A molecular phylogeographic study based on DNA sequences from individual metacercariae of Paragonimus mexicanus from Guatemala and Ecuador.

A molecular phylogeographic study of Paragonimus mexicanus collected from Guatemala and Ecuador was performed. Genomic DNA was extracted from individual metacercariae, and two gene regions (partial mitochondrial cytochrome c oxidase subunit 1 (CO1) and the second internal transcribed spacer of the nuclear ribosomal gene repeat (ITS2)) were amplified by the polymerase chain reaction (PCR). Sequences segregated in a phylogenetic tree according to their geographic origins. ITS2 sequences from Ecuador and Guatemala differed at only one site. Pairwise distances among CO1 sequences within a country were always lower than between countries. Nevertheless, genetic distances between countries were less than between geographical forms of P. westermani that have been suggested to be distinct species. This result suggests that populations from Guatemala and Ecuador are genetically differentiated perhaps at the level of subspecies.

Affinities between Asian non-human Schistosoma species, the S. indicum group, and the African human schistosomes.

Schistosoma species have traditionally been arranged in groups based on egg morphology, geographical origins, and the genus or family of snail intermediate host. One of these groups is the 'S. indicum group' comprising species from Asia that use pulmonate snails as intermediate hosts. DNA sequences were obtained from the four members of this group (S. indicum, S. spindale, S. nasale and S. incognitum) to provide information concerning their phylogenetic relationships with other Asian and African species and species groups. The sequences came from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. Tree analyses using both distance and parsimony methods showed the S. indicum group not to be monophyletic. Schistosoma indicum, S. spindale and S. nasale were clustered among African schistosomes, while S. incognitum was placed as sister to the African species (using ITS2 and 28S nucleotide sequences and CO1 amino acid sequences), or as sister to all other species of Schistosoma (CO1 nucleotide sequences). Based on the present molecular data, a scenario for the evolution of the S. indicum group is discussed.

High throughput detection of drug-metabolizing enzyme polymorphisms by allele-specific fluorogenic 5' nuclease chain reaction assay.

We have developed an allele-specific fluorogenic 5' nuclease chain reaction assay for detecting polymorphisms in the following human drug-metabolizing enzyme genes: CYP2C9 (CYP2C9*2 and *3), CYP2C19 (CYP2C19*2 and *3), CYP2D6 (CYP2D6*4, *10, *14, *18, and *21(C8)), N-acetyltransferase 2 (NAT2*5B, *6A, and *7B), thiopurine methyltransferase (TPMT*3C), and aldehyde dehydrogenase2 (ALDH2*2). This method is a marriage of two emerging technologies, the use of allele-specific amplification primers for target DNA and hybridization of the TaqMan probe. The TaqMan probe is labeled with both a fluorescent reporter dye and a quencher dye. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. All assays are performed using a single thermocycling protocol. This genotyping method is rapid and highly sensitive and yields a high throughput. It could be applied toward automated large-scale genotyping.

Detection assay of rare variants of the thiopurine methyltransferase gene by PCR-RFLP using a mismatch primer in a Japanese population.

Thiopurine methyltransferase (TPMT) catalyzes the metabolism of important drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. The identification and frequency distributions of several variant TPMT alleles (TPMT*2--*8) have been described recently in many ethnic groups. We have recently demonstrated that TPMT*3C is the most common allele in Japanese subjects; however, it remains to be elucidated whether TPMT*4--*8 variants also exist in Japanese subjects. To detect polymorphisms in the TPMT gene (TPMT*4--*8), we have developed a mismatch polymerase chain reaction and restriction fragment length polymorphism method and conducted a population study of Japanese subjects. Genotyping of these variant forms was carried out in 192 Japanese healthy volunteers. The TPMT*4, TPMT*5, TPMT*6, TPMT*7, and TPMT*8 variants were not detected in any of the samples analyzed. This study provides the first analysis of the TPMT*4--*8 variants in a sample of the Japanese population and indicates that TPMT*4--*8 variants do not occur or are rare alleles in this population.

Therapeutic effect of clarithromycin for respiratory-tract infections in children caused by Chlamydia pneumoniae. Research Group of Sapporo for Pediatric Chlamydial Infections.

Children infected with Chlamydia pneumoniae sometimes experience lower respiratory tract infections such as pneumonia and bronchitis. Although numerous anti-microbial compounds have been reported to be active against the organism, most of them have not been in a clinical trial in infants and children with C. pneumoniae infection. Clarithromycin has been shown to express anti-chlamydial effects in vitro. In this study, we evaluated the clinical anti-C. pneumoniae properties of clarithromycin in children with mainly lower respiratory tract infection. We administered clarithromycin orally to 21 infants and children at a dose of 10-15 mg/kg/day divided into two or three doses for 4-21 days. Clinical symptoms, roentgenographic and laboratory abnormal findings improved. The overall clinical efficacy rate was 85.7% (18 of 21 cases). Administration of clarithromycin was considered to be a suitable treatment for improving lower respiratory infections in infants and children caused by C. pneumoniae.

Rapid detection of CYP2C18 genotypes by real-time fluorescence polymerase chain reaction.

