Biomedical subjects

I Igarashi

Publications and source records attributed to I Igarashi.

At least 37 records · Page 2Linked to original sources

PubMed2001

Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis.

A cDNA expression library prepared from mRNA of Haemaphysalis longicornis (H. longicornis) was screened with a H. longicornis-infested rabbit serum. A cDNA encoding 27/30kDa proteins was cloned and designated P27/30 gene. The predicted amino acid sequence of the P27/30 gene shows a rather high homology (58% amino acid identities and 11% amino acid similarity) with Drosophila melanogaster troponin I clone E2. H. longicornis P27/30 possesses amino acid sequence of actin-binding domains of troponin I at the amino acid residues 128-148, suggesting that H. longicornis P27/30 is a troponin I-like protein. By immunoblot analysis, mouse anti-recombinant P27/30 serum reacted with major constituent protein bands in extracts of adult ticks, and also immunoreacted with muscle, cuticle, gut, and salivary gland in H. longicornis ticks. Moreover, immunohistochemistry using the anti-P27/30 serum showed a strong reactivity in muscle, suggesting that native P27/30 is expressed abundantly in that tissue.

Amino Acid Sequence↗

PubMed2001

Interferon-gamma-induced apoptosis in host cells infected with Neospora caninum.

Interferon-gamma (IFN-gamma) has a crucial role for host defence against parasite infection. It is not clear, however, how IFN-gamma affects the parasite-infected host cells. The effect of IFN-gamma on Neospora caninum-infected cells was investigated in murine fibroblasts and canine kidney cells in vitro. In the presence of IFN-gamma, the viability of the infected host cell was decreased and apoptotic cell death occurred, as analysed by DNA stainings with propidium iodide and a terminal deoxy-nucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) and DNA fragmentation. The percentage of apoptotic cells depended on the dose of IFN-gamma. Flow cytometric analysis indicated a significant increase of FasL expression on the IFN-gamma treated cells following N. caninum infection. Moreover, IFN-gamma treatment down-regulated Bcl-2 expression in the cells cultured with N. caninum while parasite infection up-regulated Bcl-2 expression. The present study suggests that the IFN-gamma induced increases of FasL expression and down-regulated Bcl-2 expression in N. caninum-infected cells are associated with apoptosis in vitro.

3T3 Cells↗

PubMed2001

Detection of antibodies to Babesia equi in horses by a latex agglutination test using recombinant EMA-1.

A latex agglutination test (LAT) using recombinant equi merozoite antigen 1 (EMA-1) for the detection of antibodies to Babesia equi was developed. The LAT was able to differentiate very clearly between sera from B. equi-infected horses and sera from Babesia caballi-infected horses or from normal horses. The LAT results were identical to those of a previously developed enzyme-linked immunosorbent assay. These results indicate that LAT using recombinant EMA-1 might be very useful as a routine screening method for the diagnosis of B. equi infection.

Animals↗

PubMed2001

Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.

The gene encoding the entire Babesia equi merozoite antigen 1 (EMA-1) was inserted into a baculovirus transfer vector, and a recombinant virus expressing EMA-1 was isolated. The expressed EMA-1 was transported to the surface of infected insect cells, as judged by an indirect fluorescent-antibody test (IFAT). The expressed EMA-1 was also secreted into the supernatant of a cell culture infected with recombinant baculovirus. Both intracellular and extracellular EMA-1 reacted with a specific antibody in Western blots. The expressed EMA-1 had an apparent molecular mass of 34 kDa that was identical to that of native EMA-1. The secreted EMA-1 was used as an antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA differentiated B. equi-infected horse sera from Babesia caballi-infected horse sera or normal horse sera. The ELISA was more sensitive than the complement fixation test and IFAT. These results demonstrated that the recombinant EMA-1 expressed in insect cells might be a useful diagnostic reagent for detection of antibodies to B. equi.

