Biomedical subjects

Nobuo Ohta

Publications and source records attributed to Nobuo Ohta.

At least 37 records · Page 2Linked to original sources

PubMed2004

Research on calpain of Schistosoma japonicum as a vaccine candidate.

Vaccine development by the use of calpain of Schistosoma japonicum has been tried in our laboratory. We cloned cDNA encoding the heavy chain of S. japonicum calpain, and prepared recombinant molecule of a possible vaccine region of the heavy chain. When BALB/c mice were immunized with our recombinant calpain of S. japonicum with Freund's complete adjuvant, we observed significant reduction in worm burden (41.2% reduction, P<0.05), and also significant anti-fecundity effects. In this sense, calpain of S. japonicum seems to have infection control as well as anti-disease effects. Mechanisms of vaccine effects of calpain remain to be clarified, however, several effector mechanisms are suspected. In immunized mice, raised level of iNos expression was observed, while adhesion of peritoneal exudates cells were also observed in the presence of calpain-immunized sera, suggesting the possibilities of both cellular and humoral protective mechanisms. We examined tissue distribution of calpain in various developmental stages of S. japonicum. Strong signal was observed around excretory grand of cercariae, and they secreted calpain during their migratory movement tested in vitro. Together with the findings, calpain seems to induce larvicidal effects in the immunized mice. We observed time-course kinetics of antibody production against vaccine candidates in experimental S. japonicum infection in pigs. Although significant levels of antibody production were observed for paramyosin and GST, no significant antibody production was observed for calpain. This suggests that calpain is less immunogenic, and route of immunization and/or choice of adjuvant are important in future trials of calpain vaccine.

Animals↗

PubMed2004

Molecular cloning and characterization of Trypanosoma vivax alternative oxidase (AOX) gene, a target of the trypanocide ascofuranone.

Trypanosoma vivax causes nagana disease in cattle. Since T. vivax is transmitted not only by tsetse flies but also by other biting flies (non-cyclic transmission), the parasite has been distributed to and has had a significant economic impact on wide geographical areas, including Africa and South America. Our previous study on Trypanosoma brucei brucei showed that the trypanosome alternative oxidase (TAO, TbAOX) is a promising target of chemotherapy. For this reason, we also have cloned the T vivax AOX (TvAOX) gene and characterized the recombinant enzyme. The deduced amino acid sequence (328 a.a.) of TvAOX shares 76% identity with TbAOX and contains the diiron-coordination motifs (-E-, -EXXH-) that are conserved among AOXs. The Km of recombinant TvAOX (rTvAOX) expressed in Escherichia coli for ubiquinol (87.0 +/- 0.54 microM) was significantly lower than the value for recombinant TbAOX (rTbAOX) (714 +/- 4.5 microM). Ascofuranone, the most potent inhibitor of TbAOX, was a competitive inhibitor of rTvAOX with a Ki value (0.40 +/- 0.00 nM) significantly lower than that for rTbAOX (1.29 +/- 0.00 nM). The non-cyclic transmission ability of T. vivax and the in vivo chemotherapeutic efficacy of ascofuranone against T. vivax and T. b. brucei infection are discussed in terms of these Km and Ki values.

Amino Acid Sequence↗

PubMed2003

Roles of FcgammaRIIB in nasal eosinophilia and IgE production in murine allergic rhinitis.

The low-affinity IgG Fc receptor, FcgammaRIIB, displays inhibitory potential in experimental models such as autoimmune diseases. However, whether this receptor is involved in the onset of allergic diseases remains unknown. This study examines the role of FcgammaRIIB in the initiation of allergic rhinitis in mice. Repeated intranasal sensitization with Schistosoma mansoni egg antigen (SEA) induced SEA-specific IgE and marked nasal eosinophilia in high-responder BALB/c mice. FcgammaRIIB gene-deficient (-/-) BALB/c mice displayed severe eosinophilia compared with that of wild-type counterparts. However, FcgammaRIIB -/- mice conversely produced less SEA-specific IgE. The production of interleukin (IL)-4 but not of IL-5 or IFN-gamma by nasal mononuclear cells was also decreased in FcgammaRIIB -/- mice, suggesting that the exacerbation of nasal eosinophila in FcgammaRIIB -/- mice is independent of the local IL-5 levels. The findings in low responder C57BL/6 mice were similar. In addition, nasal eosinophilia in FcgammaRIIB -/- mice passively sensitized with SEA was exacerbated, and conversely, specific IgE production was inhibited after a nasal challenge. These results suggest that FcgammaRIIB plays a regulatory role in the initiation of allergic rhinitis that is independent of either mouse strain or type of sensitization.

