Production of loach (Misgurnus anguillicaudatus) germ-line chimera using transplantation of primordial germ cells isolated from cryopreserved blastomeres.

An efficient procedure for the cryopreservation of fish blastomeres followed by restoration through germ-line chimera formation was established. Blastomeres of the loach (Misgurnus anguillicaudatus) were cryopreserved in 250-µL straws in Eagle's minimum essential medium with various concentrations of dimethyl-sulfoxide (0, 5, 10, 15, and 20%), and the best concentration was combined with glycerol (1, 2, and 4%) and external cryoprotectants (1 or 2% sucrose; 2, 5, or 10% fetal bovine serum; 1 or 2% BSA). Postthaw viability of the blastomeres was used to optimize cryopreservation conditions. Donor blastomeres were injected with zebrafish green fluorescence protein-nos1 3' untranslated region mRNA and biotin dextran before cryopreservation in the optimal freeze medium. Host embryos were injected with zebrafish DsRed-nos1 3' untranslated region mRNA and reared to the blastula stage. Donor blastomeres were thawed at 25 °C for 10 s and transplanted to the host embryos either immediately or after incubation for 16 h at 20 °C. Donor and host primordial germ cell migration was visualized with fluorescent imaging during the early stages of embryogenesis, and also by histology in 4-d-old embryos. Transplantation of blastomeres immediately after thawing gave decreased hatching rates (approximately 3%) and generated a smaller percentage of germ-line chimeras (approximately 1.1%). In contrast, incubation of a cryopreserved sample for 16 h followed by transplantation of the green fluorescence protein-positive blastomeres improved the hatching rate to 90%, and successfully produced presumable germ-line chimeras at a rate of 16.5%. The improved survival rates and germ-line chimerism may be an effective method for gene banking and subsequent reconstitution of endangered fish genotypes.

[A case of Behçet's disease presenting with Hughes-Stovin syndrome (multiple pulmonary arterial aneurysms remitting with corticosteroid therapy)].

A 32-year-old male suffering from thrombophlebitis of the right leg was admitted to our hospital because of massive hemoptysis. Chest radiography showed multiple rounded opacities in the right lung field. Pulmonary angiography revealed multiple aneurysms of the right pulmonary arteries and thromboembolism of the left pulmonary artery. Venography demonstrated obstruction of the deep veins of the right leg and the right femoral vein, and deep vein thrombosis was diagnosed. A more precise diagnosis was Hughes-Stovin syndrome, that is, venous thrombosis especially of the vena cava, accompanied by single or multiple pulmonary arterial aneurysms in young patients. Furthermore, the patient had an aphthous ulcer in the oral cavity, an ulcer in the genital region, leading to a diagnosis of the incomplete type of Behçet's disease. Chest radiographs 3 months after the initiation of corticosteroid showed complete resolution of the aneurysms. Repeated pulmonary angiography also showed partial recanalization of the occluded arteries. This report describes this very rare case of Behçet's disease presenting with Hughes-Stovin syndrome.

Association of tumor necrosis factor alpha gene promoter polymorphism with the presence of chronic obstructive pulmonary disease.

Tumor necrosis factor alpha (TNF-alpha), a potent proinflammatory cytokine, may be involved in the development of chronic obstructive pulmonary disease (COPD). The production of TNF-alpha is elevated in the airways of these patients. A polymorphism at position -308 of the TNF-alpha gene promoter (TNF-alpha-308*1/2) is known to be associated with alteration of TNF-alpha secretion in vitro. In this study we examined the differences in TNF-alpha-308*1/2 allele frequency to investigate the association of this polymorphism with the presence of smoking-related COPD. TNF-alpha-308*1/2 allele frequency in 106 patients (73 men and 33 women) was compared with 110 asymptomatic smoker/ex-smoker control subjects matched for sex and age and population control subjects consisting of 129 blood donors. Genotype was analyzed by the polymerase chain reaction-restriction fragment length polymorphism technique on genomic DNA isolated from peripheral blood lymphocytes. TNF-alpha-308*1/2 allele frequencies were significantly different among the groups: 0.835/0.165 in patients with COPD, 0.918/0.082 in smoker/ex-smoker control subjects, and 0.922/0.078 in population control subjects. These results indicate that TNF-alpha-308*1/2 alleles are significantly associated with the presence of smoking-related COPD.