Animals↗

PubMed2001

Identification and expression of a 50-kilodalton surface antigen of Babesia gibsoni and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.

A cDNA expression library prepared from Babesia gibsoni merozoite mRNA was screened with B. gibsoni-infected dog serum. cDNA encoding a 50-kDa protein was cloned and designated the P50 gene. The complete nucleotide sequence of the P50 gene was 1,922 bp. Computer analysis suggested that the sequence of the P50 gene contained an open reading frame of 1,401 bp with a coding capacity of approximately 50 kDa. The complete genomic nucleotide sequence of the P50 gene has been analyzed and shown to contain a single intron of 37 bp. Southern blotting analysis indicated that the P50 gene was present at a single copy in the B. gibsoni genome. The native P50 protein of B. gibsoni with a molecular mass of 50 kDa was identified by Western blotting with anti-recombinant P50 mouse serum. Confocal laser microscopic analysis showed that the P50 protein was located on the surface of B. gibsoni merozoites. The recombinant P50 protein expressed by baculovirus in insect cells was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Furthermore, the antibody response against the recombinant P50 protein was maintained until the chronic stage of infection in dogs experimentally infected with B. gibsoni was developed. These results demonstrate that the recombinant P50 protein might be a useful diagnostic reagent for detection of antibodies to B. gibsoni in dogs.

Amino Acid Sequence↗

PubMed2001

Diagnosis of equine piroplasmosis in Brazil by serodiagnostic methods with recombinant antigens.

Serum samples from horses in the States of Sao Paulo and Mato Grosso do Sul, Brazil were examined for diagnosis of equine piroplasmosis by both the latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens. Of the 47 samples analyzed, 38 (81%) and 42 (90%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 35 (75%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in the States of Sao Paulo and Mato Grosso do Sul, Brazil.

Animals↗

PubMed2001

Comparison of the accessory activity of murine peritoneal cavity macrophage derived dendritic cells and peritoneal cavity macrophages in a mixed lymphocyte reaction.

A detailed comparison of the accessory cell activities was carried out among murine peritoneal cavity macrophages (PEC-Mphi), peritonea] cavity macrophages stimulated with granulocyte-macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4), the most popular cytokine combination widely used to generate dendritic cells (DC) and peritoneal cavity macrophage-derived DC (PEC-DC) using a two-way mixed lymphocyte reaction (MLR). All the cell types used efficiently induced statistically significant naïve T cell proliferation at all culture time points and responder:stimulator ratios used. However, marked differences were noted in the magnitude of the proliferative responses. These variations may be attributed to the intensity of expression of MHC class II glycoproteins, as well as the actual numbers of MHC class II+ cells.

Animals↗

PubMed2001

Detection of Cryptosporidium parvum oocysts in environmental water in Hokkaido, Japan.

Control of cryptosporidiosis is important in public health. Rivers that are polluted with Cryptosporidium and drinking water that is treated for drinking water production from polluted rivers could result in the waterborne disease of cryptosporidiosis. We carried out an epidemiological study of natural water supplies in Hokkaido, one of the largest dairy prefectures in Japan. To detect Cryptosporidium oocysts in environmental water, the filtration method was used for 28 samples, which were collected from 10 rivers. A method adapted from the United States Environmental Protection Agency (U.S. EPA) filtration method using a cartridge filter has been used for the collection of samples. Oocysts were separated from a pellet by discontinuous sucrose gradient method. Twelve samples were collected from 10 rivers and parasites were purified by iron (III) flocculation method. Cryptosporidium parvum oocysts were identified with the immunofluorescence antibody technique using DIF kit (Cellabs Pty. Ltd., Sydney/Australia). We detected Cryptosporidium oocysts in 6 out of 10 rivers sampled. Fifty percentage (14/28) of the samples were Cryptosporidium-positive. The average number of Cryptosporidium oocysts was 16.73/100 L (max. 80/100 L).

