New genome type of adenovirus serotype 19 causing nosocomial infections of epidemic keratoconjunctivitis in Japan

Twelve strains of adenovirus serotype 19, isolated from cases of epidemic keratoconjunctivitis in Japan in 1992, 1993, 1997, and 1998, were analyzed by DNA restriction analysis, using restriction endonucleases BamHI, BglI, BglII, EcoRI, HindIII, KpnI, PstI, SacI, SalI, SmaI, and XhoI. Among these 11 restriction endonucleases, EcoRI, PstI, SacI, and SmaI were discriminative enzymes, showing restriction patterns different from those reported previously for the prototype and the variant 19a. This new genome type was isolated in 1997 and 1998, when an increase of epidemic keratoconjunctivitis cases caused by adenovirus serotype 19 was observed for both sporadic and nosocomial infections. Strains from 1992 and 1993 showed restriction patterns similar to those of the worldwide reported variant 19a for all enzymes used. The changes detected in strains from 1997 and 1998 could be the reason for the recent epidemic.     

Culture of chondrocytes in fibroin-hydrogel sponge

Fibroin-hydrogel sponge and collagen gel were used as scaffold for in vitro cartilage regeneration. Fibroin-hydrogel sponge was formed by phase separation from freezed fibroin solution. Chondrocytes were harvested from proximal humerus, distal femur and proximal tibia of 4-week-old Japanese white rabbits and inoculated in the fibroin-hydrogel sponge and collagen gel. Those constructs were cultured in DMEM supplemented with 10% FCS and 50 ml L-ascorbate at 37 degrees C. Histological observation, measurement of sulfated glycosaminoglycan and cell density were carried out at 3, 7, and 14 days after the cultivation. Well-defined cartilage tissue can be seen both in the fibroin-hydrogel sponge and in the collagen gel. The matrix was intensely stained by safranin-O and showed a metachromatic reaction in both group. However, the quantity of sulfated glycosaminoglycan and cell density of the fibroin-hydrogel sponge group were increased more rapidly than these of the collagen gel group. Thus, the chondrocytes proliferated in the fibroin sponge without losing their differentiated phenotype. It is possible that culture environment in the fibroin sponge was suitable for chondrocytes regeneration.     

Survey of atopic dermatitis and skin barrier functions in Japanese and Chinese school students]

BACKGROUND: The comparative studies of the prevalence of atopic dermatitis and skin barrier functions in Japanese and Chinese were performed. METHODS: Clinical investigations were performed in 68 elementary school students in Lhasa, Tibet Autonomous Region and 67 students in Yixing, Jiangsu Province in China, and 99 students in Nishinomiya, Hyogo in Japan. Transepidermal water loss (TEWL) and capacitance were measured. Questionary survey about bathing frequency was also performed for students in Lhasa, Yixing and Osaka. RESULT: The prevalence rate of atopic dermatitis was 0% in Lhasa, 2.63% in Yixing, 4.26% in Nishinomiya. TEWL of students in Nishinomiya was higher than that in Yixing and Lhasa. Capacitance of students in Lhasa was lower than that in Nishinomiya and Yixing. The frequency of taking a bath in Lhasa was about 2.2 times per month and fewer than that in Nishinomiya and Yixing. CONCLUSION: There was tendency that the prevalence of atopic dermatitis increased according to increase of TEWL. It was thought that more investigations are necessary whether the development of habitat and environment influence the prevalence of atopic dermatitis and skin barrier function.     

Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans

Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformis luciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement of N-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.     

Psychosocial factors regulating natural-killer cell activity in recurrent spontaneous abortions

PROBLEM: The preconceptional natural-killer cell (NK) activity predicts subsequent miscarriage among women with unexplained recurrent spontaneous abortion (RSA). Psycho-neuro-immuno-endocrine network has recently been proposed as a mechanism for abortions. We therefore examined which psychosocial factors influenced the NK activity among women with RSA. METHOD OF STUDY: We measured the preconceptional NK activity of 61 women with a history two consecutive unexplained first-trimester miscarriages and no live births. We also administered semi-structured interviews and a battery of self-report questionnaires to assess their social support, personality, self-esteem and psychiatric symptoms. RESULTS: The preconceptional NK activity was negatively correlated with the women's neuroticism personality trait (r= -0.32, P = 0.01) and current depressive symptoms (r = -0.26, P= 0.05), and positively correlated with their self-esteem (r = 0.34, P = 0.01). CONCLUSIONS: In addition to several substances such as transforming-growth-factor beta and granulocyte-macrophase colony-stimulating factor, we found that low neuroticism, low depression scale score and high self-esteem contributed to high NK activity among women with RSA.     

