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.

Comparison of neutralization profiles for anti-HCV reactive donor samples with or without detectable HCV RNA.

BACKGROUND AND OBJECTIVES: At Japanese Red Cross (JRC) Blood Centers, all donated blood is screened for hepatitis C virus (HCV) by serological and nucleic acid amplification testing. Donor plasma that tested reactive for anti-HCV by serological test is disqualified even if the donor tests negative for HCV RNA. These test results reflect both true-positive results because of past HCV infection and false-positive results because the cross-reactivity of plasma IgG, which current testing methods are unable to distinguish. To characterize these antibody test results, we examined the neutralizing activity of these plasma samples. MATERIAL AND METHODS: Donor plasma samples that tested reactive for anti-HCV by serological test but negative for HCV RNA (n = 43) were analysed for determining their neutralizing activities measured by the inhibition of the cellular entry of pseudoparticles harbouring HCV envelope glycoproteins (HCVpp). RESULTS: Strong and broad neutralizing activities against HCVpp entry similar to the samples that tested reactive for anti-HCV serological test and positive for HCV RNA (considered to be derived from individuals with chronic HCV infection) were observed in three of 43 plasma samples from donors who tested anti-HCV reactive but HCV RNA negative. CONCLUSION: By examining the neutralizing activities of plasma samples, we identified individuals with a past HCV infection from those in whom we were unable to confirm HCV infection according to the current testing algorithms of JRC, which do not perform anti-HCV confirmatory tests.

Establishment of a cell line panel as an alternative source of platelet antigens for a screening assay of anti-human platelet antibodies.

BACKGROUND: A panel of platelets expressing various human platelet antigens (HPAs) for a platelet antibody screening assay is difficult to prepare because some antigens are rarely expressed. Therefore, an alternative method without using platelets would be helpful in detecting HPA antibodies. This study describes the establishment of cell lines that stably express specific HPAs and their application for detecting specific antibodies. METHODS: Wild-type ß3, HPA-1b, -6b, -7b and -7 variant cDNA as well as wild-type αIIb and HPA-3b cDNA were individually co-transduced with wild-type αIIb and ß3 cDNA in the K562 cell line. We performed an immunobead monoclonal antibody immobilisation of platelet antigens (MAIPA) assay to evaluate this cell line panel for antibody detection using identified sera containing HPA antibodies, whose specificities had been determined by the mixed passive haemagglutination test. RESULTS AND CONCLUSION: Of the 12 sera containing HPA-1a (n = 2), HPA-3a (n = 6), HPA-6b (n = 3) or HPA-7 variant (n = 1) antibodies, all antibodies were detected and determined by our new method, except for two HPA-3a antibodies. One of the two antibodies was also negative for conventional platelet MAIPA, suggesting that the cell line panel might be used as an alternative source of platelet antigens in the MAIPA assay.

Clinical features of 215 stage I ovarian tumors in Japanese women.

PURPOSE: Differences of the clinical features of Stage I borderline ovarian tumors and Stage I ovarian cancer need to be clarified. METHODS: We retrospectively investigated 215 patients with Stage I ovarian tumors (67 with borderline tumors and 148 with ovarian cancer) treated between 1988 and 2001. RESULTS: Only one patient with a borderline tumor developed recurrence, while recurrence was found in 20 patients with Stage I ovarian cancer. There was a significant difference in the recurrence rate between patients with Stage Ia or Ib ovarian cancer and those with Stage Ic cancer (p = 0.007). Clear cell adenocarcinoma showed a higher recurrence rate. Among our patients with recurrence, only five in whom the recurrent tumor could be surgically resected are currently alive and disease-free. CONCLUSIONS: This study confirmed the low aggressiveness of Stage I borderline ovarian tumors and high aggressiveness of Stage Ic ovarian cancer or clear cell adenocarcinoma. In patients with recurrence, surgical resection may improve survival.

Long cellular repeats flanking a defective HTLV-I provirus: implication for site-targeted integration.

Retroviruses generally integrate as proviruses which are flanked by long-terminal repeats (LTRs) on both 5' and 3' ends. Since these LTRs are required for the efficient integration mediated by the viral integrase, it is believed that defective proviruses with a single LTR are normally formed by deletion after integration. However, we found no deletion of cellular sequences around the integration site of such a defective HTLV-1. Rather, we identified 99 bp-long direct repeats adjacent to both ends of the defective provirus. The repeated cellular sequences contained a potential poly(A) signal followed by a retroviral primer-binding-site-like sequence. The presence of the direct repeats of cellular sequences can be explained by the integration of the defective virus through homologous recombination between cellular and viral read-through sequences.

