Pretreatment of recipients with mitomycin-C-treated dendritic cells induces significant prolongation of cardiac allograft survival in mice.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
M Uchijima
Publications and source records attributed to M Uchijima.
Immunization with plasmid DNA encoding MHC class II binding peptide/CLIP-replaced invariant chain (Ii) induces specific helper T cells in vivo: the assessment of Ii p31 and p41 isoforms as vehicles for immunization.
A single helper T cell (Th) epitope-specific T cell subset was successfully induced in vivo by immunization with plasmid DNA encoding MHC class II binding peptide/class II-associated invariant chain peptide (CLIP)-replaced murine Ii molecules. Spleen cells from mice immunized by gene gun bombardment with plasmid DNA for Ii p31 and p41 molecules, whose CLIP regions were replaced with an I-A(d)-restricted Th epitope, ovalbumin (OVA) 323-336, showed the specific proliferation and interferon-gamma (IFN-gamma) production. A20-2J B cell lines having these plasmids were capable of stimulating spleen cells from the immunized mice and naïve DO10-transgenic mice bearing the epitope-specific T cell receptor (TCR) transgenes by examining the specific proliferative response and IFN-gamma production. Some mice immunized with the Ii p41-OVA323, but not with the Ii p31-OVA323 plasmid, produced the peptide-specific antibodies, suggesting the functional difference between Ii isoforms.
Animals↗
Identification of immunostimulatory DNA-induced genes by suppression subtractive hybridization.
Bacterial DNA and related synthetic immunostimulatory oligodeoxyribo-nucleotides (ISS-ODN) have stimulatory effects on mammalian immune cells through a Toll-like receptor, TLR9. Genes upregulated in ISS-ODN-stimulated immune cells are obviously significant to delineate the mechanism of the induced innate immunity. Employing suppression subtractive hybridization (SSH), we have generated a profile of genes induced by ISS-ODN in spleen cells. Sequencing of 87 clones isolated by the SSH showed 39 clones corresponding to known mouse genes in the public database. Eleven clones appeared to possess 80-90% homology with known mouse genes and the remaining 37 clones showed no significant homology with any known mouse genes. A series of known genes which have not previously been reported to be induced with ISS-ODN were confirmed to be induced in ISS-ODN-stimulated bone marrow-derived macrophages: NF-kappaB p105, IRF-1, PA28beta, IRG2, and MyD88. These genes were suggested to be involved in the molecular process of innate host defense mechanisms.
Adjuvants, Immunologic↗
Protective CTL response is induced in the absence of CD4+ T cells and IFN-gamma by gene gun DNA vaccination with a minigene encoding a CTL epitope of Listeria monocytogenes.
Our work was undertaken to learn the mechanism of induction of protective cytotoxic T lymphocytes (CTL) by gene gun DNA vaccination with p91m encoding an H-2Kd-restricted T cell epitope of listeriolysin O (LLO). Vaccination with p91m induced vigorous antigen-specific CD8+ CTL that produce IFN-gamma and was able to confer partial protection against listerial challenge. However, the p91m-induced protective immunity was revealed to be independent of the IFN-gamma and CD4+ T cell help. The CTL induction is also suggested to require neither adjuvant activity of the plasmid used nor IFN-gamma. The data may be feasible for the design of CTL inducing vaccines in various immunodeficiencies.
Animals↗
Toxic shock syndrome toxin-1 accelerated collagen-induced arthritis in mice.
