorbol ester; a strong activator of protein kinase C) or forskolin (an inducer of cyclic-AMP), or infection with feline herpesvirus type 1 (FHV-1). Although TPA or forskolin treatment moderately increased the level of both proteins that bound to AP-1 and ATF sites, FHV-1 infection markedly changed the protein-binding patterns of the sites. Furthermore, FHV-1-induced proteins that bind adjacent to the transcriptional initiation site of FIV promoter were also observed in FHV-1-infected CRFK cells, suggesting that the FHV-1-induced-proteins affects the transcription of FIV through the AP-1, ATF and leader sequences.

Persistence of high virus neutralizing antibody titers in cats experimentally infected with feline immunodeficiency virus.

The development of virus neutralizing (VN) antibody is one of the most effective host defense mechanisms against virus infection. In the present study, we developed a new VN assay against feline immunodeficiency virus (FIV) using a feline T-lymphoblastoid cell line, MYA-1 cells, based on inhibition of viral reverse transcriptase production. This assay is applicable to strains of FIV which can not infect CRFK cells. By using the assay, we examined long-term responses of VN antibody in cats experimentally infected with FIV. VN antibody titers increased progressively during first 30 weeks post inoculation and remained at high titers thereafter for 7 years of observation periods.