Biomedical subjects

H Tsujimoto

Publications and source records attributed to H Tsujimoto.

At least 73 records · Page 4Linked to original sources

PubMed2000

Induction of innate immunity by nasal influenza vaccine administered in combination with an adjuvant (cholera toxin).

Inactivated influenza vaccine was administered intranasally to BALB/c mice together with an adjuvant (cholera toxin B subunit [CTB] supplemented with a trace amount of the whole toxin, CTB*) and its ability to induce innate immunity and confer protection against influenza was examined. Nasal wash virus titres 3 days after inoculation of homologous viruses were measured as an index of the ability of the vaccine to confer protection in mice immunized with either CTB*-combined vaccine or CTB* alone 1-21 days previously. The results were as follows. (1) Partial but significant reduction of the nasal-wash virus titres (prevention) was detected beginning 3 days after the vaccination, that is, 2 days earlier than the appearance of both virus-specific antibody-forming cells (AFCs) in the nasal-associated lymphoid tissue (NALT) and virus-specific IgA antibody responses in the nasal washes of mice immunized with the CTB*-combined vaccine. (2) The protection, detected on day 3 and peaking on day 5 but lost by day 21, was also conferred in mice immunized with CTB* alone. (3) The non-specific prevention was detected at doses of more than 0.3 microg of CTB*/mouse. (4) The nonspecific protection beginning 3 days after the immunization involved the enhanced expression of cytokine mRNAs (IL-15 and IL-18), considered responsible for natural killer (NK) cell activation, by the non-T cell populations in the NALT. (5) Normal NALT cells, when cultured in vitro with CTB*, secreted IL-1beta within a few hours in culture. These results demonstrate that the CTB*-combined vaccine, when given intranasally into mice, can confer nonspecific protection against influenza beginning 3 days after the vaccination and that CTB* also possessed this ability to confer protection non-specifically and temporarily by inducing the secretion of IL-1beta, one of the most important cytokines that initiates both innate and adaptive immunity, and also NK cell activity.

Adjuvants, Immunologic↗

PubMed2000

IgE-reactivity to major Japanese cedar (Cryptomeria japonica) pollen allergens (Cry j 1 and Cry j 2) by ELISA in dogs with atopic dermatitis.

The present study investigated IgE-reactivity to two major Japanese cedar (Cryptomeria japonica, C. japonica) pollen allergens (Cry j 1 and Cry j 2) in dogs with atopic dermatitis by use of a fluorometric ELISA. The serum samples from 27 dogs that showed IgE-sensitivity to crude C. japonica pollen allergen by ELISA were tested for specific IgE to the two major allergens. All 27 dogs had anti-Cry j 1 IgE, and 10 (37%) had anti-Cry j 2 IgE. Inhibition of binding of dog specific IgE to crude C. japonica pollen allergen was carried out by addition of Cry j 1. When serum samples containing anti-Cry j 1 IgE but no anti-Cry j 2 IgE were incubated with Cry j 1, specific IgE binding to crude C. japonica pollen allergen was almost abolished. These findings suggest that Cry j 1 is a major allergen in dogs.

Animals↗

PubMed2000

Inhibitory effect of stromal cell derived factor-1 on the replication of divergent strains of feline immunodeficiency virus in a feline T-lymphoid cell line.

The effect of a CXC-chemokine, stromal cell derived factor-1 (SDF-1), on the replication of divergent strains of feline immunodeficiency virus (FIV) was examined in order to identify the mechanism of cell entry of FIV. A chemotaxis assay, using a modified Boyden chamber method, confirmed the biological activity of recombinant human (rh) SDF-1 for a feline T-lymphoid cell line (Kumi-1). The viral replication of FIV, as measured by the reverse transcriptase (RT) activity in the culture supernatant, was significantly suppressed by addition of rhSDF-1 in a dose-dependent manner in Kumi-1 cells. Furthermore, PCR analysis of the FIV proviral genome indicated that the inhibitory effect of rhSDF-1 on the replication of FIV in Kumi-1 cells was due to the inhibitory effect in the early event of replication. The inhibitory effect on viral replication by exogenous rhSDF-1 was shown for four divergent FIV isolates of subtypes A, B, and D in Kumi-1 cells.