Animals↗

PubMed2001

Ontogeny of pig discrete Peyer's patches: expression of surface antigens.

Leukocyte populations present in the discrete Peyer's patches (PP) of the pig were characterized from birth (Day 0) to day 35 after birth by immunohistochemistry and image analysis. Immediately after birth, cell membrane expression of CD2 and CD3, major histocompatibilty complex (MHC) class 11 (both SLA (swine leukocyte antigen) -DQ+ and SLA-DR+), CD21, 74-22-15 and surface immunoglobulin (sIg) were all demonstrable. Computer assisted morphometric techniques were used to confirm the significant expansion of these cell populations from birth onwards. The distribution of the cell types was not random but suggested a preferential retention of cells at specific sites. This implies a degree of organization of immunological cells within the discrete PP, enhancing the potential to mount immune responses in the most efficient manner.

Animals↗

PubMed2001

Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs.

A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.

Animals↗

PubMed2001

Characterization of phagocytic hemocytes in Ornithodoros moubata (Acari: Ixodidae).

Effects of fetal bovine serum (FBS) and complement on phagocytic activity in Ornithodaros moubata (Murray 1877) hemocytes and protease activity in the hemocytes were examined. At least three morphologically different cell types, granulocytes, plasmatocytes, and prohemocytes, were detected in hemolymph of O. moubata, and granulocytes and plasmatocytes showed phagocytic activity. FBS altered phagocytic activity of granulocytes, and complement affected phagocytic activity of plasmatocytes. Ticks were inoculated with fluorescent polystyrene beads in combination with FBS or complement. The average number of beads in granulocytes was significantly higher in the FBS injected group than the control (P < 0.01). The percentage of bead-ingesting plasmatocytes in complement inoculated ticks was significantly lower than that in heat-inactivated complement inoculated and control ticks (P < 0.05). Proteases of tick hemocytes localized in small granules in the cytoplasm not only in phagocytic hemocytes but also in prohemocytes. Results suggested modulation of tick hemocyte function through serum components, and digestion of phagocytosed foreign bodies in the hemocytes.

Animals↗

PubMed2001

Analysis of a growth-promoting factor for Babesia caballi cultivation.

Serum-free media were examined to culture Babesia caballi. Daigo's T (DT) basal medium supplemented with Daigo's GF21 (GF21) or GIT medium, which already contains GF21, supported the parasite propagation at 37 C in a humidified atmosphere under 5% CO2 in air. Growth of B. caballi was dependent of the suitable concentration (10-20%) of GF21. Therefore, GF21 was suggested as the growth-promoting factor for B. caballi. However, GIT medium did not support the growth of parasites from cryopreserved stabilates, and serum supplementation was essential for the retrieval of parasites.

Animals↗

PubMed2001

[A survey of awareness among new patients at the department of oral diagnosis and general dentistry].

The purpose of this study is to know the outline of clinical statistics of new patients at the Clinic for Initial Diagnosis/Emergency, Dental Hospital, Tokyo Medical and Dental University. We examined 1,001 new patients who visited our hospital from 19 October to 8 December 1995. The results were obtained as follows: 1. Our subjects were 1,001 patients, males were 357(35.7%) and females 644(64.3%). The ratio of male to female was 1:1.8. 2. Concerning age distribution, the majorities were in their twenties and fifties in order. 3. 71.5% of patients said it took less than one hour for them to come to our hospital. 4. The rate of introduced patients was 5.5% of all the new patients.

Adult↗

PubMed2000

Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein.

The 48-kDa Babesia caballi merozoite rhoptry protein was expressed using a pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion protein (GST-BC48), and the expressed GST-BC48 was used in an ELISA to detect specific antibodies in serum samples. No cross-reaction was observed with sera from horses experimentally infected with Babesia equi. GST-BC48 ELISA was a highly sensitive and specific test when compared with the CFT. A total of 209 horse sera obtained from Central Mongolia were examined with the GST-BC48 ELISA and 46.4% (97/209) were found to be seropositive for B. caballi, suggesting that the GST-BC48 ELISA can be successfully used for both quarantine and epidemiological studies.

