RT-PCR法によるヒトサイトメガロウイルス前初期遺伝子mRNAの検出

We have established a method for detection of human cytomegalovirus (HCMV) immediate early gene (IE)-specific mRNA by reverse transcription polymerase chain reaction (RT-PCR). Using the IE-1 specific primer fragments designed for RT-PCR, we have amplified a 232 base-pair fragment which represents IE-1 mRNA transcribed from the IE-1 gene of HCMV AD169 strain. No DNA cross reactivities to other human herpes virus DNAs or human embryonic lung cell (MRC-5 line) DNA were observed. The RT-PCR assay indicated HCMV IE-1-specific mRNA in HCMV-infected MRC-5 cells and in peripheral blood specimens from one of 3 patients examined. The results indicate that RT-PCR will be readily applicable to rapid detection of HCMV mRNA and may be useful for diagnosis of active primary or recurrent HCMV infections

Structural and Expressional Alterations of Episomal and Integrated Human Papillomavirus Type 16 in Precancerous Lesions and Carcinomas of the Cervix.

HPV infection has long been implicated in the development of cervical car-cinoma. We have analyzed the HPV 16 genome structure and expression of the viral mRNA in cervical intraepithelial neoplasias (CINs) and cervical car-cinomas by using modified polymerase chain reaction (PCR) methods. Genome structure has been determined by PCR using multi-primer sets which are located in each open reading frames and then these results have been compared with the physical state of the viral DNA determined by two-dimensional electrophoresis. Furthermore, we have analyzed the expression of HPV 16 mRNA and genome structure using DNA and RNA simultaneously extracted from CINs and cervical carcinomas using PCR and reverse transcription (RT-) PCR. Our data showed that the DNA regions from the El to Ll region were delet-ed in two of three CINs containing episomal HPV 16 and three out of seven cervical carcinomas containing integrated HPV 16. However, the E6/E7 region was conserved in all the HPV 16-positive samples. RT-PCR analysis has determined the presence of mRNA species which could encode the E6, E6*I, E6*II, E7, E2, E2 ? C, E1^E4, E1^E2 ? C, E4, E2 ? C-E5 and L2 proteins. The overall results of DNA and mRNA analyses in cervical lesions indicated that the expression patterns of the early and late transcripts studied were not specifically related to the grade of malignancy and the physical state or the deletion of the viral genome. Furthermore, alterations in the splicing pat-terns of HPV 16 transcripts may not be involved in tumor progression

The Double Polymerase Chain Reaction with Consensus Primers Permits Rapid and Sensitive Detection of Genital Human Papillomavirus Oncogenes

We have developed a sensitive procedure for the detection of relatively low copy numbers of multiple genital human papillomaviruses (HPVs) using the polymerase chain reaction (PCR). HPV DNAs were detected by agarose gel electrophoresis and ethidium bromide staining after 2 rounds of PCR amplification (double PCR) with outer and inner consensus primer pairs for HPV-6, 11, 16, 18, 31, 33, 52, and 58. The detection limit of this method (i. e., 10?? copy of HPV DNA per cell in 1 μg cell DNA) was sufficient for analysis of cervical intraepithelial neoplasia (CIN) specimens. Overall prevalence rate of HPV was 100% in 20 cases of CIN specimens. HPV typing by restriction enzyme analysis revealed that HPV-16 sequence was present in 11 cases, HPV-18 in 1 case, HPV-31 in 4 cases, HPV-33 in 1 case, HPV-52 in 2 cases, HPV-58 in 3 cases, and an unidentified type(s) in 3 cases. There were 4 cases of mixed infections. This procedure obviates the use of hybridization- based for-mat for identification of at least 8 types of HPV sequences present in a small fraction of cells within a heterogeneous population

Phase II study of S-1 on alternate days plus bevacizumab in patients aged ≥ 75 years with metastatic colorectal cancer (J-SAVER)

BackgroundAlternate-day administration of S-1 is thought to reduce toxicities. This phase II study evaluated S-1 on alternate days combined with bevacizumab as first-line treatment for elderly patients with metastatic colorectal cancer.Patients and methodsEligible patients had histologically proven colorectal adenocarcinoma, measurable metastatic lesions, age ≥ 75 years, Eastern Cooperative Oncology Group performance status ≤ 1, no previous chemotherapy, and refused oxaliplatin- or irinotecan-containing regimens. Patients received 40 mg, 50 mg, or 60 mg (body surface area ≤ 1.25 m2, > 1.25 to ≤ 1.50 m2, or > 1.50 m2, respectively) of S-1 twice orally on Sunday, Monday, Wednesday, and Friday every week. Bevacizumab (7.5 mg/kg) was administered every 3 weeks. The primary endpoint was progression-free survival.ResultsOf 54 enrolled patients, 50 patients were evaluated for efficacy and 53 for safety. The median age was 79 years (range 75–88 years). The median progression-free survival was 8.1 months (95% confidence interval (CI) 6.7–9.5 months). The median overall survival was 23.1 months (95% CI 17.4–28.8 months). The response rate was 44% (95% CI 30.2–57.8%), and the disease control rate was 88% (95% CI 79.0–97.0%). Grade 3 or higher hematologic, non-hematologic, and bevacizumab-related adverse events occurred in 9%, 11%, and 25% of patients, respectively. The most common grade 3 and 4 treatment-related adverse events were hypertension (11%), nausea (6%), fatigue (6%), anemia (6%), and proteinuria (6%). Only 6 patients discontinued treatment due to adverse events.ConclusionS-1 on alternate days combined with bevacizumab showed better tolerability and comparable survival compared with the results of similar studies