The aim of this study was to explore the roles of toxic shock syndrome toxin-1 (TSST-1) in collagen-induced arthritis (CIA). DBA/1 mice were immunized with type II collagen (CII) and treated with TSST-1. Intraperitoneal and intravenous injections of TSST-1 aggravated CIA, enhancing its incidence and severity. CIA was accompanied by an increase in anti-CII IgG Ab levels. Intraperitoneal administration with TSST-1 enhanced IFN-gamma, TNF-alpha, and IL-4 production in DBA/1 mice. We discovered the mRNA expressions of IFN-gamma, IL-2, TNF-alpha, IL-1beta, and iNOS in spleen cells stimulated with TSST-1 in vitro. However, IL-12 and IL-4 mRNA expression were seen constitutively without stimulation. Only a little increase of IL-12 and IL-4 mRNA expression was seen at 2-3 h after treatment with TSST-1. Our experiments demonstrated that CIA was aggravated by the treatment with TSST-1, which may have induced various proinflammatory cytokines and the production of both Th1 and Th2 cytokines.
Animals↗
Protective cytotoxic T lymphocyte responses induced by DNA immunization against immunodominant and subdominant epitopes of Listeria monocytogenes are noncompetitive.
Taking advantage of the fact that plasmid DNA encoding a single cytotoxic T lymphocyte (CTL) epitope can induce CTLs, we examined the influence of T-cell responses to dominant epitopes on those to a subdominant epitope derived from Listeria monocytogenes. Our data suggest that interaction between T cells against dominant and subdominant epitopes does not operate in the generation of the hierarchy. Furthermore, we found that a single dominant epitope is sufficient for the induction of protective immunity.
3T3 Cells↗
Identification of differentially expressed genes in rat hippocampus after transient global cerebral ischemia using subtractive cDNA cloning based on polymerase chain reaction.
BACKGROUND AND PURPOSE: The purpose of this study is to identify new molecules that play important roles in the phenomena that occur in the hippocampus after transient global cerebral ischemia, as clues to better understanding of the mechanisms. METHODS: A subtractive cDNA library was established by suppression subtractive hybridization of rat hippocampal tissues after transient global cerebral ischemia. With differential screening of the library, upregulated fragments were identified. The mRNA expression levels of selected genes were measured with semiquantitative reverse transcriptase polymerase chain reaction (PCR). RESULTS: Among more than 100 isolated fragments, approximately half were determined to be identical to known sequences. The rest showed high homology to known sequences, and only 2 did not exhibit homology to any known sequences. The expression of 5 genes identified in this study increased in 24 hours after ischemia to a level twice as high as that in sham-operated controls. These included furin, prosaposin, synaptotagmin IV, heat shock protein 105, and the neutral and basic amino acid transporter (NBAT). The increases in the mRNA expression levels of the genes except NBAT, as revealed by semiquantitative reverse transcription PCR, were statistically significant at both 6 and 24 hours after ischemia. CONCLUSIONS: Genes isolated are thought to be associated with production of proteins necessary for degeneration, neuroprotection, and reconstruction of neurons. How the expression of these genes relates to functional changes after ischemia remains to be determined. PCR-based subtractive cDNA cloning is demonstrated to be a useful tool for analyzing in vivo gene expression in animal ischemia models.
Amino Acid Transport Systems, Basic↗
Advantage of gene gun-mediated over intramuscular inoculation of plasmid DNA vaccine in reproducible induction of specific immune responses.
Utilizing a plasmid DNA encoding a single cytotoxic T lymphocyte (CTL) epitope and that encoding ovalbumin (OVA), we compared the reproducibility in the induction of immune responses by gene gun and intramuscular immunization. As compared to intramuscular inoculation, gene gun DNA immunization appeared to bring about highly reproducible and reliable results in the induction of specific CTL and IFN-gamma production to the CTL epitope and production of anti-OVA IgG. The results obtained by intramuscular inoculation vary significantly. Our data shown here strongly suggest that gene gun immunization of skin is a much more reliable method for DNA vaccination to induce effective immune responses in an animal model.
Animals↗
DNA vaccines.