Animals↗

PubMed2000

Positive reactions to common allergens in 42 atopic dogs in Japan.

Clinically important allergens for the diagnosis and treatment of atopic dermatitis vary geographically. In order to identify the most prevalent allergens in atopic dogs in Japan, 42 dogs with a clinical diagnosis of atopy were tested using both in vivo (intradermal skin test (IDST)) and in vitro (antigen-specific IgE assay) allergy tests. Allergens used for IDST included 26 allergen extracts from eight allergen groups: trees, weeds, grasses, house dust mites (HDM), molds, foods, epithelia, and arthropods. Immunodot assay was used to measure antigen-specific IgE against 24 allergens from these eight groups and against fish such as cod and sole. In the 42 dogs, the most common positive allergen reaction was to HDM on both IDST (29/42 dogs or 69%) and in vitro testing (23/42 or 54.8%). The second most frequent positive allergen reaction was to Japanese cedar pollen (21/42 or 50.0% for IDST and 7/42 or 16.7% for in vitro testing). In both tests, less than 20% of dogs had positive reactions to molds or foods. Positive reactions to cat epithelia were frequently found on IDST, but rarely found on in vitro testing. Agreement between the two tests was found in 26 instances: HDM (21 dogs), Japanese cedar pollen (five dogs) and wheat (one dog). In this study, the two most common allergens involved in atopic dermatitis in dogs in Japan were HDM and Japanese cedar pollen.

Allergens↗

PubMed2000

Cytokine mRNAs in the nasal-associated lymphoid tissue during influenza virus infection and nasal vaccination.

Intranasal immunization with a current inactivated influenza vaccine together with an adjuvant (cholera toxin B subunit supplemented with a trace amount of whole toxin, CTB*) was confirmed in BALB/c mice to mimic influenza virus (A/PR/8/34, H1N1) infection with respect to mucosal IgA antibody responses, in which IgA antibody-forming cell responses in the nasal-associated lymphoid tissue (NALT) were involved with a peak around 7 days after infection or vaccination. Next, the expression of various cytokine mRNAs in the NALT was compared in mice either infected with viruses or immunized with CTB*-combined vaccine, to examine Th cell and cytokine regulation of mucosal IgA antibody responses. In infected mice, strong IL-2, weak IL-4, strong IL-6 and strong IFN-gamma mRNA expressions were induced during early days of infection; especially, IFN-gamma mRNA was expressed by both CD4(+) and CD8(+) T cells around 7 days after infection. In mice given CTB*-combined vaccine, weak IL-2, strong IL-4, strong IL-6 and weak IFN-gamma mRNA expressions were induced during early days of vaccination; especially, IL-4 mRNA was expressed by CD4(+) T cells. Thus, IL-6 mRNAs were expressed strongly in both infected and vaccinated mice. The IFN-gamma-rich cytokine mRNA profiles in the infected mice were reflected upon serum IgG2a-rich Ab responses, while the IL-4-rich profiles in the vaccinated mice were reflected upon the IgG1-rich Ab responses. Thus, influenza virus infection and CTB*-combined nasal vaccine induced Th1 dominant and Th2 dominant cytokine profiles, respectively, while the similarity of mucosal IgA antibody responses between infection and vaccination could be explained by the appearance of IL-6 mRNAs.

Administration, Intranasal↗

PubMed2000

Activation of monocytes and endothelial cells depends on the severity of surgical stress.