Animals↗

PubMed2000

Delivery of Neospora caninum surface protein, NcSRS2 (Nc-p43), to mouse using recombinant vaccinia virus.

In order to develop a vaccine against Neospora caninum in dogs and cattle, we constructed a recombinant vaccinia virus expressing the N. caninum surface protein, NcSRS2 (Nc-p43). Monoclonal antibodies to NcSRS2 and anti-N. caninum tachyzoite mouse serum recognized the NcSRS2 expressed by the recombinant vaccinia virus. In addition, recombinant NcSRS2 was transported to the cell surface. Mice infected with the recombinant virus predominantly produced IgG1 antibody (Ab) to N. caninum, rather than producing IgG2a Ab. Moreover, splenocytes from mice infected with the recombinant virus proliferated in the presence of the N. caninum antigen. Mice immunized with the recombinant virus gave rise to humoral and cellular immune responses to N. caninum tachyzoites. This study showed that a recombinant vaccinia virus expressing NcSRS2 might be useful for the production of a live vaccine against N. caninum infection.

Animals↗

PubMed2000

Expression of SAG-1 of Toxoplasma gondii in transgenic mice.

We describe the expression of SAG-1 cDNA in B6C3F1 mice by microinjecting a 3.3 kbp DNA fragment, consisting of the cytomegalovirus enhancer-chicken beta-actin hybrid promoter and SAG-1 into the pronucleus of a fertilized egg at the one-cell stage. Offspring derived from this microinjection were analyzed for the integration and functional expression of the SAG-1 transgene. Steady-state expressions of both the mRNA for SAG-1 and SAG-1 protein product were detected in the brain, thymus, spleen and liver. Approximately 50% of F1 and F2 progeny inherited the SAG-1 transgene from SAG-1 transgenic mice in Mendelian fashion. These results indicated that SAG-1 transgenic lines were established. Transgenic mice harboring the SAG-1 gene will contribute a critical tool of defining the molecular mechanisms of SAG-1 in pathogenesis and host immune response.

3T3 Cells↗

PubMed2000

Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells.

Monoclonal antibodies were produced against Neospora caninum tachyzoites to identify antigens which may play a role during invasion of host cells. Confocal laser microscopy showed that most antigens recognised by the mAb were located on the surface, but one mAb, 1A5, reacted to the apical end of the parasite. Some mAbs, which recognised 70, 42 and 36kDa parasite proteins, significantly inhibited the invasion of the parasite in vitro. The mAbs which recognised 42 and 36kDa parasite protein, reacted with Nc-p43 and Nc-p36 expressed by vaccinia virus and Escherichia coli, respectively. These results suggest that a 70kDa protein, Nc-p43 and Nc-p36 are involved in the invasion of the parasite into host cells.

Animals↗

PubMed2000

Ontogeny of pig discrete Peyer's patches: distribution and morphometric analysis.

We investigated the development of lymphoid and non-lymphoid cells in discrete Peyer's patches (PP) of the pig using immuno-histology and image analysis. In newborn piglets discrete PP were mainly populated by CD2+, CD3+ T cells, and major histocompatibility complex class II+ cells, many of which were of macrophage and dendritic cell lineage. Four days after birth, cells were localized in defined regions: the follicle; the inter-follicular area and the dome region. Compartmentalization within the follicle started about 6 days after birth. The first signs of secondary follicles were seen from about 14 days. The pig discrete PP attained their mature structure at about 3 weeks after birth. Here we show that despite the demonstration at birth of the cell types that support antigen processing and presentation, PP did not fully differentiate morphologically until at least this time when antigen can be handled in an efficient manner.

Animals↗