[A case of miliary tuberculosis with multiple cerebral tuberculoma and spinal tuberculosis owing to total delay].

We reported a case of miliary tuberculosis with multiple cerebral tuberculoma and spinal tuberculosis. The case was a 37 year old man. In the last sixteen months to the first visit to a hospital, he has been suffering from low grade fever, cough, and back pain, but he bared his symptoms without any therapy. At a hospital he first visited, he was told that he might have a malignant disease, which prevented him to visit the hospital because of a fear for his disease. Six months later, he was admitted to other hospital because of severe back pain. At last, he was diagnosed as tuberculosis and referred to our hospital. By the examinations on admission he was diagnosed as multiple cerebral tuberculoma and spinal tuberculosis, and anti-tuberculous therapy was started. It is thought that his severe disease status on admission is caused by the total delay, namely the delay in visiting a doctor and the doctor's delay in making diagnosis.

[A case of cutaneous tuberculosis associated with steroid therapy for mixed connective tissue disease].

Patients receiving immunosuppressive therapy, such as adrenocorticosteroids, are high risk groups of tuberculosis. We report a case of cutaneous tuberculosis associated with steroid therapy for mixed connective tissue disease. A 63-year-old female was hospitalized after 6 months' treatment with prednisolone for connective tissue disease and bilateral abnormal shadows were revealed on her chest X-ray films. As her sputum smear was positive for acid-fast bacilli, the patient was transferred to our hospital for isolation and treatment. After three months' treatment with INH, RFP and EB, she complained the swelling of her left palm, left arm, and right leg, and skin puncture was performed. As smears of fluid aspirated from the swelling showed acid-fast bacilli, and fluid PCR tests showed positive for M. tuberculosis, she was diagnosed as cutaneous tuberculosis (scrofuloderma). In spite of administration of antituberculous agents, the swelling showed little improvement. Therefore, the dose of prednisolone was reduced and cutaneous lesions were resected by surgery. High risk of tuberculosis should be considered when a patient administered immunosuppressive drugs, such as adrenocorticosteroids.

[A preventable case who died of miliary tuberculosis after receiving contact examination].

A 30-year-old-man was admitted to our hospital because of headache and fever. His consciousness on admission was clouding. Sputum examination was positive for acid fast bacilli which later identified as Mycobacterium tuberculosis. Chest-X-ray and computed tomogram on admission showed multiple cavitary lesions on bilateral upper lung fields and bilateral diffuse nodular shadow. He was diagnosed as miliary tuberculosis with tuberculous meningitis. His mother admitted because of pulmonary tuberculosis four months ago, and her sputum examination was smear positive for acid fast bacilli, Gaffky 4, and she complained of cough for 6 months before admission. Because of this situation, he rapidly underwent the contact examination with chest X-ray, but not examined by tuberculin skin test because he was 30-year-old. As then chest X-ray was normal, he was not indicated of chemoprophylaxis, and he died of miliary tuberculosis and tuberculous meningitis 4 months after the contact examination.

[A case of thymic carcinoma producing granulocyte colony-stimulating factor].

A 73-year-old man was admitted complaining of chest pain and weight loss. A chest X-ray film and chest CT scan showed a tumorous mass in the anterior mediastinum with moderate pleural effusion. Percutaneous needle biopsy specimens from the mass resulted in a diagnosis of poorly differentiated squamous cell carcinoma of the thymus. Laboratory findings on admission showed marked leukocytosis with no evidence of infection or bone marrow metastasis. The level of serum granulocyte colony-stimulating factor (G-CSF) was abnormally high (79.2 pg/ml, normal < 30). Carcinoma cells in the specimen showed positive staining with anti G-CSF monoclonal antibody, thus indicating that they produced G-CSF. Because multiple metastatic lesions on the chest wall and liver were detected, we performed combination chemotherapy with cisplatin, vindesine and mitomycin C. After 2 courses of chemotherapy, a decrease in tumor size was verified by CT scan. As the tumor size decreased, the patients white blood cell count and serum G-CSF concentration fell to normal levels.

