We developed a new quantitative system for diagnosis of invasive pulmonary aspergillosis (IPA) using real-time automated polymerase chain reaction (PCR). Intra-assay and interassay precision rates for in vitro examination were 2.53% and 2.20%, respectively, and the linearity of this assay was obtained when there were >20 copies/well. We examined 323 samples taken from 122 patients with hematological malignancies, including 33 patients with IPA and 89 control patients. Blood samples were subjected to PCR antigen detection methods, using enzyme-linked immunosorbent assay (ELISA) and determination of plasma (1-->3)-beta-D-glucan (BDG) concentration. The sensitivities of PCR, ELISA, and BDG measurement for diagnosis of IPA were 79%, 58%, and 67%, respectively; the specificities were 92%, 97%, and 84%. Positive findings on PCR preceded those of computed tomography by -0.3+/-6.6 days, those of BDG measurement by 6.5+/-4.9 days, and those of ELISA by 2.8+/-4.1 days. Real-time PCR was sensitive for IPA diagnosis, and quantitation was accurate.

Interindividual variability of the activity of CYP1A2 may be expected to affect cancer susceptibility, since the enzyme is capable of activating several carcinogens. In the present study, we found three new polymorphisms in the 5′ ′ ′ ′-flanking region (CYP1A2/B) and intron 1 (CYP1A2/C and CYP1A2/D) of CYP1A2 in Japanese by using polymerase chain reaction-single strand conformation polymorphism. We developed methods to detect these polymorphisms by polymerase chain reaction-restriction fragment length polymorphism and performed a population study (159 subjects) to estimate the frequencies of the alleles. Cytochrome P450 (CYP) enzymes play an important role in the metabolism of endogenous and exogenous substrates. Human CYP1A2 has been shown to be responsible for the 3-demethylation of caffeine, the initial major step in the biotransformation of caffeine in humans. 1) CYP1A2 is also known to be involved in the metabolic activation of numerous carcinogens such as 2-aminofluorene, 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP).2-4) Several studies on the CYP1A2-dependent metabolism of caffeine or phenacetin have demonstrated that this enzyme is expressed in human livers at a variety of levels among individuals.5, 6) Considerable interindividual variability in the activities of CYP1A2-dependent N-oxidation of 2-naphthylamine, 7) 2-acetylaminofluorene 8) and 4-aminobiphenyl has also been noted.9) The sequence analysis of Japanese DNA samples in our previous study suggested that the considerable variation in the level of CYP1A2 expression was not due to mutation of the exonic, intronic, or 5′-flanking regions.10) However, our recent study clarified that genetic polymorphism existed in the 5′-flanking region of the human CYP1A2 gene in Japanese subjects. 11)This mutation affects the CYP1A2 inducibility. Further, we discovered three additional polymorphisms of the CYP1A2 gene by using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method in the 5′-flanking region and intron 1 of the CYP1A2 gene (data not shown). The four CYP1A2 mutations are summarized in Fig. 1. CYP1A2/A was already reported by our group. 11) CYP1A2/B, CYP1A2/C and CYP1A2/D are new polymorphic alleles.In this report, we describe methods to detect the three mutated alleles by PCR-restriction fragment length polymorphism (PCR-RFLP) and we present an estimate of the allele frequencies in a Japanese population.The use of human blood for this study had been approved by the Hokkaido University Ethics Committee. The 159 subjects were all healthy Japanese. Genomic DNA was extracted from peripheral leukocytes with phenol-chloroform, followed by ethanol precipitation.12) DNA was dissolved in 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA, and stored at 4°C until PCR reactions. Four CYP1A2 genotypes were detected by PCR-RFLPs. The sequences of the primers for PCR are shown in Table I. 13,14) PCR was performed to detect the CYP1A2/A genotype using primers P1 and P2, 1...

CYP2E1, XPA and ERCC1 polymorphisms may affect the risk of OSCC.

Microsatellite Instability (MSI) status is an established predictive biomarker for the treatment of the anti‐programmed death 1 (PD‐1) antibody. The current approach to determine the MSI status in tumours requires matched normal DNA. Some mononucleotide microsatellite markers are known to have few variant alleles in both Caucasians and Asians. Therefore, the length of these microsatellite makers is almost confined within the quasi‐monomorphic variation range (QMVR). Considering the application of MSI testing for various types of cancers, a simple, sensitive and inexpensive method is desired. This study assessed the clinical utility of the QMVR for determining the MSI status in patients with unresectable metastatic colorectal cancer (mCRC). The study enrolled 435 patients with mCRC. The concordance of the MSI status in mCRC between the standard method using tumour DNA plus matched normal DNA and the testing method using only tumour DNA was evaluated. Eleven (2.5%) MSI‐high cases were detected by both the standard and testing methods. The sensitivity and specificity of the testing method were both 100%, indicating complete concordance between the methods. Among the mononucleotide markers, three and two patients showed discordance for NR‐21 and BAT‐25, respectively. Results from MSI testing with normal tissue indicated that four of five patients had rare germline variants outside the QMVR. For BAT‐26, NR‐24 and MONO‐27, all patients showed complete concordance. Using the QMVR, the MSI status of mCRC can be determined without matched normal DNA.