In man, CYP2C19, a liver enzyme, plays an important role in the metabolism of several drugs. Mutation of the CYP2C19 gene results in a poor metaboliser phenotype. S-Mephenytoin hydroxylation genetic polymorphism is due to two mutations of the CYP2C19 gene, namely CYP2C19*2, located in exon 5, and CYP2C19*3, located in exon 4. CYP2C18 is also polymorphically expressed. The mutant alleles of this enzyme are CYP2C18m1, located in exon 2 and CYP2C18m2, located in the 5'-flanking region. We have developed an allele-specific TaqMan polymerase chain reaction (PCR) assay with which to detect CYP2C18 mutant alleles. This assay combines hybridization of the TaqMan probe and allele-specific amplification primers to the target DNA. The TaqMan probe is labelled with 6-carboxyfluorescein at the 5' end and 6-carboxytetramethylrhodamine together with a phosphate at the 3' end. Genotypes are separated according to the different threshold cycles of the wild type and mutant primers. We applied this procedure to DNA extracted from the blood or saliva of 144 healthy Japanese volunteers. The wt/wt, wt/m1, wt/m2, m1/m1, m1/m2 and m2/m2 genotypes of the CYP2C18 alleles detected by the assay were consistent with the results obtained from restriction enzyme cleavage. In accordance with a previous report, the genotypes of CYP2C18m1 and CYP2C18m2 coincided with those of CYP2C19*3 and CYP2C19*2, respectively. Therefore, detection of CYP2C18 mutant alleles also allows that of CYP2C19 mutant alleles. Among 19 poor metabolisers, eight showed the homozygous CYP2C19*2/CYP2C19*2, two the homozygous CYP2C19*3/CYP2C19*3 and nine the compound heterozygous CYP2C19*2/CYP2C19*3 genotype. We found the allele-specific TaqMan PCR assay rapid, simple and cost-effective, as well as suitable for high-throughput applications in a routine laboratory. This assay allows the fast and reliable detection of inherited disorders that might influence diagnosis and treatment.

Genetic analysis of thiopurine methyltransferase polymorphism in a Japanese population.

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathiopurine. Several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. The molecular basis for the genetic polymorphism of TPMT has been established for European Caucasians, African-Americans, Southwest Asians and Chinese, but it remains to be elucidated in Japanese populations. The frequency of the four allelic variants of the TPMT gene, TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3C (A719G) were determined in Japanese samples (n=192) using polymerase chain reaction (PCR)-RFLP and allele-specific PCR-based assays. TPMT*3C was found in 0.8% of the samples (three heterozygotes). The TPMT*2, TPMT*3A and TPMT*3B alleles were not detected in any of the samples analyzed. This study provides the first analysis of TPMT mutant allele frequency in a sample of Japanese population and indicates that TPMT*3C is the most common allele in Japanese subjects.

Rapid detection of CYP2C9*3 alleles by real-time fluorescence PCR based on SYBR Green.

CYP2C9 catalyzes the metabolism of important drugs such as phenytoin, S-warfarin, tolbutamide, losartan, and nonsteroidal anti-inflammatory drugs. A functional polymorphism of the CYP2C9 gene has been described. The single-base mutation of A1061C (Ile359Leu) in the CYP2C9 gene termed CYP2C9*3 was found at a frequency of about 2.1% in Japanese. We developed a rapid mutation analysis method for detecting the CYP2C9*1 genotype. This method is a marriage of two emerging technologies: allele-specific amplification primers for target DNA and a new double-stranded DNA-selective fluorescent dye, SYBR Green. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. We applied this procedure to DNA extracted from the blood of healthy Japanese volunteers. The CYP2C9 wild-type CYP2C9*1/CYP2C9*1 and heterozygous CYP2C9*1/CYP2C9*3 genotypes of the CYP2C9 alleles detected by the assay were consistent with the results obtained from restriction enzyme cleavage. No genotype of CYP2C9*3/CYP2C9*3 was found in these samples. Using plasmid DNA containing a point mutation of CYP2C9*3 as template, the assay separated the three genotypes. We conclude that this simple, rapid, and inexpensive procedure is applicable to routine high-throughput assays.

Carcinoma of the colon in childhood; report of 2 cases expressing p53 without K-ras mutation.

We report on 2 children with colonic carcinoma and also review 62 cases of Japanese children with colonic carcinoma including ours. Although the dismal prognosis in colonic cancer in children is possibly due to the predominance of poorly differentiated carcinoma, there is no significant difference in the 5-year survival rates among well, moderately and poorly differentiated carcinomas in children. Positive staining with p53 in tumor cells was observed in each, but K-ras mutations were not detected in any. Therefore, these carcinomas possibly developed from de-novo carcinoma. The development pathway of colonic carcinoma may relate to the prognosis in children, and be different from that in adults.

Severe neonatal nemaline myopathy--histological and histochemical studies of respiratory muscles.

The histological and histochemical findings in the respiratory muscles of a patient with severe neonatal nemaline myopathy are described. The patient suffered from frequent pneumonia associated with vomiting due to gastroesophageal reflux and died at 3 months from respiratory failure. The diaphragm was moderately involved and the intercostal muscles mildly involved. Core/targetoid structures were observed in the diaphragm and intercostal muscles.