Surgical injury not only induces a systemic endocrine-metabolic response but also influences the function of the leukocytes and endothelial cells leading to various systemic responses. These responses appear to depend on the severity of surgical stress, which differs according to the surgical procedures. In this study, we investigated the response of monocytes and endothelial cells, and the development of systemic inflammatory response syndrome (SIRS) in relation to the severity of surgical stress. The postoperative clinical course was evaluated between patients undergoing an esophagectomy (ER group) and a distal gastrectomy (DG group). The tumor necrosis factor alpha (TNF-alpha) production of monocytes, the serum interleukin 6 (IL-6) levels, the CD11b expression on either monocytes or granulocytes, and the intercellular adhesion molecule-1 (ICAM-1) expression on human umbilical vein endothelial cells (HUVECs) stimulated with culture supernatants of monocytes were compared between the 2 groups. The development of SIRS was observed in all patients in the ER group, whereas no patients demonstrated SIRS in the DG group. The serum IL-6 levels, TNF-alpha production of monocytes, and CD11b intensity on monocytes or granulocytes in the ER group were higher than those in the DG group. In the ER group, the ICAM-1 intensity on HUVECs with monocytes immediately after operation significantly increased compared with before the operation. In conclusion, both the CD11b expression on monocytes and the TNF-alpha production of monocytes are considered to reflect the degree of surgical stress, and the activation of endothelial cells stimulated with these activated leukocytes may therefore lead to both tissue and organ injury.

Adenocarcinoma↗

PubMed2000

Increased levels of circulating soluble CD14 in Kawasaki disease.

The CD14 molecule, which is known to be a receptor for endotoxin, is expressed on monocytes and neutrophils. It is found as a soluble CD14 (sCD14) in circulation, and the plasma level has been shown to be increased in some infectious diseases, including sepsis. To investigate the potential significance of circulating sCD14 in Kawasaki disease (KD), the plasma level of sCD14 was measured using ELISA in patients with KD, patients with a Gram-negative bacterial infection (GNBI) including sepsis, patients with viral infection (VI), and healthy controls. We also analysed CD14 receptor expression in monocytes and neutrophils using flow cytometry and a semiquantitative reverse transcription-polymerase chain reaction. Although KD patients had significantly lower counts of peripheral neutrophils and monocytes than GNBI patients, KD patients had significantly higher levels of sCD14 than GNBI. No significant correlations were observed between sCD14 levels and clinical laboratory values or the cytokine (interferon-gamma, tumour necrosis factor-alpha) levels in the acute phase. The mean intensity of CD14 receptor expression on neutrophils markedly increased in the acute phases of KD and GNBI compared with that in their convalescent phases, while that on monocytes decreased. The expression of CD14 mRNA in neutrophils increased in the acute phases of KD and GNBI, while that in monocytes did not decrease but instead remained quite abundant. The present findings suggest that the elevated level of circulating sCD14 appears to be an important parameter for KD and that sCD14 shedding is accompanied by different kinetics regarding the expression of CD14 antigen and CD14 gene between monocytes and neutrophils.

Adult↗

PubMed2000

Characterization of the cyclophilin of Trichophyton mentagrophytes.

A genetic approach to cyclophilins in a dermatophyte, Trichophyton mentagrophytes, was carried out. The nucleotide and deduced amino acid sequences of the cyclophilin of T. mentagrophytes shared about 70% sequence similarity with those of Schizosaccharomyces pombe, Saccharomyces cerevisiae and Candida albicans. However, the first 21 amino acid and the C-terminal amino acid regions of 188 to 226 of the T. mentagrophytes cyclophilin were distinct from those of the other fungal cyclophilins. The recombinant glutathione S-transferase (GST)-T. mentagrophytes cyclophilin fusion protein produced by Escherichia coli was purified. The protease digest of the fusion protein had a molecular weight of about 13 kDa and peptidyl-prolyl cis-trans isomerase (PPI) activity. This digest protein from T. mentagrophytes was confirmed to be cyclophilin by proving PPI activity.

Amino Acid Sequence↗

PubMed2000

Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-oprM efflux pumps in Pseudomonas aeruginosa.

To find the exact substrate specificities of three species of tripartite efflux systems of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems. Comparison of the susceptibilities of the constructed mutants to 52 antimicrobial agents belonging to various groups suggested the following substrate specificities. All of the efflux systems extrude a wide variety of antimicrobial agent groups, i.e., quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (all but carbenicillin and sulbenicillin), most cephems (all but cefsulodin and ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin B or imipenem. Extrusion of aminoglycosides is specific to MexXY-OprM, and extrusion of a group of the beta-lactams, i.e., carbenicillin, sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to MexAB-OprM. Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin, cefsulodin, and flomoxef, while MexXY-OprM does not. These substrate specificities are distinct from those reported previously.

