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We performed haptoglobin (Hp) genotyping by polymerase chain reaction using allele-specific primer-pairs. The major six genotypes of Hp were identified using this method. Among Japanese individuals living in Ehime and Okayama Prefectures, the allele frequencies were estimated to be Hp2 = 0.723 and Hp1s = 0.277. Genotyping of Hp was possible with 0.3 ng of DNA and with 0.125 microliter of blood. It was also possible with whole blood left at room temperature for a month and also with the bloodstains left at room temperature for three years. In the heated blood samples, both alleles, Hp2 and Hp1s, were detected in those heated at 100 degrees C for 2 h. In bloodstains, Hp2 and Hp1s were detected in samples heated at 100 degrees C for 2 h and 120 degrees C for 30 min. In addition, the genotype could be detected in samples other than blood such as saliva, hair roots, tissue sections and dental pulps. The present method for Hp genotyping is expected to become a useful method in forensic analysis.

Recently, factors predicting the response to interferon (IFN) therapy against hepatitis C virus (HCV) have received much attention. To evaluate the usefulness of the quantitation of intrahepatic HCV RNA as a predictive marker of the response to IFN therapy, we compared the amount of intrahepatic HCV RNA with serum levels in 16 patients. Eleven patients who had 10(10) copies/g or more of intrahepatic HCV RNA had increased level of serum alanine aminotransferase (ALT) after IFN therapy, while 4 of 5 patients who had less than 10(10) copies/g of intrahepatic HCV RNA achieved sustained normalization of serum ALT level and were designated as complete responders. Four complete responders possessed significantly less HCV RNA in the liver parenchyma than partial and nonresponders (P = 0.010, Mann-Whitney U-test), but the amount of HCV RNA in the serum was not significantly different between those groups. In conclusion, the results suggest that the quantitation of intrahepatic HCV RNA is a better indicator of the response to IFN therapy than serum HCV RNA.

Hepatitis C virus (HCV)-RNA in the blood was measured by polymerase chain reaction (PCR) in 37 subjects from eight families in which 2 or more persons tested seropositive for antibodies against C100-3 or CP9. HCV-RNA was positive in 17 of 37 subjects. Two or more HCV-RNA-positive subjects were observed in six of the families. Intrafamilial HCV infection was studied by determining the HCV-RNA type (I, II, III or IV) by PCR using type-specific primers. In two families, all of the subjects showed type III infection, and in three other families, all of the subjects showed type II infection, with different types of HCV infections being observed in only one family. The HCV type was uniform in all but one. These findings suggest a possibility of intrafamilial infection between husbands and wives and between members of the same household.

The sensitivity and specificity of single cell polymerase chain reaction (PCR) were studied. Its high sensitivity enabled detection of a single-copy gene, such as human T-lymphotropic virus type I genome in paraffin sections. The rate of obtaining positive signals with this method was affected by the number of copies of the gene in the target cell. Specificity was satisfactory if the procedure was properly and carefully followed. Since the single cell PCR is a time-consuming method which requires skill and experience to pick up the target cells accurately, the applicability of this method is limited. It works best when it is used to analyze a single or a few copy genes in histologically identified cells.

We analyzed nucleotide changes in 3 genes, ARIX, PHOX2B, and KIF21A, in 6 patients of 3 families with congenital superior oblique muscle palsy. Three exons of ARIX, 3 exons of PHOX2B, and exons 8, 20, and 21 of KIF21A were amplified by polymerase chain reaction from genomic DNA isolated from the peripheral blood. The DNA fragments were directly sequenced in both directions. In 2 different families, a heterozygous nucleotide change, ARIX 153G>A, in the 5’-untranslated region was found in common between a father and daughter with muscle palsy and between a mother and daughter with muscle palsy (Family No. 1 and No. 3). In the other family (Family No. 2), a heterozygous 15-nucleotide deletion, PHOX2B 1124del15, resulting in loss of 5 alanine residues in the alanine repeat of the protein, was found in the daughter with muscle palsy and her father with normal traits, but was not found in the mother with muscle palsy. No KIF21A nucleotide change was found in any patients. The ARIX 153G>A polymorphism might be a genetic risk factor for the development of congenital superior oblique muscle palsy.

The aim of this study was to determine the optimal temperature of graft preservation after ex vivo gene transfer to rat lung isografts. Left lungs were harvested and infused with cationic lipid/LacZ-DNA complex via the pulmonary artery, and the grafts were stored for 4h. The grafts (n＝7) were allocated into groups IﾝIV according to the storage temperature:4℃, 10℃, 16℃, and 23℃, respectively. Forty-eight h after orthotopic transplantation, the arterial blood gas was analyzed and the peak airway pressure (PAP) and the level of LacZ protein production in the grafts were measured by reverse transcription polymerase chain reaction. After reperfusion, the grafts were stained with hematoxylin and eosin. The grafts in groups III and IV showed more deterioration as evidenced by decreased arterial oxygen tension, increased PAP, and predominant infiltration of inflammatory cells compared with groups I and II. The level of LacZ production was significantly lower in group I than in groups IIﾝIV. The optimal temperature of lung graft preservation after ex vivo gene transfer was determined to be 10℃, balancing considerations of lung injury and efficiency of transgene expression.

