Efficient and accurate homologous recombination in hESCs and hiPSCs using helper-dependent adenoviral vectors.

Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7-81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23-57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.

Pelodera strongyloides infestation presenting as pruritic dermatitis.

Pelodera strongyloides is a free-living soil nematode of the order Rhabditida. We report an 18-year-old man with P strongyloides skin infestation. In this case, pruritic follicular papulopustules developed on the buttocks, then the right flank. Skin scrapings revealed many live rhabditiform larvae that were cultured adult worms and hatched ova, identified as P strongyloides . The eruption was treated effectively with topical 1% gamma-hexachlorocyclohexane ointment.

Common antigenicities between Angiostrongylus cantonensis and various species of snails.

Studies on common antigenicities were carried out by using rabbit sera immunized with Angiostrongylus cantonensis adult worms or the third stage larvae and antigens of various species of snails and vice versa by the immunoblotting technique. The results obtained are summarized as follows: (1) Common antigenicities between A. cantonensis adult worms and snails susceptible to A. cantonensis were observed in a range of molecular weights of 14.3 to 200 kDa. In Puerto Rican pigmented Biomphalaria glabrata and Achatina fulca, which had high infection rates with A. cantonensis, we recognized 15 to 16 bands against the adult worm, especially the band with a molecular weight of 29 kDa, which had a more intense reaction. (2) Common antigenicities between A. cantonensis third stage larvae and snails susceptible to A. cantonensis, were observed in a range of molecular weights of 14.3 to 97.4 kDa, especially A. fulica and B. glabrata, where we detected many bands in molecular weight range of 18.4 to 43 kDa. Based on the common antigenicities between A. cantonensis and snails susceptible to A. cantonensis, it is possible that the common antigenicities are one of the factors defining the different susceptibilities of various species of snails to A. cantonensis, and more bands are seen with increasing infection rates with A. cantonensis. Of those bands, the protein with the molecular weight of 29 kDa may be the main common antigen between the A. cantonensis adult worm, the third stage larvae and the snails susceptible to A. cantonensis.

Biological characteristics and control of intermediate snail host of Schistosoma japonicum.

Except for imported cases, we have had no new Schistosoma japonicum infection in Japan since 1977. But there are still two habitats of the intermediate snail host: Oncomelania nosophora in the previous endemic areas of Kofu Basin and Obitsu. O. nosophora from Kofu Basin and Obitsu are susceptible to Chinese and Philippine strains of S. japonicum. The number of immigrants from current endemic areas in China or the Philippines is increasing. In order to prevent re-emerging of S. japonicum infections in Japan, we should continue monitoring on those existing snail hosts and investigate an adequate quarantine system. In Japan, elimination of schistosomiasis has been mainly accomplished by control of the snail host. As measures of snail control, cement-lining of ditches and chemical mollusciciding were most effective in Japan. But the cost of this joint program is too expensive compared with health budget in almost developing countries. In endemic areas of Japan, land reformation from paddy field to fruit farm was also effective. The intermediate snail host in the Philippines, Oncomelania quadrasi is much more aquatic than O. nosophora. For control of O. quadrasi, small drainage of the water and land reclamation from swampy field to rice-field were effective. Based on biological characteristics of Oncomelania spp., we can modify the past successful snail control program in Japan to be adapted ecologically and economically to each endemic area of developing countries.

In vitro cultivation of cells from ovotestis tissue of pigmented Biomphalaria glabrata.

Cells derived from ovotestis tissue of pigmented Biomphalaria glabrata, Puerto Rican strain were cultured in double diluted GIT medium supplemented with modification of amino acids components of pigmented B. glabrata, ovotestis and mid-gut region and 3% inactivated fetal calf serum. As a result, two types of cells, epithelial and fibroblastic like cells increased in number during the cultivation. It seem that the medium used in this study is a suitable medium for cultivation of cells from ovotestis of pigemeted B. glabrata. These two types of cells have been maintained by successive transplantation for over 3 passages.