Bacterial Outer Membrane Proteins↗

PubMed2000

Contribution of the MexX-MexY-oprM efflux system to intrinsic resistance in Pseudomonas aeruginosa.

To test the possibility that MexX-MexY, a new set of efflux system components, is associated with OprM and contributes to intrinsic resistance in Pseudomonas aeruginosa, we constructed a series of isogenic mutants lacking mexXY and/or mexAB and/or oprM from a laboratory strain PAO1, and examined their susceptibilities to ofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin. Loss of either MexXY or OprM from the MexAB-deficient mutant increased susceptibility to all agents tested, whereas loss of MexXY from the MexAB-OprM-deficient mutant caused no change in susceptibility. Introduction of an OprM expression plasmid decreased the susceptibility of the mexAB-oprM-deficient-/mexXY-maintaining mutant, yet caused no change in the susceptibility of a mexAB-oprM- and mexXY-deficient double mutant. Immunoblot analysis using anti-MexX polyclonal rabbit serum generated against synthetic oligopeptides detected expression of MexX in the PAO1 cells grown in medium containing tetracycline, erythromycin, or gentamicin, although expression of MexX was undetectable in the cells incubated in medium without any agent. These results suggest that MexXY induced by these agents is functionally associated with spontaneously expressed OprM and contributes to the intrinsic resistance to these agents.

Anti-Bacterial Agents↗

PubMed2000

Clonality analysis of various hematopoietic disorders in cats naturally infected with feline leukemia virus.

The clonality analysis of the bone marrow cells was carried out by detecting the integrated proviruses of feline leukemia virus (FeLV) to understand the pathogenesis of FeLV-associated hematopoietic disorders in cats. Bone marrow cells from 4 cases with acute myeloid leukemia (AML), 9 cases with myelodysplastic syndromes (MDS), 2 cases with pure red cell aplasia (PRCA) and 3 healthy carriers infected with FeLV were subjected to Southern blot analyses using an exogenous FeLV probe. Clonal hematopoiesis was found in all the cases with AML and in 6 of the 9 cases with MDS, but not in the cases with both PRCA and healthy carriers infected with FeLV. In the 2 cases with MDS, it was thought that the same clones of the hematopoietic cells might proliferate before and after the progression of the disease irrespective of the changes of the hematological diagnoses by cytological examination. This study indicates that MDS in cats is a disease manifestation as a result of clonal proliferation of hematopoietic cells and can be recognized as a pre-leukemic state of AML.

Animals↗

PubMed2000

Experimental sensitization with Japanese cedar pollen in dogs.

Japanese cedar pollinosis is a type I allergic disease mediated by immunoglobulin E (IgE) antibodies to Japanese cedar (Cryptomeria japonica) pollen antigen (CPAg). By using 22 dogs consisting of 20 dogs aged 3 months and 2 dogs aged 3 years, immunization was performed by subcutaneous injections of CPAg with aluminum hydroxide gel. Variable levels of CPAg-specific IgE antibody response were detected in 21 of the 22 immunized dogs two weeks after the second immunization. This study provided an experimental sensitization system with CPAg in dogs, which will be useful for further immunological studies on Japanese cedar pollinosis.

Animals↗

PubMed2000

In vivo and in vitro tests showing sensitization to Japanese cedar (Cryptomeria japonica) pollen allergen in atopic dogs.

Using both in vivo and in vitro tests, dogs with atopic dermatitis were examined for sensitization with Japanese cedar (Cryptomeria japonica, CJ) pollen allergen. Ten dogs with clinical manifestation of atopic dermatitis were shown to be sensitized to CJ pollen based on the results of intradermal skin test and serum antigen-specific IgE test. In vitro lymphocyte stimulation test showed blastogenic response after stimulation with crude antigen of CJ pollen in all of the 5 cases examined. The peripheral leukocytes showed increased histamine release after stimulation with crude antigen of CJ pollen in 2 cases examined. These data indicate that a proportion of dogs with atopic dermatitis is sensitized to CJ pollen in a cell-mediated manner and show immediate phase reaction of type I hypersensitivity.

