Mitochondrial DNA divergence in populations of the tapeworm Diphyllobothrium nihonkaiense and its phylogenetic relationship with Diphyllobothrium klebanovskii.

Diphyllobothrium nihonkaiense [Y. Yamane, H. Kamo, G. Bylund, J.P. Wilkgren. Diphyllobothrium nihonkaiense sp. nov (Cestoda: Diphyllobothriidae)- revised identification of Japanese broad tapeworm. Shimane J Med Sci 1986;10:29-48.] and Diphyllobothrium klebanovskii [I.V. Muratov, P.S. Posokhov. Causative agent of human diphyllobothriasis - Diphyllobothrium klebanovskii sp. n. Parazitologiia. 1988;22:165-170.] are two major species of human diphyllobothriasis in Japan and Far East Russia, respectively, but their taxonomical relationship remains unclear. In this study, we analysed the DNA sequences of 16 clinical isolates of D. nihonkaiense from Japanese people, 3 isolates of D. klebanovskii from a bear in Kamchatka, and 4 clinical isolates of D. klebanovskii from native Udygeyci people in Russia, as well as 4 plerocercoids from Oncorhynchus spp. 18S rDNA and internal transcribed spacer 1 (ITS1) sequences from D. nihonkaiense and D. klebanovskii showed a high level of similarity, indicating synonymy of the two species. Analyses of mitochondrial DNA (mtDNA) sequence polymorphisms in the cox1 and nad3 genes of D. nihonkaiense (D. klebanovskii) revealed two deeply divergent lineages, A and B, with genetic distances (Kimura-2 parameter) of 0.018-0.022. Furthermore, the distinct monophyletic groupings of cox1 haplotypes corresponded to the distinct monophyletic groupings of nad3 haplotypes. The two lineages were neither distinguished by morphological features nor defined by the localities of the samples. These results suggest that the two morphologically cryptic lineages have diverged and coexisted over a long period of time.

Diplogonoporiasis in Japan: genetic analyses of five clinical isolates.

Infection of the whale tapeworm Diplogonoporus balaenopterae (Diphyllobothriidae) is occasionally found in humans, especially among Japanese. In the present study, we analysed the nucleotide sequences of the 18S rDNA, ITS1 and cox1 genes of the immature and mature proglottids of Diplogonoporus species recovered from five Japanese patients. The nucleotide sequences of 18S rDNA, ITS1 and cox1 showed little, if any, intraspecific divergence. Phylogenetic analyses of several diphyllobothriid species revealed a close relationship of Diplogonoporus isolates with the cetacean tapeworm Diphyllobothrium stemmacephalum. The results suggest that the genus Diphyllobothrium is paraphyletic and raise a question regarding the validity of the genus Diplogonoporus.

Unconstrained Monitoring of Biological Signals Using an Aortic Pulse Wave Sensor.

This paper proposes a system to extract biological signals from aortic pulse waves which are measured by a microphone type pulse wave sensor. Theproposed system enables extraction of three biological signals corresponding to respiratory rate, pulse pressure wave, and RR interval simply by sitting on the seat on which the sensor is laid. Experiment results demonstrated that the mean absolute errors between the signals measured by the proposed system and the conventional sensors are as low as 0.38 times per minute for the respiratory rate, 11.2 mmHg for the pulse pressure wave, and 16.6 ms for the RR interval. The proposed system thus may be applied to monitor the physiological state of a human subject to prevent accident caused by health condition.

T cell-dependent and -independent expression of intestinal epithelial cell-related molecules in rats infected with the nematode Nippostrongylus brasiliensis.

To determine how T cells of thymic origin regulate the intestinal mucous response induced by nematode infection, mucin production and goblet cell-specific secretory peptide expression were examined in euthymic rnu/+ and athymic rnu/rnu rats infected with the nematode Nippostrongylus brasiliensis. Euthymic rats showed transient goblet cell hyperplasia and upregulation of mucin production, which returned to preinfection levels by 21 days postinfection, when nematodes had been rejected from the intestine. In athymic rats, which failed to reject nematodes, goblet cell hyperplasia and accelerated mucin production continued at least until 21 days postinfection. Gene transcription of mucin-core peptide (MUC)-2 and -3 and trefoil factor (TFF)-2 and -3 in the jejunal epithelium was upregulated parallel to the levels of goblet cell hyperplasia in both euthymic and athymic rats. On the other hand, resistin-like molecule (Relm)beta, sialyltransferase Siat4c and sulfotransferase 3ST1 showed significantly higher transcription levels in euthymic than in athymic rats at 7 and/or 10 days postinfection. These results suggest that the induction of intestinal mucin production occurs without the activation of thymus-derived T cells, while the expression of Relmbeta, Siat4c and 3ST1 in the intestinal epithelial cells seems to be regulated at least partly by thymus-dependent mechanisms.