Animals↗

PubMed2003

Purification of active recombinant trypanosome alternative oxidase.

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain in long slender bloodstream forms of African trypanosomes. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase. These characteristics are distinct from those of the bacterial quinol oxidases, proteins that belong to the heme-copper terminal oxidase superfamily. The inability to purify stable TAO has severely hampered biochemical studies of the alternative oxidase family. In the present study, we were able to purify recombinant TAO to homogeneity from Escherichia coli membranes using the detergent digitonin. Kinetic analysis of the purified TAO revealed that the specific inhibitor ascofuranone is a competitive inhibitor of ubiquinol oxidase activity.

Animals↗

PubMed2003

NADPH-dependent reductases and polyol formation in human leukemia cell lines.

Because of the limited availability of human tissues, leukemia cell lines are often utilized as the models for human leukocytes. In this study, we investigated the NADPH-dependent reductases and polyol pathway in commonly utilized human leukemia cell lines. The relative amounts of aldose and aldehyde reductases were estimated by separating two enzymes with chromatofocusing. The flux of glucose through the polyol pathway was examined by 19F-NMR using 3-fluoro-3-deoxy-D-glucose (3FG) as substrate. Sugar alcohol analysis was conducted by gas chromatography. In myelocytic leukemia cells, the major reductase was aldehyde reductase, and levels of aldose reductase were extremely low. Although lymphocytic cells also contained both aldose and aldehyde reductases, the levels of aldose reductase appeared to be higher in lymphocytic cells than myeolcytic cells. In two lymphocytic cells MOLT-4 and SKW6.4, aldose reductase is clearly dominant. When incubated in medium containing D-galactose, all cell lines quickly accumulated galactitol. There was correlation between galactitol levels and aldose reductase levels. The aldose reductase inhibitor FK 366 significantly reduced the formation of galactitol. 19F-NMR of the cells cultured with 3FG as substrate demonstrated the formation of 3-fluoro-3-dexoy-sorbitol in all the cell lines examined in this study. The relative amounts of sorbitol and fructose varied significantly among the cells. The data confirm that the polyol pathway is present in both myelocytic and lymphocytic leukemia cell lines. However, there is a large variation among the cell lines in the levels of enzymes and flux of glucose through the polyol pathway.

Chromatography, Gas↗

PubMed2003

Adult worms of the rodent intestinal nematode, Strongyloides venezuelensis, successfully invade chick intestinal mucosa.

In order to study the mucosal invasion of a rodent intestinal nematode in bird intestine, chicks were infected with the intestinal nematode of rodents, Strongyloides venezuelensis, by subcutaneous larva inoculation and adult worm implantation. No evidence was obtained for larvae reaching the lungs or the intestine after infective larva inoculation. Adult worms implanted in the small intestine invaded the mucosa and remained there at least for 24 h, whereas those implanted in the caecum were trapped by mucus, and did not invade the mucosa. Mucosal invasion of adult worms in the small intestine was confirmed by histological examination. The number of adult worms in the intestinal mucosal tissue dropped rapidly within the first 24 h, which was associated with infiltrating granulocytes around the worms. The present study suggests that S. venezuelensis adult worms are able to invade the intestinal tissue of chicks, which do not belong to the vertebrate class of its normal definitive host, but that they are eliminated rapidly by mucosal defense system of the bird.

Animals↗

PubMed2003

Intraepithelial infiltration of eosinophils and their contribution to the elimination of adult intestinal nematode, Strongyloides venezuelensis in mice.