DNA vaccination or genetic immunization is a rapidly developing technology that offers new approaches for the prevention and therapy of disease. Regarding the inoculation method of DNA vaccine, we recommend the gene gun delivery system, which is a highly reliable method compared to intramuscular inoculation. DNA vaccines could have potential advantages over other types of vaccines in that these vaccines can induce strong cellular immune responses, cytotoxic T lymphocytes and type 1 helper T cells, without resorting to live organisms or complicated protein formulation. The cellular immune responses are especially required for the protection against infections with intracellular pathogens such as viruses and Mycobacterium tuberculosis and protection against cancers, suggesting that they seem to be suitable targets of DNA vaccines. We describe here that their application to bacterial infections requires optimization of codon usage in the DNA vaccines to the host animal to improve translational efficiencies of the bacteria genes. DNA vaccines for a variety of pathogens and cancers have now entered phase I/II human clinical trials.
Animals↗
Codon optimization effect on translational efficiency of DNA vaccine in mammalian cells: analysis of plasmid DNA encoding a CTL epitope derived from microorganisms.
Interspecific difference of codon usage is one of the major obstacles for effective induction of specific immune responses against bacteria and protozoa by DNA immunization. Using genes encoding major histocompatibility complex class I-restricted cytotoxic T-lymphocyte (CTL) epitopes, derived from an intracellular bacterium, Listeria monocytogenes and a mouse malaria parasite, Plasmodium yoelii, we report here that the codon optimization level of the genes is not precisely proportional to, but does correlate well with the translational efficiency in mammalian cells, which is concomitantly associated with the induction level of specific CTL response in the mouse. These results suggest that DNA immunization using the gene codon-optimized to mammals through the entire region is very effective.
Animals↗
Decreased expression of Th2 type cytokine mRNA contributes to the lack of allergic bronchial inflammation in aged rats.
Sensitized Brown Norway rats are known to develop eosinophilic bronchial inflammation and airway hyperresponsiveness after Ag exposure. However, we have previously observed that sensitized aged rats of the same strain failed to develop such allergic inflammation. In the present study, we investigated age-associated changes of cytokine mRNA expression in bronchoalveolar lavage (BAL) cells. Both young (8- to 10-wk-old) and aged (100- to 120-wk-old) Brown Norway rats were sensitized with OVA, and BAL was performed 24 h after OVA inhalation challenge. Semiquantitative RT-PCR analysis of BAL cells showed that the cells from aged rats preferentially expressed Th1 type cytokine (IFN-gamma) mRNA, whereas cells from young animals expressed more Th2 type cytokine mRNAs including those for IL-4 and IL-5. Decreased expression of Th2 type cytokine transcripts in aged animals was further confirmed by quantitative analysis, competitive RT-PCR of BAL cells, and in situ hybridization. The age-associated changes of cytokine profile were not restricted to BAL cells but were a general feature of lymphocytes, as shown by examination of popliteal lymph nodes draining the site of sensitization. These findings suggest that decreased allergic inflammation in aged animals is attributable to age-dependent impairment of Th2 generation in response to Ag.
Age Factors↗
Biased T cell receptor Vbeta gene expression in bronchoalveolar lavage fluid from Japanese patients with sarcoidosis.
OBJECTIVE: Sarcoidosis is believed to be one of the T cell-mediated granulomatous diseases with unknown aetiology. We attempt to search for the causative T cell clones of sarcoidosis. METHODS: We study T cell receptor beta-chain variable region (Vbeta) repertoire in peripheral blood (PB) and bronchoalveolar lavage fluid (BALF) from patients with sarcoidosis, using semi-quantitative reverse transcriptase-polymerase chain reaction method. The expression of 22 kinds of Vbeta genes is examined in 17 patients with sarcoidosis and nine normal subjects. RESULTS: Compared with control subjects, the group with sarcoidosis exhibits significantly high expressions of the Vbeta2 (P < 0.005, Wilcoxon's test) and Vbeta6 (P = 0.005) genes in BALF. In each BALF sample, the Vbeta2 (P < 0.01, chi2 test) and Vbeta6 (P < 0.01) genes were overexpressed (> 2 SD above the mean value for each Vbeta observed in control subjects) in 11 and 10 of 17 patients with sarcoidosis, respectively. Furthermore, the amino acid sequences of Vbeta6+ complementarity determining region 3 were conserved in one of three patients. There is, however, no disposition of Vbeta gene usage in PB from patients with sarcoidosis compared with control subjects. CONCLUSIONS: The T lymphocytes with Vbeta2 and/or Vbeta6 are associated with the pathogenesis of sarcoidosis. The possibility exists that these T lymphocytes might be capable of recognizing the restricted antigens, thereby inducing oligoclonal expansion.