Partial response to biotin therapy in a patient with holocarboxylase synthetase deficiency: clinical,biochemical, and molecular genetic aspects

We report the clinical course and biochemical findings of a 10-year-old, mentally retarded girl with late-onset holocarboxylase synthetase (HCS, gene symbol HLCS) deficiency and only partial response to biotin. On treatment, even with an unusually high dose of 200mg/day, activities of the biotin-dependent mitochondrial carboxylases in lymphocytes remained below 50% of the mean control values. Not only urinary 3-hydroxyisovaleric acid excretion has been persistently elevated, but also plasma and, with even higher concentrations, cerebrospinal fluid 3-hydroxyisovaleric acid have not normalized. The unusual and insufficient response of this patient to biotin treatment can be explained by the effect of the combination of the common HLCS allele IVS10 +5 g>a on one chromosome and a truncating mutation on the other. This case illustrates mechanisms involved in the genotype-phenotype correlation that unequivocally exists in HCS deficiency.     

Ras P21 EXPRESSION IN NEPHROBLASTOMA GROUP TUMORS

Ras p21 expression in 30 cases of nephroblastoma and related tumors was evaluated by the immunoperoxidase method using monoclonal anti- ras p21 antibody (RAP-5) raised against a synthetic polypeptide fragment of ras p21. In normal renal tissue, the epithelial cells of convoluted and collecting tubules and arterial wall leiomyocytes were positively stained. In nephroblastoma tumors, some of the epithelial cells forming primitive tubular and glomerular-like structures in the nephroblastic-type tumors and the cells with including intimal features were positive. However, the epithelial cells in more differentiated glandular structures of epithelial-type tumors, the rhabdoid cells in rhabdoid tumors and the cells in congenital mesoblastic nephroma failed to show positive staining. Nephroblastic cells, stromal cells and sarcomatous cells were also negative.     

Enhancement of the B-cell response to Staphylococcus aureus Cowan strain 1 by natural human gamma interferon.

The requirements for a primary, antigen-specific in vitro immunization of human peripheral lymphocytes using haemocyanin, a T-cell dependent antigen, have been studied. In order to obtain a specific response in vitro the peripheral lymphocytes had to be separated into B, T, accessory (A) and dendritic (D) cells. These cells were activated and reconstituted to give a population with a B:T ratio of 1:2. If the induction was supported by MHC-restricted, radioresistant T cells, this cell population could then be antigen-specifically activated using haemocyanin. The immunization had also to be supported by cytokines, such as B-cell growth and differentiation factors, interleukin-2 and gamma-interferon. A 5-day in vitro immunization using 2 micrograms haemocyanin/ml resulted in 200-300 cells secreting anti-haemocyanin-specific antibodies per 10(6) B cells.     

Mechanism of immune interferon production in vitro: interaction between immune interferon-producing cells and antigenic cells.

Various processes of in vitro immune interferon production by sensitized spleen cells stimulated with allogeneic cells were investigated. When L cells, an interferon-inducing antigen, were fixed with methyl alcohol or paraformaldehyde, the ability to induce immune interferon disappeared. In this immune interferon production system, the majority of sensitized spleen cells adhered to target cells within 1 h of cocultivation. Adherence of immune interferon-producing cells to target cells was observed only when L cell-sensitized spleen cells were cocultured with L cells or with mouse embryo cells derived from C3H mice. Fixation of antigenic cells with methyl alcohol or paraformaldehyde significantly reduced cell adherence. When L cells alone or sensitized spleen cells alone were pretreated separately with cytochalasin D, neither cell type could bind to partner cells. Specific adherence did not take place at 4 degrees C, nor in the presence of dinitrophenol or sodium azide. Continuous protein synthesis in both cells was not required for immune cell adherence. Divalent cations, Ca2+ or Mg2+, were required for this immune specific adherence to take place. However, once stable adherence was established, treatment with cytochalasin D, ethylenediaminetetraacetic acid, or sodium azide, or simple reduction of temperature, did not disrupt the binding. Interaction between immune interferon-producing cells and antigenic cells can be subdivided into two phases according to the requirement for divalent cations: (i) lymphocytes and antigenic cells interact transiently, and divalent cations are required to maintain the binding; (ii) lymphocytes and antigenic cells form a stable interaction, and deprivation of divalent cations does not disrupt the binding. Colchicine showed an inhibitory effect in the period after cell-to-cell adherence. Colchicine did not inhibit the release of interferon. On the other hand, vinblastine, another antimicrotubule agent inhibited the secretion of immune interferon. Since interferon synthesis was not stopped immediately after addition of cycloheximide, continued protein synthesis of sensitized spleen cells was not required for interferon secretion. The present study showed that adherence of immune interferon-producing cells to antigenic cells was a complex phenomenon involving a series of successive events.