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Kazunari Kamachi

Publications and source records attributed to Kazunari Kamachi.

17 recordsLinked to original sources
PubMed2006

Development of safer pertussis DNA vaccine expressing non-toxic C180 polypeptide of pertussis toxin S1 subunit.

A toxic N-terminal 180-amino-acid fragment (C180) of pertussis toxin S1 subunit has the most potent ability to induce protective immunity against pertussis toxin (PT) following DNA-based immunization [Kamachi K, Arakawa Y. Infect Immun 2004;72:4293-6]. For the development of a safer pertussis DNA vaccine, three plasmids encoding mutant C180 (C180-R9K, C180-E129G and C180-R9K/E129G) were constructed and tested for their protective immunogenicity and cytotoxicity. All of the gene gun delivery of the plasmid, performed by inserting the mutant C180 gene into a mammalian expression vector pcDNA3.1, successfully induced anti-PT IgG antibody production without the loss of immunogenicity in mice. The immunizations of mice with the plasmids significantly inhibited leukocytosis-promoting activity by PT. Among stably transfected Chinese hamster ovary (CHO) cells expressing mutant C180, the expression of C180-R9K and C180-R9K/E129G was non-toxic to the transfectants, confirming that these mutant C180s have no cytotoxicity to mammalian cells. These results indicate that C180-R9K and C180-R9K/E129G genes, especially C180-R9K/E129G, are candidates for safe and effective antigen DNAs in the development of pertussis DNA vaccine.

Animals↗
PubMed2006

Plasmid pBP136 from Bordetella pertussis represents an ancestral form of IncP-1beta plasmids without accessory mobile elements.

The complete 41,268 bp nucleotide sequence of the IncP-1beta plasmid pBP136 from the human pathogen Bordetella pertussis, the primary aetiological agent of whooping cough, was determined and analysed. This plasmid carried a total of 46 ORFs: 44 ORFs corresponding to the genes in the conserved IncP-1beta backbone, and 2 ORFs similar to the XF1596 and XF1597 genes with unknown function of the plant pathogen Xylella fastidiosa. Interestingly, pBP136 had no accessory genes carrying genetic traits such as antibiotic or mercury resistance and/or xenobiotic degradation. Moreover, pBP136 had only two of the kle genes (kleAE) that have been reported to be important for the stability of IncP-1 plasmid in Pseudomonas aeruginosa. Phylogenetic analysis of the Kle proteins revealed that the KleA and KleE of pBP136 were phylogenetically distant from those of the present IncP-1 plasmids. In contrast, IncC1 and KorC, encoded upstream and downstream of the kle genes respectively, and the replication-initiation protein, TrfA, were closely related to those of the IncP-1beta 'R751 group'. These results suggest that (i) pBP136 without any apparent accessory genes diverged early from an ancestor of the present IncP-1beta plasmids, especially those of the R751 group, and (ii) the kle genes might be incorporated independently into the backbone region of the IncP-1 plasmids for their stable maintenance in various host cells.

Bacterial Proteins↗
PubMed2006

Development and evaluation of a loop-mediated isothermal amplification method for rapid diagnosis of Bordetella pertussis infection.

We developed a loop-mediated isothermal amplification (LAMP) method to detect Bordetella pertussis infection. This LAMP assay detected B. pertussis with high sensitivity, but not other Bordetella species. Among nasopharyngeal swab samples from subjects with suspected pertussis, LAMP results showed a high level of agreement with results of conventional PCR. This method is a rapid, sensitive, and specific method for diagnosis of B. pertussis infection even in clinical laboratories with no specific equipment.

Base Sequence↗
PubMed2006

Genetic and phenotypic characterization of Haemophilus influenzae type b isolated from children with meningitis and their family members in Vietnam.

To investigate Haemophilus influenzae type b (Hib) infection in Vietnamese children under the age of 5 years, cerebrospinal fluid (CSF) samples from patients with meningitis were screened for Hib, and isolates were subjected to evaluation of susceptibility to 12 antibiotics, biotyping, and genotyping with pulsed-field gel electrophoresis (PFGE). The major biotype was type II (68.3%), followed by type I (22.8%). Among 79 Hib isolates, 45 (57%) were beta-lactamase-producing and ampicillin-resistant (44 and 1 isolates produced TEM-1- and ROB-1-type beta-lactamases, respectively), and 34 isolates (43%) were beta-lactamase-nonproducing and ampicillin-sensitive. No beta-lactamase-nonproducing and ampicillin-resistant isolates were found. The PFGE patterns of Hib isolates were highly divergent, but most could be classified into three clusters. We also investigated Hib colonization in household contacts of patients, and found that Hib isolates from the CSF of patients and from nasopharyngeal cavities of household contacts showed the same PFGE patterns. This observation suggested that household contacts of patients are a possible reservoir of Hib.