Eosinophils were examined for the capacity of attacking Strongyloides venezuelensis adult worms in the intestinal mucosa by using interleukin (IL)-5 transgenic mice. In IL-5 transgenic mice, most of the subcutaneously inoculated infective larvae were killed during migration, and only a few worms could reach the small intestine. When the same number of adult worms were surgically implanted in the small intestine of IL-5 transgenic and control mice, fecal egg output as well as the number of adult worms recovered from the intestine was significantly lower in IL-5 transgenic mice. In the intestinal mucosa of IL-5 transgenic mice, large number of eosinophils was present in the lamina propria even before adult worm implantation. The number of eosinophils increased significantly as early as 24 h after implantation and tripled by day 3, whereas mucosal eosinophilia remained low in wild-type mice. Most notably, eosinophils infiltrated into the intestinal epithelium and surrounded adult worms in IL-5 transgenic mice, which was never seen in wild-type control mice. However, IL-5 transgenic mice required the same period as normal mice to completely expel implanted adult worms. The amount of specific IgA as well as total IgA in the stool was high in IL-5 transgenic mice before adult worm implantation, and dropped rapidly after adult worm implantation. The present study suggests that eosinophils are capable of attacking adult nematodes in the intestinal epithelia, probably in conjunction with secretory IgA, although they are not enough for the complete worm expulsion.

Animals↗

PubMed2003

Overproduction of highly active trypanosome alternative oxidase in Escherichia coli heme-deficient mutant.

Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in humans and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs, because it does not exist in the host. In this study, we established a system for overproduction of highly active TAO in Eschericia coli heme-deficient mutant. Kinetic analysis of recombinant enzyme and TAO in Trypanosoma brucei brucei mitochondria revealed that recombinant TAO retains the properties of native enzyme, indicating that recombinant TAO is quite valuable for further biochemical study of TAO.

Animals↗

PubMed2003

The efficacy of ascofuranone in a consecutive treatment on Trypanosoma brucei brucei in mice.

Consecutive administration of ascofuranone without glycerol was found to have therapeutic efficacy against Trypanosoma brucei brucei infection in mice. A suspension of ascofuranone (25-100 mg/kg) was administrated intraperitoneally every 24 h for 1-4 consecutive days to trypanosome-infected mice and efficacy was compared with oral treatment. With intraperitoneal administration, all mice treated with 100 mg/kg ascofuranone for 4 consecutive days were cured. On contrary, with oral treatment a higher dose of ascofuranone (400 mg/kg) was needed for 8 consecutive days to cure the mice. With intraperitoneal treatment, parasitemia was strongly suppressed, with almost all long slender bloodstream forms of the parasite changed to short stumpy forms by day 3 and the parasites have been eliminated 4 days after the start of treatment. These ascofuranone-induced short stumpy forms were morphologically analogous to the stumpy forms 2 days after peak parasitemia of pleomorphic clone of T. b. brucei GUTat 3.1. However, the properties of ubiquinol oxidase activity, which is the target of ascofuranone, in mitochondria isolated from before and after treatment, were almost same. The enzymatic activities of ubiquinol oxidase were only decreased to approximately 30% within a day after treatment, and then kept at nearly the same level. In the present study, we have improved regimen for administration of ascofuranone without glycerol, and demonstrated that consecutively administrated ascofuranone showed trypanocidal effects in T. b. brucei infected mice. Our present results strongly suggest that consecutive administration of ascofuranone may be an effective chemotherapy for African trypanosomiasis.

Administration, Oral↗

PubMed2003

Historical view of schistosomiasis japonica in Japan: implementation and evaluation of disease-control strategies in Yamanashi Prefecture.

We summarized historical aspects of disease control activities targeting schistosomiasis japonica in Kofu basin, Yamanashi Prefecture, Japan. Kofu Basin was one of the biggest endemic foci of schistosomiasis japonica in Japan, and the last place where transmission of Schistosoma japonicum was confirmed in Japan. Because of the most severe endemic situations in Yamanashi, intensive control measures had been implemented by the central as well as the local government. The last human case in Japan was in 1977, which is just before praziquantel being available. Therefore, the main efforts were focused on snail control. Mass examination and mass chemotherapy were implemented, however, the compliance was not so good because of the severe side effects due to the available therapeutics, Stibnal. Along with socioeconomic development after World War II, big changes in land use, life style, and farming led drastic reduction in the disease prevalence in Kofu Basin in the 1960s. A large amount of budget was also used for disease control. Cementing water canals covered more than 95% of paddy fields in Kofu Basin, and this resulted in ecological changes. After elimination of schistosomiasis, environmental repair is the urgent subject in Kofu Basin. Our experiences in Yamanashi contain both good influences and also a lot of reflection. It is important to evaluate each activity in our history before we give intensive cooperation with countries where endemic foci is still present.