Analysis of a novel defective HTLV-I provirus and detection of a new HTLV-I-induced cellular transcript.

HTLV-I generally integrates at least one full-length copy in adult T-cell leukemia (ATL) cells. A group of patients without full-length provirus have a unique conserved truncation of the provirus which retains env-pX-3'LTR. Tumor cells of a patient from this group were genetically analyzed. Analysis of the 5' and 3' cellular flanking region adjacent to the provirus suggest that the defective provirus was integrated immediately downstream of a promoter of an unknown cellular gene. The activity of the promoter was weak but was responsive to Tax-like HTLV-I LTR. The provirus may have utilized it as a substitute for the 5'LTR and thus 3'LTR may have become an alternative promoter for the cellular gene, which may give similar viral-cellular interactions to that of general cases with full-length proviruses. Surprisingly, the 3' cellular flanking region which is thought to be controlled originally by the promoter is constitutively expressed specifically in an HTLV-I producing ATL cell line HUT1O2G, in which the corresponding region is not modified by provirus. The detection of this HTLV-I-induced transcript provides a probe to find an HTLV-I inducible unknown cellular gene that may be related to the pathogenesis of ATL.

Rev-dependency of expression of human immunodeficiency virus type 1 gag and env genes.

Structural gene expression of human immunodeficiency virus type 1 (HIV-1) requires a viral regulatory protein, Rev transactivator. We investigated Rev-dependency of HIV-1 gene expression by various reporter systems. Expression of unspliced and single-spliced viral mRNAs was demonstrated to be differentially dependent on the Rev function. This difference of Rev-dependency was found not to be determined by cis-elements in gag, pol, and env coding sequences reported so far, and was lost when the reporter constructs containing minimum elements for Rev-responsiveness such as splice signals and rev responsive element were used for experiments. These findings indicated that the fundamental structure of HIV-1 mRNA was critical for the differential regulation of gene expression by Rev transactivator.

HIV-1 capsid mutants inhibit the replication of wild-type virus at both early and late infection phases.

In-frame mutations were introduced into various portions of the human immunodeficiency virus type 1 (HIV-1) gag gene, and potentials of the mutants to suppress the replication of wild-type HIV-1 were monitored. In contrast to results obtained with matrix and nucleocapsid mutants, almost all capsid mutants blocked HIV-1 replication completely in single-round replication assays. A capsid mutant designated C6b was demonstrated to be one of the most efficient inhibitors for HIV-1 reported to date, and to be effective at both early and late viral replication phases. T-cells, which are engineered to express the C6b Gag in response to HIV-1 infection, were perfectly resistant to HIV-1.

Differential determination of recombinant human interleukin-1 alpha and its deamidated derivative by two sandwich enzyme immunoassays using monoclonal antibodies. Comparison with a polyclonal antibody-based competitive enzyme immunoassay.

Using three different monoclonal antibodies (McAb no. 374, no. 964 and no. 1190) to human interleukin-1 alpha (rHu-IL-1 alpha), we have established two sandwich enzyme immunoassays (EIA) to differentiate rHu-IL-1 alpha and its deamidated derivative (rHu-Asp36-IL-1 alpha) where the asparagine at position 36 (counting from the N-terminus) of rHu-IL-1 alpha is converted to Asp. The McAb no. 1190 reacts specifically with rHu-IL-alpha and not with the rHu-Asp36-IL-1 alpha whereas both no. 374 and no. 964 can react with the two different forms of rHu-IL-1 alpha. The first EIA (S-EIA I) which uses the McAb no. 964 labelled with horse-radish peroxidase and the McAb no. 1190 fixed to the microtiter plate, only measure rHu-IL-1 alpha. The second EIA (S-EIA II) which uses enzyme labelled no. 964 and no. 374 fixed to the plate, can detect both rHu-IL-1 alpha and rHu-Asp36-IL-1 alpha and this assay of total rHu-IL-1 alpha is comparable to a competitive EIA using an enzyme-labelled rHu-IL-1 alpha and an anti-rHu-IL-1 alpha polyclonal antibody. Thus, the level of rHu-Asp36-IL-1 alpha in the samples containing the two IL-1 alpha s can be calculated by subtracting the level measured by S-EIA I from that measured by S-EIA II. The two EIA systems with an assay range of 1.5-100 ng/ml do not recognize IL-1 beta, IL-2, rHu-TNF alpha, IFN-alpha and IFN-gamma of human origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon-11-labeled KF15372: a potential central nervous system adenosine A1 receptor ligand.