Adolescent↗
PubMed2004

Augmenting effect of antibiotics on endotoxin activity may cause a safety problem.

Enhancing/interfering effect of antibiotics on endotoxin was evaluated using the endotoxin test and the cell line assay in 28SC cells that has a responsiveness consistent with that of human peripheral blood. When a total of 21 products of seven different kinds of antibiotics were tested, none showed any significant effect on the endotoxin test at its therapeutic dose. However, aminoglycosides showed a significant augmenting effect on IL-6 induction of endotoxin in 28SC cells. Detailed examination of the augmenting effect was made on spectinomycin in the in vitro cell line assay and also in the lethal endotoxin challenge assay in D -galactosamine-treated mice. Spectinomycin also enhanced the endotoxin lethality in D -galactosamine-treated mice. A kinetic analysis in endotoxin-sensitized 28SC cells revealed that the augmentation takes place as quickly as 10 min after spectinomycin treatment. Accordingly, a special caution concerning the augmenting effect was assumed necessary for the safety control of antibiotic products as well as for selecting antibiotics for the therapeutic use.

Animals↗
PubMed2004

Expression of a C terminally truncated form of pertussis toxin S1 subunit effectively induces protection against pertussis toxin following DNA-based immunization.

Four plasmids encoding different C terminally and N terminally truncated pertussis toxin S1 subunits of Bordetella pertussis were constructed and tested for inducibility of protection against pertussis toxin in mice after DNA-based immunization. The region encoding an N-terminal 180-amino-acid fragment of the S1 subunit had the most potent ability to induce protective immunity.

Animals↗
PubMed2004

Antigenic divergence suggested by correlation between antigenic variation and pulsed-field gel electrophoresis profiles of Bordetella pertussis isolates in Japan.

Antigenic divergence has been found between Bordetella pertussis vaccine strains and circulating strains in several countries. In the present study, we analyzed B. pertussis isolates collected in Japan from 1988 to 2001 using pulsed-field gel electrophoresis (PFGE) and sequencing of two virulence-associated proteins. The 107 isolates were classified into three major groups by PFGE analysis; 87 (81%) were type A, 19 (18%) were type B, and 1 (1%) was type C. Sequence analysis of the S1 subunit of pertussis toxin (ptxS1) and adhesion pertactin (prn) genes revealed the presence of two (ptxS1A and ptxS1B) and three (prn1, prn2, and prn3) variants, respectively, in the isolates. Among those isolates, 82 (95%) of the 87 type A strains and the type C strain had the same combination of ptxS1B and prn1 alleles (ptxS1B/prn1) as the Japanese vaccine strain. On the other hand, 17 (90%) of 19 type B strains had an allele (ptxS1A/prn2) distinct from that of the vaccine strain. A correlation was found between the antigenic variation and the PFGE profile in the isolates. In addition, the frequency of the type B strain was 0, 27, 0, 42, and 37% of the isolates in the periods 1988 to 1993, 1994 to 1995, 1996 to 1997, 1998 to 1999, and 2000 to 2001, respectively. In contrast, the number of reported pertussis-like and pertussis cases decreased gradually from 1991 on, suggesting that the antigenic divergence did not affect the efficacy of pertussis vaccination in Japan.

Amino Acid Sequence↗
PubMed2004

Endotoxin content in Haemophilus influenzae type b vaccine.

Haemophilus influenzae type b (Hib) is a major cause of bacterial meningitis among children. Hib conjugate vaccines have effectively prevented Hib infection, and routine immunization with Hib conjugate vaccine has diminished the incidence of the disease in the United States and European countries. Introduction of Hib conjugate vaccines is also required in Japan. However, endotoxin that can carry over from Gram-negative H. influenzae with a purified component may contribute to adverse events following Hib vaccination. In the present study, we examined the endotoxin content in Hib conjugate vaccines. The Hib conjugate vaccine batches, which were produced by a European vaccine manufacturer, were shown to have considerably high endotoxin activity and to vary from 13.9 to 173.7 endotoxin units/dose. These results suggest that it is necessary to monitor the endotoxin content of the vaccine batches to ensure the quality and safety of the vaccines.