Periodontal conditions in older age cohorts aged 65 years and older in Japan, measured by CPITN and loss of attachment.

Periodontal conditions were assessed by CPITN and loss of attachment in 601 elderly people randomly selected in Kitakyushu, Japan. The percentage of edentulous persons was 21 per cent, 38 per cent and 65 per cent in the 65-74 year, 75-84 year and 85 years and older groups, respectively. In dentate persons, over 60 per cent had pocket probing depths of 4mm or more; in the majority loss of attachment did not exceed 5mm. The mean numbers of sextants with pocket probing depths of 4-5mm (CPITN 3) and probing depths of 6mm and more (CPITN 4) were similar in each age group. The mean numbers of sextants with both pockets and loss of attachment showed no clear difference among groups. Only the mean number of excluded (no, or only one tooth) sextants increased with increasing age from 1.9 to 3.1. It could therefore be suggested that in the population examined the progress of periodontal destruction with increasing age might not be shown by an increase in pocket probing depth, loss of attachment or gingival recession, but only by an increase in tooth loss. The results for the remaining teeth show that a) severe loss of periodontal attachment (6mm or more) was not frequently encountered, b) recession (attachment loss without pocketing) was present at only very low levels, c) the main periodontal problem seemed to be pocketing, which could be treated. It is therefore tempting to suggest that some of the teeth already lost, could have been saved if proper self- and professional care had been applied.

Correlation between volatile sulphur compounds and certain oral health measurements in the general population.

Oral malodor was measured using a portable sulphide monitor in 2,672 individuals aged 18 to 64 years. In addition, dental (DMFT) and periodontal conditions (CPITN and attachment loss), dental plaque, and tongue coating status were assessed. Before clinical examination, subjects were interviewed about their oral health habits, smoking habits, and medical history. Data on volatile sulphur compounds (VSC) were analyzed by gender, age group, and time of measurement. There were no significant differences observed in the VSC between males and females in any age group. In each age group, the measured values of oral malodor were highest in the late morning group (58.6 ppb in average), followed by the late afternoon group (52.1 ppb), while lowest values were shown in the early afternoon group (39.4 ppb). Significant correlation was observed only between the VSC value and periodontal conditions and tongue coating status. The results also suggest that oral malodor might be caused mainly by tongue coating in the younger generation and by periodontal diseases together with tongue coating in older cohorts in the general population. Age was not a risk factor for increasing VSC.

Relationship between dental caries experience of a group of Japanese kindergarten children and the results of two caries activity tests conducted on their saliva and dental plaque.

To establish a dental caries preventive programme in 4- and 5-year-old children, caries activity tests were used to assess salivary levels of mutans streptococci (using the Mucount test, Showa Yakuhin, Japan) and the acidogenic ability of dental plaque bacteria (using the Cariostat test, Sankin, Japan) in 260 kindergarten children. The relationships between the results of these two tests and the dental caries experience (dfs) of the children was evaluated. There was a significant positive correlation between the results of the Mucount and Cariostat tests in the total group of subjects. However, 91 subjects (35%) gave conflicting results with the two tests. These subjects were divided into two groups: one group (group B) included Mucount-negative and Cariostat-positive subjects, and the other group (group C) included Mucount-positive and Cariostat-negative subjects. Group B had a significantly higher mean dfs score than a group that was negative on both tests (group A). On the other hand, no significant difference in mean dfs score was observed between group C and group A. These findings suggested that mutans streptococci levels alone are an insufficient indicator for assessing dental caries activity of children at these ages and that the role of lactobacilli and other aciduric bacteria should be considered. It was concluded that assessment of caries activity using both Mucount and Cariostat tests is useful for a dental caries preventive programme for children of these ages.