Allergens↗

PubMed2000

Molecular analysis of multidrug resistance in feline lymphoma cells.

OBJECTIVE: To evaluate the mechanism of multidrug resistance in feline lymphoma cell lines. SAMPLE POPULATION: A feline lymphoma cell line (FT-1) and its adriamycin (ADM)-resistant subline (FT-1/ADM). PROCEDURES: The FT-1 cell line was cultivated in the presence of a gradually increasing concentration of ADM to generate its ADM-resistant subline (FT-1/ADM). Susceptibility of cells from the parental FT-1 cell line and the FT-1/ADM subline to antineoplastic drugs was determined. From the complementary DNA (cDNA) template of FT-1/ADM cells, feline MDR1 cDNA was amplified by use of polymerase chain reaction (PCR) and sequenced. Reverse transcription (RT)-PCR and Western blot analyses were performed to assess expression of the MDR1 gene and P-glycoprotein (P-gp) in FT-1/ADM cells, compared with that in FT-1 cells. RESULTS: A drug sensitivity assay revealed that FT-1/ADM cells were much more resistant to ADM and vincristine than the parental FT-1 cells. The feline MDR7 cDNA amplified by use of PCR was 3,489 base pairs long, corresponding to approximately 90% of the whole open reading frame of human MDR1 cDNA; its amino acid sequence was 91.5, 87.0, and 79.4% identical to that of human MDR1, mouse mdr1a, and mdr1b cDNA, respectively. By RT-PCR analysis, expression of MDR1 messenger RNA was clearly detected in FT-1/ADM cells but not in the parental FT-1 cells. Western blot analysis also revealed the expression of P-gp encoded by the MDR1 gene in FT-1/ADM cells but not in FT-1 cells. CONCLUSIONS: The basic structure of the feline MDR1 gene was essentially the same as that of multidrug-resistance genes of other species. Expression of P-gp appeared to be one of the mechanisms responsible for the development of multidrug resistance in feline lymphoma cell lines in vitro.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Quantification of viral ribonucleic acid in plasma of cats naturally infected with feline immunodeficiency virus.

OBJECTIVE: To assess plasma viral RNA concentration in cats naturally infected with feline immunodeficiency virus (FIV). ANIMALS: 28 FIV-infected cats. PROCEDURE: Cats were categorized into 1 of the 3 following stages on the basis of clinical signs: asymptomatic (nonclinical) carrier (AC; n = 11), acquired immunodeficiency syndrome-related complex (ARC; 9), or acquired immunodeficiency syndrome (AIDS; 8). Concentration of viral RNA in plasma (copies per ml) was determined by use of a quantitative competitive polymerase chain reaction (QC-PCR) assay. Total lymphocyte count, CD4+ cell and CD8+ cell counts, and the CD4+ cell count-to-CD8+ cell count ratio were determined by use of flow cytometry. RESULTS: Plasma viral RNA concentration was significantly higher in cats in the AIDS stage, compared with cats in AC and ARC stages. Most (5/7) cats in the AIDS stage had low total lymphocyte, CD4+ cell, and CD8+ cell counts. CONCLUSIONS AND CLINICAL RELEVANCE: Concentration of plasma viral RNA is a good indicator of disease progression in FIV-infected cats, particularly as cats progress from the ARC to the AIDS stage. Determination of CD4+ and CD8+ cell counts can be used as supportive indicators of disease progression.

Animals↗

PubMed2000

Epitope analysis and utility of monoclonal antibodies to native and recombinant human thymidylate synthase.