Adult↗
[DNA vaccines for infections with intracellular bacteria].
Explore the source record for details and available documents.
Animals↗
Optimization of codon usage of plasmid DNA vaccine is required for the effective MHC class I-restricted T cell responses against an intracellular bacterium.
In an attempt to study codon usage effects of DNA vaccines on the induction of MHC class I-restricted T cell responses against an intracellular bacterium, Listeria monocytogenes, we designed two plasmid DNA vaccines encoding an H-2Kd-restricted epitope of listeriolysin O (LLO) of L. monocytogenes, LLO 91-99. One DNA vaccine, p91wt, carries the wild-type DNA sequence encoding LLO 91-99, and the other one, p91mam, possesses the altered DNA sequence in which the codon usage was optimized for murine system. Our read-through analyses with LLO 91-99/luciferase fusion genes confirmed that the optimized 91mam DNA sequence showed extremely higher translation efficiency than the wild-type sequence in murine cells. Consistent with this, i.m. injections of p91mam, but not of p91wt, into BALB/c mice were capable of inducing specific CTL- and IFN-gamma-producing CD8+ T cells able to confer partial protection against listerial challenge. Taken together, these observations suggest that optimization of codon should be taken into consideration in the construction of DNA vaccines against nonviral pathogens.
Animals↗
Reduced susceptibility to collagen-induced arthritis in mice deficient in IFN-gamma receptor.
Collagen-induced arthritis (CIA) is an arthritic model that was developed after immunization with type II collagen (CII). Apparently, contradictory results have been reported regarding the role of IFN-gamma in the development of CIA. Therefore, we employed IFN-gamma R-deficient mice to study the role of IFN-gamma. To introduce the CIA susceptibility gene (H-2q), IFN-gamma R-deficient (H-2b/b/IFN-gamma R-/-) mice were mated with DBA/1 (H-2q/q/IFN-gamma R+/+) mice; next, the F1 mice were interbred to yield F2 offspring bearing different combinations of H-2 (H-2q/q, H-2q/b, and H-2b/b) and IFN-gamma R (IFN-gamma R+/+, IFN-gamma R+/-, and IFN-gamma R-/-) genes. Although the H-2q allele appeared to confer susceptibility to CIA, mice that were homozygous for the IFN-gamma R mutation showed a substantially decreased incidence and severity of CIA. The CII-specific IgG levels of serum samples, which are known to be involved in the development of CIA, were remarkably reduced in IFN-gamma R-/- mice. Furthermore, the anti-CII IgG2a levels controlled by IFN-gamma R were significantly reduced in IFN-gamma R-/- F2 mice compared with those seen in IFN-gamma R+/+ and IFN-gamma R+/- mice, although the levels of all IgG subclass Abs examined were lower in IFN-gamma R-/- mice than in IFN-gamma R+/+ mice. No clear evidence of the imbalance of Th1/Th2 cytokines was observed in CII-immunized, IFN-gamma R-deficient mice. Taken together, these results suggest that IFN-gamma exacerbates CIA by affecting, at least, levels of CII-specific IgG Ab rather than the imbalance of Th1/Th2 cells.
Animals↗
Effect of staphylococcal enterotoxin B-induced anergy on cytokine gene expression: anergy-sensitive and resistant mRNA expression.
