Kazuo Yasuraoka

Oral Itraconazole and Topical Miconazole With Debridement for Acanthamoeba Keratitis

We treated three patients who had Acanthamoeba keratitis with oral itraconazole, a new antifungal agent, topical miconazole, and surgical débridement of the lesion. In these patients, healing and regression of the keratitis began six or seven days after initiation of oral itraconazole and miconazole 0.1% eyedrops (every hour during the day). The clinical signs of corneal infection disappeared after nine weeks in Patient 1, after five weeks in Patient 2, and after eight weeks in Patient 3. Visual acuities improved markedly from hand motions to 20/30 in Patient 1, from counting fingers to 20/16 in Patient 2, and from hand motions to 20/40 in Patient 3. In these patients, no systemic or topical signs of toxicity or adverse reactions were noted during the course of treatment.

Integration and expression of murine retrovirus-related sequences in schistosomes

Antibodies against the retrovirus envelope glycoprotein (gp70) of mouse xenotropic retrovirus, BALB virus 2 (Bv2) reacted with the adult worms of Schistosoma japonicum and S. mansoni. This reaction was completely inhibited after adsorption of the antibodies with virions of retrovirus. The reactive schistosome antigen was located in the subtegumental layer of the adult male fluke and in the vitelline gland of the adult female of S. japonicum and S. mansoni. Proteins extracted from both parasites were examined by immunoblot analysis. Anti-Bv2 gp70 antiserum reacted with those proteins from both schistosomes and the band patterns were different among sexes and species. Southern hybridization of the DNA extracted from adults of S. japonicum and S. mansoni demonstrated the presence of sequences homologous to the env gene of mouse ecotropic and xenotropic retroviruses. DNA sequences homologous to the gag and pol regions of the ecotropic murine leukaemia virus were also detected in the DNAs of schistosomes.

Existence of host-related DNA sequences in the schistosome genome

DNA sequences homologous to the mouse intracisternal A particle and endogenous type C retrovirus were detected in the DNAs of Schistosoma japonicum adults and S. mansoni eggs. Furthermore, other kinds of repetitive sequences in the host genome such as mouse type 1 Alu sequence (B1), mouse type 2 Alu sequence (B2) and mo-2 sequence, a mouse mini-satellite, were also detected in the DNAs from adults and eggs of S. japonicum and eggs of S. mansoni. Almost all of the sequences described above were absent in the DNAs of S. mansoni adults. The DNA fingerprints of schistosomes, using the mo-2 sequence, were indistinguishable from each other and resembled those of their murine hosts. Moreover, the mo-2 sequence was hypermethylated in the DNAs of schistosomes and its amount was variable in them. These facts indicate that host-related sequences are actually present in schistosomes and that the mo-2 repetitive sequence exists probably in extra-chromosome.

Dynamic changes of DNA sequences in Schistosoma mansoni in the course of development

Deletion and/or amplification of DNA sequences in Schistosoma mansoni were demonstrated by Southern blot analysis. Total cellular DNAs and genomic clones derived from S. mansoni miracidia, adult males and females were used as probes. Endonuclease BamHI-restricted DNAs from miracidia, adult males and females of both S. mansoni and S. japonicum were reacted to each probe. Hybridization with a total cellular DNA from S. mansoni miracidia as a probe showed elimination of signals in S. mansoni adults. On the other hand, blot analysis using a total cellular DNA from S. mansoni adult males as a probe revealed elimination of hybridization signals in S. mansoni miracidia. Hybridization with a clone SmE15 DNA from S. mansoni miracidia as a probe showed no signal in the DNAs from S. mansoni adults, indicating these sequences deleted in adults. Hybridization experiments using the probes SmF25 and SmM51 which are 1.3 and 2.2 kb fragments cloned from S. mansoni adult females and males respectively, demonstrated no signal to DNA from S. mansoni miracidia. Our data suggested the existence of stage-specific DNA sequences in S. mansoni. We propose a model for multiple-step rearrangement of DNA sequences in S. mansoni during the course of development.

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.

Degenerative changes in the reproductive organs of female schistosomes during maintenance in vitro.