The purpose of this study was to assess the usefulness of real-time automated PCR as a quantitative, highly reproducible, and sensitive method to detect cytomegalovirus (CMV) DNA in blood specimens. Intra- and interassay precision rates were 0.89% (small number of copies [L]), 1.43% (middle number of copies [M]), and 1.12% (high number of copies [H]), and 4.46% (L), 1.51% (M), and 2.28% (H), respectively. The linearity of this assay was obtained between 10 and 107 copies/well, with a minimum detection limit of 20 copies/well. Specimens from 55 of 70 healthy subjects were found to be positive for CMV antibody, but CMV DNA was not detected in any of them. In the qualitative assessment of each specimen, the results of the CMV antigenemia assay and those of the real-time PCR assay agreed in 80% (plasma specimens), 79% (all nucleated cells), and 86% (blood) of the cases examined. For eight patients diagnosed as having CMV infection or disease, no sample was positive in the antigenemia assay earlier than in the real-time PCR assay. Furthermore, the results of this assay could be obtained within 8 h. We concluded that the real-time PCR assay is useful for rapid diagnosis of CMV infection and monitoring of clinical courses.

Human herpesvirus 6 (HHV-6) infection in recipients of cord blood stem cell transplants (CBSCTs) was estimated by semiquantitative and real-time quantitative polymerase chain reaction (PCR) and reverse-transcription PCR. Of the CBSCT recipients, 7 (70%) of 10 had active HHV-6 infection after transplantation, and all 7 were inferred from their age to have already had a primary infection. Because HHV-6 DNA is seldom detected in cord blood, these cases were considered likely to represent reactivation. In contrast, the 3 patients without HHV-6 infection were all believed to be naive regarding HHV-6 primary infection because of their age and the results of PCR assays given before the transplantation procedure. The incidence of HHV-6 infection after transplantation was significantly higher (P < .05) than after bone marrow (BM) transplantation and peripheral blood stem cell (PBSC) transplantation, when recipients without primary HHV-6 infection prior to transplantation were excluded (CBSCT, 100%; BMT/PBSCT, 56.3%). Real-time PCR revealed a higher level of viral DNA in the peripheral blood mononuclear cells from CBSCT recipients than from BMT/PBSCT recipients or patients with exanthem subitum (P < .05). HHV-6 mRNA of the U79/80gene was also detected by reverse-transcription PCR in all analyzed patients with HHV-6 infection. Its detection was correlated with the emergence of viral DNA in the plasma and symptoms such as fever and rash. Thus, HHV-6 infection was more frequent and the viral load was higher in CBSCT recipients with prior primary infection.

In our study, 5'-nucleotidase was released from bovine liver by the treatment with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and purified to a homogeneous state by concanavalin A-Sepharose and (diethylaminoethyl)-Toyopearl column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified 5'-nucleotidase were then cleaved by cyanogen bromide (CNBr), and then inositol phosphoglycan-containing C-terminal peptides (IPG peptides) were separated by C18 reverse-phase liquid chromatography and analyzed by peptide sequencer, amino acid analyzer, gas chromatography (GC), and GC-mass spectrometry (MS). Ser523 of the amino acid sequence deduced from 5'-nucleotidase cDNA [Suzuki et al. (1993) J. Biochem. (Tokyo) 113, 607-613] is revealed to be the C-terminal amino acid to which a glycosylphosphatidylinositol is anchored. Separated peaks of CNBr-cleaved IPG peptides were then analyzed by electron spray ionization (ESI)-MS. Eight different molecular weight (MW) species of CNBr-cleaved IPG peptides were detected. Three fractions of CNBr-cleaved IPG peptides were separately treated by trypsin, and trypsinized IPG peptides were purified by C18 reverse-phase liquid chromatography. Finally, five different MW species of trypsinized IPG peptides (1629.4, 1752.7, 1791.8, 1832.8, and 1994.5) were detected by ESI-MS. Together with sequential exoglycosidase treatment and quantitative analysis of sugar moieties by GC and GC-MS, microheterogeneity in the structures of these five glycosylphosphatidylinositol (GPI) anchor species was determined. The common core structure was ethanolamine phosphate-mannose-mannose-mannose(-ethanolamine phosphate)-glucosamine-myoinositol phosphate. Variations observed in additional mannose, N-acetylhexosamine, and ethanolamine phosphate moieties form this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

β-tricalcium phosphate (β-TCP) was grafted into rat mandibular bone defects to assess its potential as a scaffold material for bone regeneration. For this purpose, β-TCP (TCP), allogenic bone (Allograft), and allogenic bone combined with β-TCP (Combined) were employed as graft materials. To the left side of the graft materials in the bone defects, platelet-rich plasma (PRP) was added. The rats were sacrificed at one, three, and five weeks. Bone formation rate (BFR), remaining β-TCP rate (RTR), β-TCP absorption rate (TAR), whole amount of β-TCP (WTCP), and total rate of BFR and RTR (TBR) were measured. Combined showed equivalent BFR to Allograft at five weeks, and showed higher RTR at one week and higher BFR at five weeks than TCP. Combined with PRP showed higher TAR than that without PRP at three weeks. Therefore, combination with allogenic bone showed reduced β-TCP absorption, hence enhancing the role of β-TCP in bone regeneration. These findings suggested that β-TCP is a better scaffold for bone regeneration if its early absorption is reduced when used in combination with an osteogenic material.