Animals↗

PubMed2003

Treatment of ranula wth intracystic injection of the streptococcal preparation OK-432.

Intracystic injection of OK-432 was developed as a therapy for operatively difficult lymphangioma (cystic hygroma) and is currently becoming a treatment of first choice for this disease. We tried this therapy in 32 patients with ranula (oral floor type in 21 cases and plunging type in 11 cases). Disappearance or marked reduction of the lesion was observed in 31 patients (97%) who had this therapy, and local scarring did not occur in any patient. As side effects, local pain at the injection site and fever (37 degrees C to 39 degrees C) were observed in almost half of the patients who had this therapy, but such problems resolved within a few days. We treated the initial 4 cases in the hospital for 4 to 5 days, but after the safety of this method had been confirmed, we treated the other 28 cases on an outpatient basis. Thus, we confirmed that intracystic injection therapy with OK-432 is relatively safe and can be used as a substitute for surgery in the treatment of ranulas.

Adolescent↗

PubMed2003

Identification of suberization-associated anionic peroxidase as a possible allergenic protein from tomato.

A 45 kDa protein, which is recognized by IgE antibodies in sera of food-allergic patients, was purified and characterized as an allergenic protein from the tomato. The IgE-binding protein purified from tomato extract was found to be a glycoprotein with a molecular weight of approximately 45,000, an isoelectric point of 4.2, and no free N-terminal amino group. Furthermore, it was shown that the purified protein had peroxidase activity. From the amino acid sequence of a peptide fragment prepared by lysylendopeptidase digestion, the allergenic protein was identified to be the tomato suberization-associated anionic peroxidase 1 known as one of the pathogenesis-related proteins widely distributed in plants. These properties suggested the protein isolated from tomato to be a new allergenic protein in plant foodstuffs.

Adolescent↗

PubMed2003

Sensitive enzyme-linked immunosorbent assay with urine samples: a tool for surveillance of schistosomiasis japonica.

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Schistosoma japonicum soluble egg antigens (SEA) in un-concentrated urine was developed. The urine ELISA was applied to samples collected in a schistosomiasis-endemic village in China. The levels of anti-SEA antibodies detected in urine correlated well with those obtained with paired serum samples (r = 0.694, p<0.0001). Among 129 serum ELISA positives, 112 (86.8%) were positive by urine ELISA, while all 40 serum ELISA negatives from a non-endemic area were negative. The levels of anti-SEA in urine samples were stable up to 8 weeks of storage at 37 degrees C, with sodium azide as a preservative. Therefore, ELISA with urine samples can be used for the surveillance of schistosomiasis.

Antibodies, Helminth↗

PubMed2002

Strongyloides venezuelensis: longitudinal distribution of adult worms in the host intestine is influenced by mucosal sulfated carbohydrates.

Mechanisms for the longitudinal distribution of parasitic females of Strongyloides venezuelensis in the host intestine were investigated in mice. Adult worms were mostly recovered from the anterior-most one-third of the small intestine throughout the infection after infective larvae inoculation. Surgically implanted adult worms established well in the small intestinal mucosa, either in the duodenum or in the ileum, whereas a few worms could establish in the large intestine. Implanted worms in the small intestine remained where they were implanted until expelled. Mucosal mast cells were induced in the whole small intestine after the worm implantation. In the large intestine, a considerable number of adult worms settled in the mucosa of mutant mice, whose goblet cell mucins were undersulfated because of a mutation in sulfate-activating enzymes. In these mice, the degree of sulfation of goblet cell mucins in the large intestine was significantly reduced to the level of normal small intestine goblet cell mucins. Our results suggest that sulfated glycoconjugates, either from mucosal mast cells or goblet cells, have important effects on the longitudinal distribution of parasitic females of S. venezuelensis.