Altered expression of goblet cell- and mucin glycosylation-related genes in the intestinal epithelium during infection with the nematode Nippostrongylus brasiliensis in rat.

Intestinal nematode infection induces marked goblet cell hyperplasia and mucus secretion, but the mechanisms of regulation of the changes still remain to be elucidated. In the present study, epithelial cells were isolated from the rat small intestine at various times after Nippostrongylus brasiliensis infection, and the levels of expression of goblet cell- and mucin glycosylation-related genes were estimated by semi-quantitative reverse transcription (RT)-PCR. Among the genes investigated, mucin core peptide (MUC) 2, sialyltransferase (Siat) 4c and trefoil factor family (TFF) 3 were upregulated as early as 2-4 days post-infection, suggesting that they are associated with an early innate protective response. Seven days post-infection and thereafter, when the nematodes reached maturity, significant upregulation of MUC3, MUC4, resistin-like molecule beta (Relmbeta) and 3O-sulfotransferase (3ST)1 was observed, while 3ST2 expression levels increased after the majority of the worms were expelled from the intestine. Similar alterations of glycosylation-related gene expression were also observed in mast-cell-deficient Ws/Ws rats, suggesting that mast cells in the epithelium are not relevant to the upregulation of these genes. The present finding that the expression level of each goblet cell- or glycosylation-related gene was altered differently during the time course of infection indicates the progression of sequential qualitative changes in the mucus layer after infection.

Alterations in hexose, amino acid and peptide transporter expression in intestinal epithelial cells during Nippostrongylus brasiliensis infection in the rat.

Infection with the nematode Nippostrongylus brasiliensis induces various types of cytological alterations in the intestinal villus epithelium. The aim of this study was to analyse the expression of hexose, peptide and amino acid transporters in the small intestinal epithelium after infection. Brown-Norway rats were infected with 2000 N. brasiliensis L3 larvae and villus epithelial cells were isolated at various time points after infection. Expression of hexose transporters Na(+)/glucose cotransporter SGLT1 and glucose transporter GLUT-1, -2 and -5, a peptide transporter (PepT1) and an amino acid transporter (LAT2) was examined by reverse transcription-PCR, Western blotting or immunohistochemistry. Semi-quantitative reverse transcription-PCR studies of separated jejunal epithelial cells showed that expression levels of GLUT5, PepT1 and LAT2 were significantly decreased 7 and 14 days after infection, while these changes were not observed in the ileal epithelium. Although the apical surface glucose transporter SGLT1 showed no significant alteration in mRNA expression, Western blotting analyses of jejunal epithelial cell lysate showed a marked decrease. Contrary to SGLT1, GLUT5, PepT1 and LAT2, expression of GLUT1, which is essential in maintaining high rates of glucose influx, was significantly up-regulated in the jejunal epithelium 7 and 14 days after infection in reverse transcription-PCR as in Western blotting analyses. Immunohistochemical studies showed that GLUT1 immunoreactivity was localised to the basolateral membrane of intestinal epithelial cells 7 days after infection. These results show that N. brasiliensis infection results in an increase in GLUT1 and a decrease in various hexose, amino acid and peptide transporter expression in jejunal epithelial cells. Up-regulation of GLUT1 might be a compensatory response in injured epithelial cells.

Ascariasis in Japan: is pig-derived Ascaris infecting humans?

Human ascariasis is caused by infection with the common roundworm Ascaris lumbricoides, although the pig roundworm Ascaris suum has also been reported to infect humans and develop into the adult stage. To elucidate whether pig-derived Ascaris infects humans in Japan, 9 Ascaris isolates obtained from Japanese patients and a further 9 Ascaris isolates of pig origin were analyzed to determine their internal transcribed spacer-1 sequences. Six of the 9 clinical isolates showed the Ascaris genotype which predominantly infects humans in endemic countries, while the other 3 clinical isolates and 9 pig-derived isolates showed the genotype predominant in pigs worldwide. These results suggest that at least some cases of human ascariasis in Japan are a result of infection with pig-derived Ascaris.

Alteration of the expression profiles of acidic mucin, sialytransferase, and sulfotransferases in the intestinal epithelium of rats infected with the nematode Nippostrongylus brasiliensis.