Animals↗
PubMed2003

DNA vaccine encoding pertussis toxin S1 subunit induces protection against Bordetella pertussis in mice.

Pertussis toxin (PT) is the major virulence factor of Bordetella pertussis, and detoxified PT is a crucial antigen of acellular pertussis vaccine. Here, plasmid DNA expressing the pertussis toxin S1 subunit (pcDNA/S1) of B. pertussis was evaluated for immunogenicity and for the ability to induce protection against PT challenge or B. pertussis infection in mice. The gene gun delivery of pcDNA/S1, performed by inserting the S1 gene into a mammalian expression vector, successfully induced anti-PT IgG antibody production. Immunization of mice with pcDNA/S1 significantly inhibited leukocytosis-promoting activity caused by PT or B. pertussis. In addition, pcDNA/S1 induced significant protection against intracerebral challenge with a lethal dose of B. pertussis. The results of the present study demonstrated that a DNA vaccine encoding the PT-S1 subunit induced protection against B. pertussis infection in mice. Thus, this vaccine preparation is potentially applicable for the production of novel vaccines against B. pertussis infection.

Animals↗
PubMed2003

Evaluation of endotoxin content of diphtheria-tetanus-acellular pertussis combined (DTaP) vaccines that interfere with the bacterial endotoxin test.

Applicability of the endotoxin test to diphtheria-tetanus-acellular pertussis combined (DTaP) vaccines was examined. We found some DTaP vaccines that strongly interfered with Limulus amoebocyte lysate (LAL) activity of endotoxin without affecting lethal activity of endotoxin in D-galactosamine-treated mice. LAL activity that was interfered in such vaccines increased apparently after the treatment with phosphate buffer at 4 degrees C for a week. The DTaP vaccines that interfered with the endotoxin test showed no significant effect on endotoxin activity in inducing tumor necrosis factor-alpha (TNF-alpha) in rabbit peripheral blood. The in vitro TNF-alpha induction assay was, therefore, suggested to be an appropriate assay method for the quantitative detection of the endotoxin activity in DTaP vaccines.

Animals↗
PubMed2003

A novel apoptosis-inducing protein from Helicobacter pylori.

Helicobacter pylori infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis-inducing protein that functions as a leading factor in H. pylori-mediated apoptosis induction. We purified the protein from H. pylori by separating fractions that showed apoptosis-inducing activity. This protein induced apoptosis of AGS cells in a dose-dependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N-terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited gamma-glutamyl transpeptidase activity, the inhibition of which by 6-diazo-5-oxo-l-norleucine resulted in a complete loss of apoptosis-inducing activity. To the best of our knowledge, the apoptosis-inducing function is a newly identified physiological role for bacterial gamma-glutamyl transpeptidase. The apoptosis-inducing activity of the isogenic mutant gamma-glutamyl transpeptidase-deficient strain was significantly lower compared with that of the parent strain, demonstrating that gamma-glutamyl transpeptidase plays a significant role in H. pylori-mediated apoptosis. Our findings provide new insights into H. pylori pathogenicity and reveal a novel aspect of the bacterial gamma-glutamyl transpeptidase function.

Apoptosis↗
PubMed2003

A quantitative in vitro assay method for detecting biological activity of endotoxin using prostaglandin E2 induction in rabbit peripheral blood.

The pyrogen test and the endotoxin test (the LAL test) have been playing crucial roles in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a large number of animals or an inadequacy in evaluation of in vivo endotoxin activity. We attempted to establish a new assay method that can overcome the shortcomings of the current methods. We standardized the in vitro assay method by the use of prostaglandin E2 (PGE2) induction from peripheral blood of rabbits for detecting endotoxin activity. A linear dose-response regression was attained from approximately 0.15 to 5 endotoxin units/ml of Japanese national reference standard endotoxin by the in vitro assay. The assay showed a fine correlation with the pyrogen test but not with the LAL test, when endotoxins from various bacterial sources were tested. The in vitro assay was also shown to have the capability of detecting a synergistic effect of endotoxin and parenteral drugs. The in vitro PGE2 induction test using rabbit blood was, therefore, suggested to be the appropriate test method for guaranteeing the same level of safety of parenteral drugs as the pyrogen test does.

Animals↗
PubMed2003

A new TEM-derived extended-spectrum beta-lactamase (TEM-91) with an R164C substitution at the omega-loop confers ceftazidime resistance.