Degenerative alteration of the reproductive organs of female schistosomes in correlation with the change in egg-laying rate of schistosome pairs in vitro was studied by electron microscopy. The production of normal eggs by adult S. japonicum pairs decreased after 4 days in vitro followed by an increase of abnormal egg laying up to day 8. In S. mansoni, the yield of both normal and abnormal eggs decreased gradually from the start of maintenance in vitro in spite of a much higher pairing rate than in S. japonicum. The vitelline gland of 14-day in vitro-maintained S. japonicum stained with Fast red B, while that of S. mansoni did not. The ovary of both species exhibited regressive features after 14 days of maintenance in vitro. Ultrastructural examination showed that the vitelline cells and oocytes of S. japonicum and S. mansoni had already lost their structural integrity after 2 days in vitro and continued to exhibit signs of structural degeneration throughout the 14-day in vitro maintenance period. The regressive changes in reproductive potential of female S. mansoni maintained in vitro for 4 days could be reversed by surgically implanting the parasites into mouse mesenteric veins.

Comparison of protein composition between schistosomula of Schistosoma japonicum and S. mansoni

SDS-PAGE analysis revealed that the protein composition of mechanically transformed schistosomula (ms) of a Japanese strain of Schistosoma japonicum was essentially similar to that of a Philippine strain of S. japonicum. However, the protein components of S. mansoni ms were remarkably different from those of S. japonicum. Specific proteins of Mr 60-65 and 30 kDa were found in ms of S. japonicum and S. mansoni, respectively. In skin-penetrated schistosomula (ss) of the two species, a common protein of 67 kDa was detected. Using two-dimensional electrophoresis, strain-specific polypeptides (9 in a Japanese and 11 in a Philippine strain) were identified in ms of S. japonicum. Mechanically transformed and skin-penetrated schistosomula of S. japonicum (Japanese) had, respectively, 7 and 10 specific proteins. Four and 1 specific polypeptides were also detected in two-dimensional profiles of ms and ss of S. mansoni, respectively.

Schistosoma japonicum: In vitro killing of schistosomula by mouse neutrophils

Since Dean et al. (1974) reported that Schistosoma mansoni schistosomula were ensheathed and killed by rat neutrophils in the presence of rat antiserum, in vitro studies on the role of neutrophils have been carried out by many investigators. However, results reported to date not agree. Some groups claimed that neutrophils from various host species participated in the antibodyand/or complement-dependent killing of the S. mansoni schistosomula (Dean et al. 1975; Anwar et al. 1979; Kassis et al. 1979; Incani and McLaren 1981; Kazura et al. 1981; McKean et al. 1981; McLaren and Incani 1982; Incani and McLaren 1983). Contrary to these results, other groups stated that human neutrophils did not damage antibody-coated S. mansoni schistosomula (Butterworth et al 1977, 1979 a, b; Vadas et al. 1979; David et al. 1980; Dessein et al. 1983). Hsfi et al. (1977) concluded from the results in the rhesus monkey system using S. mansoni and S. japonicum, that both normal and sensitized neutrophils increased the schistosomulicidal effect of either noninactivated or inactivated immune serum. The present study attempted to clarify the effect of peritoneal neutrophils derived from normal uninfected mice or from mice infected with S. japonicum on antibodyand complement-dependent damage to S. japonicum schistosomula. A Japanese strain (Yamanashi) of S. japonicum was maintained in Oncomelania nosophora and male ICR mice (Shizuoka Laboratory Animal Centre, Japan). Schistosomula were obtained in vitro by allowing freshly shed cercariae to penetrate mouse skins (Clegg and Smithers 1972). Sera were prepared by cardiac puncture from ICR age-matched mice 8 weeks after S. japonicum infection. Considerable resistance developed in mice which had received the primary infection of 30 S. japonicum cercariae 8 weeks prior to challenge infection (Maeda et al. 1982). Sera were heatinactivated before use by incubation at 56~ for 30 rain. Fresh serum of ICR mice was used as a source of complement. Neutrophil-rich cell popula-