Acidic mucins such as sialomucin and sulfomucin produced by intestinal epithelial cells have been implicated in the protection of the mucosa from pathogens. In the present study, we analyzed the alteration of acidic mucins in the jejunum of euthymic and athymic rats infected with the nematode Nippostrongylus brasiliensis using alcian blue staining and a high iron-diamine method. The numbers of sialomucin+ goblet cells increased markedly 7 and 10 days post-infection and decreased gradually thereafter in euthymic rats, while athymic rats did not show sialomucin+ goblet cell hyperplasia at least until 28 days post-infection, suggesting that sialomucin production might be regulated by thymus-derived T cells. On the other hand, the numbers of sulfomucin+ goblet cells increased markedly 28 days post-infection in both euthymic and athymic rats despite the fact that sulfomucin+ goblet cell numbers in uninfected athymic rats were significantly smaller than in euthymic rats. Real-time polymerase chain reaction studies on the gene transcription levels of O-glycan sulfotransferases Gal3ST1, Gal3ST2, Gal3ST3, and Gal3ST4 in the jejunal epithelium increased gradually toward day 28 post-infection in euthymic and athymic rats. These results suggest that the production of sulfomucin and expression of Gal3STs are inducible by nematode infection without the activation of thymus-derived T cells.

Diphyllobothriasis nihonkaiense: possibly acquired in Switzerland from imported Pacific salmon.

A 5-year-old Japanese boy passed tapeworm strobila while he was living in Switzerland. During a short visit to Japan, he was successfully treated with a single dose of praziquantel. DNA sequences of ITS1, cox1 and nd3 genes from the tapeworm were compatible with those of Diphyllobothrium nihonkaiense rather than Diphyllobothrium latum, which is prevalent in Europe. The patient consumed imported salmon in Switzerland. This case highlights the globalization of D. nihonkaiense, which was once restricted to the Far East, and reflects the worldwide demand for seafood.

A case of human fasciolosis: discrepancy between egg size and genotype of Fasciola sp.

In human fasciolosis, differential diagnosis of the causative flukes, Fasciola hepatica and Fasciola gigantica, is problematic. We report a rare case of human fasciolosis in which an adult worm was recovered from the bile duct of a Japanese man. Morphometric data of the worm were consistent with those of F. hepatica, whereas the size of eggs in the stool indicated infection with F. gigantica. Nucleotide sequences of ITS-1 and -2 and CO1 genes of the DNA extracted from the eggs revealed that the genotype was that of F. hepatica. These findings suggest that the size of eggs is not a suitable marker for species identification in human fasciolosis, especially in settings such as the East Asian region where different karyotypes and hybrid genotypes of F. hepatica and F. gigantica have been found.

Up-regulation of Fas (CD95) and induction of apoptosis in intestinal epithelial cells by nematode-derived molecules.

Infection by the intestinal nematode Nippostrongylus brasiliensis induces acceleration of apoptosis in the small intestinal villus epithelial cells in vivo. In the present study, we examined whether worm extract or excretory-secretory product induces apoptosis in the rat intestinal epithelial cell line IEC-6 in vitro. In the presence of worm extract or excretory-secretory product (> or =6 microg/ml), IEC-6 cell growth was significantly suppressed, and there was a concomitant increase in the number of detached cells in culture dishes. Detached cells showed nuclear fragmentation, activation of caspase-3, and specific cleavage of poly(ADP-ribose) polymerase, suggesting that apoptosis was induced in these cells. Semiquantitative reverse transcription-PCR showed that expression of Fas (CD95) mRNA was up-regulated as early as 6 h after addition of excretory-secretory product, while Fas ligand expression and p53 expression were not up-regulated. Fluorescence-activated cell sorter analyses revealed a significant increase in Fas expression and a slight increase in FasL expression in IEC-6 cells cultured in the presence of excretory-secretory product, while control IEC-6 cells expressed neither Fas or FasL. These results indicated that N. brasiliensis worms produce and secrete biologically active molecules that trigger apoptosis in intestinal epithelial cells together with up-regulation of Fas expression, although the mechanism of induction of apoptosis remains to be elucidated.

Environmental pollutant tributyltin promotes Th2 polarization and exacerbates airway inflammation.

It has been shown that a relatively high dose of tributyltin (TBT), which is recognized as a particularly notable environmental pollutant, exerts immunotoxic effects such as thymic atrophy via induction of T cell apoptosis. However, the effect of low doses of TBT on the immune responses remains unknown. Here we show that environmentally relevant doses of TBT promoted strong Th2 polarization via suppression and augmentation of Th1 and Th2 development, respectively, from naive CD4(+) T cells primed with anti-CD3 and splenic antigen-presenting cells (APC). TBT-induced Th2 polarization was indirect, working through APC via suppression of IL-12 production by macrophages/DC and the augmentation of IL-10 production by B cells. Th2 polarization was also induced in mice treated with TBT and immunized with OVA or infected with Nippostrongylus brasiliensis. Furthermore, airway inflammation in mice sensitized and challenged with OVA was exacerbated by the administration of TBT with concomitant augmentation of Th2-type immunity. Our results highlight the fact that an important environmental pollutant TBT may present significant risk for the induction of allergic diseases via promotion of Th2 polarization.