Malignant lymphoma was induced in Japanese (JWY), New Zealand (NZY) and Dutch (DUY) white rabbits by oral spray of cell-free pellets of culture fluid (crude virus fraction) of Ts-B6 cells (cynomolgus monkey B-lymphoblastoid cells harboring Epstein Barr virus-related simian herpesvirus or Cyno-EBV). Nine of 11 inoculated rabbits developed malignant lymphomas within 42-160 days after oral inoculation (JWY, 2/3; NZY, 5/6; DUY, 2/2). In contrast, none of the control rabbits inoculated in the same fashion with B95-8 (EBV-producing marmoset cell line) cell-free pellets developed malignant lymphoma. Most rabbits showed increased anti-VCA IgG and anti-EA-DR IgG antibody titers after inoculation by oral spray of Ts-B6 cell-free pellets. EBV-encoded RNA-1 was revealed in the tumor cells by in situ hybridization. EBV DNA was detected in the rabbit peripheral blood leukocytes (PBL) by polymerase chain reaction; the earliest positive result was obtained only two days after oral inoculation. These data suggest that orally administered Cyno-EBV in Ts-B6 cells infects PBL and then induces malignant lymphoma in rabbits. The availability of this animal model promises to clarify the role of EBV in human lymphoma and provides a means for studying prophylactic and therapeutic regimens.

A simian cell line, Si-IIA, harboring Epstein-Barr-virus (EBV) -related herpesvirus (Si-IIA-EBV), produces malignant lymphoma in rabbits when administered by intravenous inoculation. In this study, we analyzed the Si-IIA-EBV genome and compared it with human EBV and herpesvirus macaca fascicularis 1 (HVMF 1 ), which is associated with B-cell lymphoma developing in SIV-infected immunosuppressed monkeys. DNA from Si-IIA-EBV was amplified by the polymerase chain reaction using three different primer pairs complementary to human EBV (B95-8) DNA; two of the primer pairs covered part of the long internal repeat 1 region (IR 1) and the third covered part of the BRRF 1 region. Direct sequencing of the three PCR products revealed that Si-IIA-EBV DNA had about 82% nucleotide homology to the human EBV DNA in all three regions and 92.4% homology to HVMF1 in the IR1 region. The blotting pattern by Southern blot analysis was different between Si-IIA-EBV and human EBV.

A method of genotyping IgA2 alleles in the human immunoglobulin alpha 2 heavy chain constant region (C alpha 2 gene) was developed by using the polymerase chain reaction (PCR). By this method, the genotype was determined by discriminating base substitution in the 3'-flanking region of alleles, A2m*1 and A2m*2, which manifest A2m serum types, by nested PCR using allele-specific primers. Three types, IgA2*1/IgA2*1, IgA2*2/IgA2*1, and IgA2*2/IgA2*2, were detected from DNA extracted from lymphocytes. Genotyping was possible from 100 pg of DNA by this method. The estimated allele frequency in 318 Japanese subjects was 0.561 for IgA2*1 and 0.439 for IgA2*2. Analysis of 29 cases of paternity tests suggested that the data follow Mendel's law of inheritance. This genotype could also be detected in whole blood, blood stains, saliva stains, and various organs and tissues. These results suggest the usefulness of the present method for paternity testing and individual identification in forensic medicine.

To study the virological and serological characteristics of asymptomatic hepatitis C virus (HCV) carriers, 165 blood donors positive for antibody against HCV proteins by the second generation assay, were analyzed for their clinical backgrounds, serological reactivity against antigens derived from HCV by recombinant immunoblot assay, and the amount and genotype of HCV by the polymerase chain reaction. Compared with blood donors having abnormal levels of alanine aminotransferase (ALT), sera from the donors with normal levels of ALT reacted less frequently against NS4 antigens (anti-5-1-1: 34.4% vs. 54.5%, P = 0.0609; anti-c100-3: 34.4% vs. 56.1%, P < 0.05). Also the positivity for antibodies against these antigens were more frequent in sera from donors with genotype 1b HCV-RNA than other genotypes (anti-5-1-1: 61.0% vs. 23.5%, P < 0.01; anti-c 100-3: 61.0% vs. 26.5%, P < 0.01). The prevalence of each genotype in blood donors with normal ALT levels was different from that in patients with advanced liver disease (P < 0.05), genotype 1b being less and genotype 2a being more frequent. The number of HCV-RNA copies/0.5 ml in donors with normal ALT was 10(7.9 +/- 1.0) (n = 27) and that in patients with chronic liver disease was 10(7.4 +/- 0.8) (n = 116), the difference being statistically significant (P < 0.05). In conclusion, the results of this study suggest that asymptomatic blood donors carrying HCV have the serological and virological characteristics different from the patients with advanced liver disease.

The presence of high-risk types of human papillomavirus (HPV) 16, 18 and 33 in cell lines established from several malignancies including 5 of cervical cancer and 6 of head and neck cancer was studied. HPV DNA, either type 16 or 18, was detected by polymerase chain reaction, and by Southern blot hybridization in all of the cell lines derived from cervical cancers. The hybridization patterns of HPV DNA after endonuclease digestion differed among cell lines, suggesting that all of these cell lines were independent isolates. Accordingly, high-risk types of HPV DNA seem to be ubiquitous in cervical cancer. HPV DNA was not detected in the cell lines derived from head and neck cancers or from any other malignancies besides cervical cancer in this study.