UNLABELLED: The carbon-11-labeled selective adenosine A1 antagonist KF15372 ([1-propyl-11C]8-dicyclopropylmethyl-1,3-dipropylxanthine) was elevated in vivo as a PET ligand for mapping CNS adenosine A1 receptors. METHODS: The regional brain distribution of [11C]KF15372 and the effects of adenosine antagonists on the distribution were determined in mice by tissue sampling. In rats, in which the retinal projection fibres to the superior colliculus had degenerated due to unilateral eye removal, the brain distribution of [11C]KF15372 was visualized by ex vivo autoradiography. RESULTS: The mouse brain uptake of [11C]KF15372 was 1.8% i.d./g at 5 min and then it gradually decreased. The uptake was high in the hippocampus, cerebral cortex, striatum and cerebellum, and was significantly reduced by A1 antagonists but not by A2 antagonists. The brain distribution of 11C assessed by the tissue sampling and autoradiography was compatible with that of the A1 receptors. Autoradiography clearly visualized unilaterally decreased A1 receptor binding in the superior colliculus. CONCLUSION: The results demonstrated that [11C]KF15372 is a selective and high-affinity adenosine A1 receptor ligand and is useful for detecting the degeneration of presynaptic neurons.

High K-ras mutation rates in goblet-cell-type adenocarcinomas of the lungs.

Adenocarcinomas of the lungs show a variable histology. We have subclassified such lesions into five cell types: hobnail, columnar, polygonal, mixed and goblet cell types, and investigated their relationships with K-ras mutations. Codons 12, 13 and 61 of the K-ras gene in 120 surgically resected pulmonary adenocarcinomas were examined by the mutation-allele-specific amplification method. Point mutations were observed in 10% of the adenocarcinomas limited to K-ras codon 12 and the commonest base substitution (nine cases) was a G to T transversion. Of the five types, goblet cell lesions demonstrated the highest mutation index, which at 100% (6/6) was significantly different from that of all other cell types. No relationship between K-ras mutation and cigarette smoking was observed. From these findings, it appears that development of goblet-cell-type adenocarcinomas of the lung may involve different carcinogenic mechanisms from adenocarcinomas of other subtypes.

Production and characterization of recombinant human neutrophil chemotactic factor.

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.

Evaluation of carbon-11 labeled KF15372 and its ethyl and methyl derivatives as a potential CNS adenosine A1 receptor ligand.

We prepared [11C]KF15372 ([1-propyl-11C]8-dicyclopropylmethyl-1,3-dipropylxanthine, refs 10, 13) as well as its 11C-ethyl and 11C-methyl derivatives ([11C]EPDX and [11C]MPDX), and examined the potential of the three compounds as PET ligands for CNS adenosine A1 receptors. The three compounds had high affinity for the A1 receptors in vitro in the following order; [11C]EPDX > [11C]KF15372 > [11C]MPDX. In mice, the highest initial brain uptake was found in [11C]MPDX followed by [11C]EPDX and [11C]KF15372, but the level of [11C]MPDX decreased faster than those of the other two compounds. The uptake of each compound was decreased by carrier KF15372, but not by an A2A antagonist, indicating the selective affinity for the A1 receptors. Autoradiography with [11C]MPDX ex vivo demonstrated decreased A1 receptor binding in the superior colliculus of rats deprived of retino-collicular fibers by contralateral eye enucleation. These results show that three compounds have potential as PET ligands for CNS adenosine A1 receptors.

Synthesis and evaluation of 5-HT3 receptor antagonist [11C]KF17643.

For imaging CNS 5-HT3 receptors by PET, a high affinity 5-HT3 receptor ligand, endo-8-methyl-8-azabicyclo[3.2.1]oct-3-yl 2-(n-propyloxy)-4-quinolinecarboxylate (KF17643), have been labeled with 11C. N-Methylation of the desmethyl compound with [11C]methyl iodide followed by HPLC separation produced [11C]KF17643 with the decay-corrected radiochemical yield of 19-28%, the specific activity of 7.5-49 GBq/mumol and the radiochemical purity of > 99% at 35-40 min from EOB. After i.v. injection of [11C]KF17643 into mice, it was taken by the brain at a high level and was stable for metabolism, but no sign for the 5-HT3 receptor selectivity was found in the brain tissues by the tissue sampling and autoradiography, probably because of large non-specific binding. The [11C]KF17643 was not suitable as a PET ligand for mapping the CNS 5-HT3 receptors.

Determination of organic acids in urine by capillary zone electrophoresis.