Animals↗

PubMed2002

T-cell clonotypes specific for Dermatophagoides pteronyssinus in the skin lesions of patients with atopic dermatitis.

T-cell mediated immune response, toward the house dust mite (HDM) antigens in particular, has been reported to be involved in the pathogenesis of atopic dermatitis (AD). On the other hand, studies on the infiltrating lymphocytes in the skin lesion of AD revealed oligoclonal T-cell accumulation. However, it is not clear exactly what antigen(s) the accumulating T cells are exactly recognize in situ. Therefore, this study attempted to determine whether or not the clonally expanded T-cell clones in the diseased skin recognize HDM. Specifically, peripheral blood mononuclear cells (PBMC) obtained from six patients with AD, who revealed high titers of anti-HDM IgE, were stimulated with HDM antigens purified from Dermatophagoides pteronyssinus (Dp). T-cell clonotypes expanded by the stimulation were then identified by the analysis of their T-cell receptor (TCR) B-gene sequences using a combination of the reverse transcription-polymerase chain reaction and subsequent single strand conformation polymorphism separation. The Dp-responding T-cell clonotypes were compared with those that accumulated in the AD skin lesion in vivo. Nucleotide sequences of the TCR were also determined. As a result, the Dp stimulation induced oligoclonal T-cell expansion from the originally heterogeneous peripheral T-cell population of AD patients. However, only a small part of the Dp-reacting T-cell clonotypes detected in PBMC was identical to those accumulated in the AD skin lesion in vivo, and vice versa. This indicates that the frequency of the clonal expansion of Dp-specific T-cell clonotypes in the skin lesion of AD would be rather limited.

Allergens↗

PubMed2002

Strain-specific difference in amino acid sequences of trypanosome alternative oxidase.

Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in human and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs because it does not exist in the host. The cDNA for TAO has been cloned from Trypanosoma brucei brucei EATRO110 strain and has been used for further characterization. In this study, we found amino acid sequence of the C-terminal part of TAO from the strain that we are using, T. b. brucei TC221, is considerably different from that of the EATRO110 strain.

Amino Acid Sequence↗

PubMed2002

Enhanced UVfemale1 tumor growth in CBF1 mice infected with Schistosoma mansoni due to modulation of Th1-like responses.

Effects of Schistosoma mansoni infection on anti-tumor immunity were examined in CBF1 mice with ultraviolet-induced UVfemale1 fibrosarcoma cells. Although many laboratory established tumor cells had rejection mechanisms independent of CD4(+) T cells, we confirmed that CD4(+) cells had significant roles in rejection of UVfemale1 cells in the syngeneic CBF1 mice. When we prepared two CBF1 mouse groups, S. mansoni-infected and schistosome-free, the former group showed up-regulation of Th2-like response to UVfemale1 cells, whereas the latter group mice showed rather type 1-dominant patterns. Cytotoxic activity against UVfemale1 cells tested in vitro, which was attributed to CD8(+) cells, was significantly weaker in S. mansoni-infected mice compared with infection-free mice. In tumor challenge experiments in vivo, we observed that rapid and complete rejection of UVfemale1 cells required the presence of CD8(+) T cells. Under only CD4-depleted situation, survival of tumor cells in schistosome-free mice was prolonged up to 1 month or more. Under the presence of both CD4(+) and CD8(+) cells, S. mansoni infected mice rejected the challenged UVfemale1 cells as was seen in normal mice. However, when CD8(+) cells were depleted from S. mansoni-infected mice, inoculated UVfemale1 cells grew more rapidly than in infection-free mice. Our results suggest that functionally polarized cytokine patterns in schistosome-infected hosts promote rapid tumor growth.

Animals↗

PubMed2002

Leishmanicidal effect of curcumin in vitro.

From a study to find anti-parasitic agents from natural resources, we found that curcumin showed the cytotoxicity against leishmania in vitro. The LD50 value of this activity was 37.6+/-3.5 microM.

Animals↗