A family case of nephrogenic diabetes insipidus.

Two brothers, patient 1 with fever and vomiting, and patient 2 with failure to gain weight were studied. After 4 hr of water deprivation test, the urinary osmolality of the patient 1 was only 105 mOsm/liter and his body weight showed a 4.6% reduction. In response to desamino-8-D arginine vasopressin intranasal administration, no significant elevation of urinary osmolality of patient 1 occurred. After low dose vasopressin tests, the maximal urinary osmolality of their father was in the normal range, but that of their mother was below the normal range. Moreover, the patients showed no significant increase of urinary osmolality after the same tests. The brothers were diagnosed as nephrogenic diabetes insipidus (NDI) and their mother was diagnosed as a carrier. An early diagnosis of NDI is important, since adequate managements such as low-solute diet with restricted protein and salt intake or such as water intake at frequent intervals can prevent the hyperosmolality which would develop the delayed mental and physical developments. The usefulness of the combination of indomethacin with thiazide diuretics is described.

Monosodium urate test: a new analgesic test by crystal-induced monoarthritis in rats.

The analgesic effects of various agents were evaluated in knee joint monoarthritic rats where arthritis was induced by monosodium urate (MSU) crystals injected into the knee joint cavities. The weight supported by each hind limb was measured quantitatively using pressure transducers, which revealed dose-dependent recovery produced by various analgesics including morphine but no changes in value by sedatives such as chlorpromazine. Relative potency obtained from the value of ED50 of each analgesic agent was congruent with that in clinical use. The MSU test may be quite suitable for quantitative and objective evaluation of the analgesic effects of various agents, particularly for spontaneous and tonic pain.

Neonatal hyperammonemia associated with carnitine deficiency.

We report a case of neonatal hyperammonemia associated with secondary carnitine deficiency. She suffered from hyperammonemia soon after the birth, and then presented severe metabolic acidosis at 2 months of age. She was successfully treated for acidosis with oral administration of L-carnitine (100 mg/kg/day). Since hyperammonemia recurred with the increase of protein intake, it was necessary to increase the dose of carnitine to 150 mg/kg/day. Urea cycle enzymopathies were excluded from the laboratory data. The urinary organic acid profiled by gas chromatography mass spectrometry revealed no abnormalities. It was found that the carnitine contents in serum urine and muscle were decreased. After we investigated the carnitine status in other members of the family, the brother of this patient, who had died of metabolic acidosis and hyperammonemia of unknown etiology in the neonatal period, was also revealed to have carnitine deficiency. Since specific enzyme defects which caused secondary carnitine deficiency could not be detected in our patients, further biochemical characterization would be necessary to clarify the cause of hyperammonemia.

Granulocytic sarcoma presenting as a mediastinal tumor. Report of a case and cytological and cytochemical studies of tumor cells in vivo and in vitro.

An unusual case of granulocytic sarcoma in a 23-year-old man is reported. The patient initially presented with mediastinal tumor and was diagnosed clinically as having thymoma. The patient was treated by radiotherapy and surgical removal of the tumor. Histology of the excised tumor had been nondiagnostic because of extensive fibrous changes. Eight months later, the patient developed pleural effusion on the right, which soon was followed by blood and bone marrow pictures consistent with acute promyelocytic leukemia. In vitro culture of pleural effusion cells unexpectedly gave rise to a continuously growing peroxidase-positive myeloid cell line. Autopsy revealed the recurrent mediastinal tumor to be positive for intracytoplasmic naphthol AS-D chloroacetate esterase and lysozyme activity. From these findings, the patient retrospectively was diagnosed as having mediastinal granulocytic sarcoma, which terminated in pleural effusion and acute promyelocytic leukemia.

Cell-mediated immunity to cytomegalovirus in pregnant women.

Employing the techniques of in vitro lymphocyte transformation (LTF) and complement fixation, cell-mediated immunity (CMI) and antibody to cytomegalovirus (CMV) were studied in pregnant and nonpregnant women. The LTF activity was determined by the whole blood microassay using four strains of CMV (AD-169 and its early antigen [EA], Davis, Veca, and Towne strains), and phytohemagglutinin (PHA). Lymphocyte transformation response to specific CMV antigens at 11-30 weeks of gestation and to nonspecific mitogen (PHA) in all pregnant and postpartum women were found to be significantly depressed compared with the nonpregnant women. The lower LTF responses to CMV antigen and PHA were found in specimens taken from pregnant women at 21-30 weeks of gestation. There were no significant differences in the mean complement-fixing (CF) antibody titers and the percentage of E-rosette-forming T lymphocytes between subjects in various stages of pregnancy. In addition, concanavalin A (Con A)-generated suppressor T cell activity was evaluated in pregnant and nonpregnant women. The suppressor effect of Con A-activated lymphocytes in the pregnant women was somewhat higher than in nonpregnant women. These observations suggest that CMV-specific suppression of cellular immunity may play an important role in reactivation of CMV in pregnancy.