The simultaneous measurement of organic acids was studied using capillary electrophoresis with direct measurement of UV absorption at 185 nm. The organic acids studied were oxalic, formic, malonic, fumaric, succinic, alpha-ketoglutaric, citric, acetic, pyruvic, lactic, isovaleric and hippuric acid. They were separated in a fused-silica capillary (100 cm x 75 microm I.D.) filled with 50 mM borax buffer (pH 10.0) containing cationic surfactant as the electroosmotic flow modifier. The method was successfully applied to the determination of organic acids in urine in comparison with an organic acid analyser.

Evaluation of oral anticoagulant therapy by measuring plasma prothrombin fragment 1 + 2.

To assess the degree of haemostatic system activation, plasma levels of prothrombin fragment 1 + 2 (F1 + 2), a direct indicator for thrombin generation in vivo, were measured in 49 patients with thrombotic disease undergoing long-term warfarin therapy (Thrombotest values < or = 40%). In these patients, vitamin K dependent coagulation factors (factors II, VII, IX and X) were decreased together with the anticoagulant proteins C and S, but the mean plasma concentration of F1 + 2 was significantly decreased compared with 48 healthy subjects. In warfarin-treated patients, F1 + 2 was positively correlated with the Thrombotest value, factors II, VII, IX and X. When analysed according to the intensity of anticoagulation, patients with Thrombotest values less than 30% showed a significant decrease in F1 + 2, but the mean F1 + 2 level was normal in patients with Thrombotests higher than 30%. These findings indicate that long-term oral anticoagulant therapy suppresses thrombin generation approximately in parallel to the decrease in coagulation factors, and levels of F1 + 2 lower than healthy subjects are observed when Thrombotest values are less than 30%.

Antibiotic A-16316-C, a new water-soluble basic antibiotic.

A new water-soluble basic antibiotic named antibiotic A-16316-C was isolated together with antibiotic A-396-I and hygromycin B from a streptomyces strain identified as Streptoverticullium eurocidicus. The properties of the antibiotic A-16316-C were similar to those of destomycin B. But it was found that the antibiotic A-16316-C was not identical with destomycin B on the basis of NMR analysis. On acidic degradation antibiotic A-16136-C gave N, N'-dimethyl-2-deoxystreptamine, destomic acid and D-mannose. The gross structure for antibiotic A-16136-C was deduced from chemical reactions and spectral data.

Synthesis and preliminary evaluation of [11C]KF15372, a selective adenosine A1 antagonist.

As a radioligand for mapping the presynaptic adenosine A1 receptors in the central nervous system by PET, [1-propyl-11C]8-dicyclopropylmethyl-1,3-dipropylxanthine ([11C]KF15372), a selective adenosine A1 antagonist, was prepared by the reaction of 8-dicyclopropylmethyl-3-propylxanthine and [11C]propyl iodide with decay-corrected radiochemical yield of 5% based on the [11C]propyl iodide, radiochemical purity of > 99%, sp. at. of 10-56 GBq/mumol and preparation time of 45-55 min. Another 11C-labeled A1 antagonist with much lower affinity for the A1 receptors, 7-[11C]methyl-KF15372 ([11C]KF17109), was also prepared using [11C]methyl iodide with a decay-corrected radiochemical yield of > 50%. In mice, the brain uptake of [11C]KF15372 (1.91% ID/g at 5 min) decreased gradually with time. Carrier KF15372 competitively reduced the brain uptake to a level (43% of the control) comparable to the brain uptake of [11C]KF17109. On the other hand, an A2 antagonist 3,7-dimethyl-1-propargylxanthine showed no effect on the brain uptake of [11C]KF15372. The results show that [11C]KF15372 has potential as a PET radioligand for mapping the adenosine A1 receptors and that [11C]KF17109 may be a reference compound reflecting the non-specific uptake of the [11C]KF15372.

Multitracer study on uptake and excretion of trace elements in rats.

The multitracer technique was first applied to the investigation of the uptake and excretion behaviour of trace elements in rats. A multitracer solution, prepared by irradiation of a gold target with a 14N-beam from the RIKEN Ring Cyclotron, was orally administered to male Wistar rats. The uptake and excretion rates of 23 elements, Be, Mn, Co, Zn, As, Rb, Sr, Y, Zr, Ce, Pm, Eu, Gd, Tb, Er, Tm, Yb, Lu, Hf, W, Re, Ir and Pt, were simultaneously determined under strictly identical experimental conditions. For some of the elements, the results obtained were consistent with previous reports on uptake and excretion of the elements in animals. For the other elements, unique behaviour was revealed for the first time as described in the present work. These results show that the multitracer technique has excellent reliability and versatility for a comparative study of the uptake and excretion of many different elements in animals.