A new plasmid-mediated TEM-derived extended-spectrum beta-lactamase, TEM-91, was identified in a ceftazidime-resistant (MIC, >128 microg per ml) Escherichia coli strain isolated in 1996 in Japan. TEM-91 has three amino acid substitutions, R164C, M184T, and E240K, compared with TEM-1 penicillinase. The isoelectric point (pI), K(m), and k(cat) of TEM-91 for ceftazidime were 5.7, 179 microM, and 29.0 s(-1), respectively. The K(i) of clavulanic acid for ceftazidime hydrolysis was 30.3 nM.

Amino Acid Substitution↗
PubMed2003

A cell line assay system for predicting the response of human blood to endotoxin.

Some parenteral drugs augment the in vivo action of endotoxin. It is necessary to regulate the overall toxic action of contaminating endotoxin by developing a clinically relevant test method for the safety control of such drugs. Although the responses of human peripheral blood cells (hPBC) to endotoxins to produce tumour necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and IL-1beta showed considerable variation depending on the endotoxin and also on the individuals used as sources of blood, the responses to each of the endotoxins evaluated relative to that to the Japanese reference standard endotoxin were found to be highly reproducible irrespective of the sources of hPBC. The evaluation procedure based on the relative responsiveness to various endotoxins was shown to be highly effective to detect differences in responsiveness among the endotoxin test, the pyrogen test, and the cytokine induction in hPBC. When eight human monocytoid cell lines were examined, only THP-1 and 28SC cells showed a significant dose-dependent IL-6 production. However, THP-1 failed to show consistency with hPBC in responses to the panel of endotoxins. 28SC cells showed appropriate consistency with hPBC not only in terms of respective responses to the endotoxins but also with regard to detection of the effect of human interferons to augment endotoxin to induce IL-6.

Animals↗
PubMed2002

Distribution of pertussis antibodies among different age groups in Japan.

We examined the distribution of antibody levels against pertussis toxin (PT) and filamentous hemagglutinin (FHA) among a healthy Japanese population aged from 0 to 77 years. Levels of both antibodies in 1108 serum samples collected in 1994 from nine prefectures were assayed using polystyrene ball ELISA. The ratio of individuals positive (>or=10 ELISA U/ml) for anti-PT and anti-FHA antibodies at ages ranging from 0 to 3 years increased rapidly with the increase in the population vaccinated over three times with acellular pertussis (aP) vaccine. However, the ratio of those positive for anti-PT antibody tended to decrease until 6-8 years of age and to increase again from 9 to 19 years among the vaccinated population, although the ratio of individuals positive for anti-FHA antibody remained constant at 80-100% in children and adolescents over 3 years old. Moreover, positivity for anti-PT antibody was high (>or=50 ELISA U/ml) in some serum samples collected from adolescents and young adults, suggesting recent symptomatic or asymptomatic infection with circulating Bordetella pertussis. On the other hand, 50-60% of infants below 12 months of age was below the detection limit (1.0 ELISA U/ml) for anti-PT and anti-FHA antibodies, and most early infants were not vaccinated for pertussis. Since intermittent circulation of B. pertussis remains among the Japanese population, complete vaccination with aP vaccine for all infants should be highly recommended to prevent pertussis.

Adhesins, Bacterial↗
PubMed2002

Characterization of a novel plasmid-mediated cephalosporinase (CMY-9) and its genetic environment in an Escherichia coli clinical isolate.

An Escherichia coli strain, HKYM68, which showed resistance to broad-spectrum cephalosporins was isolated from a sputum specimen in Japan. The high-level resistance of the strain to ceftazidime, cefpirome, and moxalactam was carried by a self-transferable plasmid. The beta-lactamase gene responsible for the resistance was cloned and sequenced. The deduced amino acid sequence of this gene product, CMY-9, had a single amino acid substitution (E85D), the residue reported to be part of the recognition site for the R1 side chain of beta-lactams, compared with the amino acid sequence of CMY-8 and also had 78% identity with the amino acid sequence of CepH, a chromosomal cephalosporinase of Aeromonas hydrophila. A sul1-type class 1 integron containing an aacA1-orfG gene cassette was identified upstream of bla(CMY-9) and ended with a truncated 3' conserved segment. The following 2.1 kb was almost identical to the common region of integrons In6 and In7 and the integron of pSAL-1, except that orf513 encoding a putative transposase was identified instead of orf341 due to addition of a single nucleotide. bla(CMY-9) was closely located downstream of the end of the common region. These observations are indicative of the exogenous derivation of bla(CMY-9) from some environmental microorganisms such as aeromonads.

Anti-Bacterial Agents↗