The expression of thymidylate synthase (TS) in human cancer tissues has been suggested to be a prognostic factor for patients receiving 5-fluorouracil-based chemotherapy. We generated monoclonal antibodies to both recombinant and native TS and analyzed the epitopes on the TS molecule. Two monoclonal antibodies were obtained from recombinant human TS proteins (RTSMA1 and RTSMA2) and two monoclonal antibodies raised to native TS in human cancers (NTSMA1 and NTSMA2) were obtained, and were found to have a high affinity and specificity for TS proteins. To identify the human TS epitope that these monoclonal antibodies recognized, we constructed plasmids to produce full-length and two partially deleted TS proteins fused to glutathion-S-transferase (GST). Western blot analysis showed that RTSMA1 and 2 only reacted with full-length TS and NTSMA1 and 2 reacted with all three recombinant TS proteins, suggesting that the epitopes of the former were located at C-terminal sites (D186-V313), and those of the latter were at N-terminal sites (M1-M61). The RTSMA 1 and 2 epitopes were more accurately mapped using 8 oligopeptides to the region of I267 to L282 which was situated at the surface of the TS protein. Highly sensitive detection of human TS was also possible by sandwich ELISA using a combination of different types of antibody rather than a single type. In conclusion, different type monoclonal antibodies to human TS protein may contribute to the detection of TS in cancer patients.

Amino Acid Sequence↗

PubMed2000

Different mechanisms of acquired resistance to fluorinated pyrimidines in human colorectal cancer cells.

5-Fluorouracil (5-FU), 5-fluoro-2'-deoxyuridine (FdUrd) and 5-trifluorothymidine (F3(d)Thd) are antimetabolites which are metabolized to their corresponding active forms which inhibit DNA synthesis via inhibition of thymidylate synthase (TS). To investigate ways of overcoming 5-FU-resistance, we established acquired-resistant colorectal cancer cell lines against these three drugs by continuous and step-wise escalation of drugs, and analyzed the cytotoxicity and the mechanism of resistance to the drugs. When cells were incubated with the 3 drugs for 72 h, the resistance ratio to parental DLD-1 human colorectal tumor cells was 65.2 for DLD-1/5-FU, 9.7 for DLD-1/FdUrd and 448.6 for DLD-1/F3(d)Thd cells. DLD-1/5-FU cells did not show any cross-resistance against FdUrd and F(3)dThd. However, DLD-1/FdUrd cells showed 3- and 9-fold increased resistance to 5-FU and F3(d)Thd, respectively, and DLD-1/F3(d)Thd cells also showed about 90-fold resistance to FdUrd. Analysis of enzyme activities and gene expression associated with pyrimidine metabolism indicated that a significant decrease in orotate phosphoribosyltransferase activity in DLD-1/5-FU cells, a 7-fold increase of TS mRNA in DLD-1/FdUrd cells, and a 37-fold decrease in thymidine kinase activity of DLD-1/F3(d)Thd cells were the major mechanisms of drug resistance. These findings were closely associated with the cytotoxicity of 5-FU, FdUrd and F3(d)Thd against the established 5-FU-, FdUrd- or F3(d)Thd-resistant cells. When DLD-1/FdUrd cells expressing increased TS mRNA were treated with FdUrd and F3(d)Thd for only 4 h, the resistance ratios of DLD-1/FdUrd cells to parental DLD-1 cells were markedly different for FdUrd and F3(d)Thd, suggesting that the cytotoxicity with short-time exposure to F3(d)Thd is due to a mechanism other than TS inhibition, although the cytotoxicity of F3(d)Thd in the short-time is low compared to that of long-time exposure. In conclusion, F3(d)Thd, an antimetabolite that inhibits TS activity, may be effective against 5-FU and/or FdUrd-resistance in colorectal cancer cells caused by amplification of TS and/or deletion of orotate phosphoribosyltransferase.

Antimetabolites, Antineoplastic↗

PubMed2000

Intelligent system for topic survey in MEDLINE by keyword recommendation and learning text characteristics.

We have implemented a system for assisting experts in selecting MEDLINE records for database construction purposes. This system has two specific features: The first is a learning mechanism which extracts characteristics in the abstracts of MEDLINE records of interest as patterns. These patterns reflect selection decisions by experts and are used for screening the records. The second is a keyword recommendation system which assists and supplements experts' knowledge in unexpected cases. Combined with a conventional keyword-based information retrieval system, this system may provide an efficient and comfortable environment for MEDLINE record selection by experts. Some computational experiments are provided to prove that this idea is useful.

Artificial Intelligence↗