We examined the effect of staphylococccal enterotoxin B (SEB)-induced anergy on expression of six different cytokine genes in T cells restimulated with SEB in vitro. We found that although IL-2, IL-3, and IL-4 mRNA levels are substantially reduced in anergic T cells, mRNAs for IL-6, IL-10, IFN-gamma, and TNF-alpha are expressed normally. Thus, there appeared both anergy-sensitive and resistant cytokine mRNA expression in restimulated anergic T cells. The same pattern of cytokine mRNA responses was observed in anergic CD4+ T cells, indicating that the preferential induction of anergy in Th1-like cells is not evident in this in vivo model. Employing TCR V beta 8.2 transgenic mice in which almost all T cells become anergic, we found that the TCR/CD3 complex can transduce both anergy-sensitive and resistant signals. Furthermore, a series of experiments using FK506, A23187, and PMA suggests that signals between TCR and activation of calcineurin and protein kinase C may be blocked in anergic T cells. This is supported by our gel mobility shift assays indicating that calcineurin and/or PMA-inducible NF-ATp, OAP40, and AP-1, but not calcineurin-independent Oct-2, are repressed in anergic spleen T cells upon restimulation with SEB. Taken together, these results suggest that, among signals elicited by stimulation of TCR with SEB, a Ca2+/calcineurin-NF-ATp pathway and other signals, including protein kinase C, are repressed in anergic T cells upstream of their activation, which are essential for the cytokine mRNA expression of the anergy-sensitive type but are dispensible for those of the anergy-resistant type.
Animals↗
Dissection of strain difference in acquired protective immunity against Mycobacterium bovis Calmette-Guérin bacillus (BCG). Macrophages regulate the susceptibility through cytokine network and the induction of nitric oxide synthase.
Protection against infection with intracellular pathogens operates in two stages, early innate resistance and late acquired protective immunity (API), in inbred mouse strains. Although both C57BL/10 (B10) and BALB/c mice bear the susceptible phenotype of innate resistance, Calmette-Guérin bacillus (BCG) vaccination generated efficient API in B10 but not in BALB/c mice. Employing a specific nitric oxide (NO) synthase inhibitor, we revealed that NO production plays a pivotal role in the API of B10 mice. Consistent with this, expressions of the inducible isoform of NO synthase (iNOS) protein and mRNA were significantly higher in the spleen of B10 mice than in that of BALB/c mice. Furthermore, IFN-gamma, a potent inducer of iNOS, and mRNAs for IL-12 (p40); an inducer of IFN-gamma and IL-2 were also vigorously expressed in the spleen of B10 mice compared with that of BALB/c mice. In an attempt to clarify the mechanism by which the different capacities for API are generated, we analyzed the cytokine network between T cells and macrophages in both B10 and BALB/c mice. We found that multiple functions of macrophages, which include capacities to express IL-12 (p40) mRNA in response to BCG and to express mRNAs for iNOS and IL-12 (p40) in response to IFN-gamma, were impaired in BALB/c mice as compared with B10 mice. However, T cells appeared to express comparable level of IFN-gamma mRNA in both strains when stimulated with IL-12. Taken together, these results indicate that the macrophage functions play a pivotal role in both the induction and effector phases of API to determine the susceptibility of mice to BCG infection.
Amino Acid Oxidoreductases↗
[Analysis of T cell receptor V beta gene expression in bronchoalveolar lavage fluid lymphocytes from patients with idiopathic interstitial pneumonia].
T lymphocytes in bronchoalveolar lavage (BAL) from idiopathic interstitial pneumonia (IIP) patients are considered to recognize unknown antigens, such as dust, fume, virus or degenerated autoantigens. To analyse the nature of these T lymphocytes, we investigated T cell receptor (TCR) V beta gene usage (22 kinds) in BAL Lymphocytes and Peripheral blood lymphocytes from 10 11P patients and 9 normal controls, using reverse transcriptase-polymerase chain reaction method. In BAL lymphocytes, predominant usage of V beta genes (> 15% of the sum of all V beta transcripts) was recognized in 7 of 10 IIP patients, which appeared to vary in individuals (Case 1: V beta 14, case 2: V beta 3, V beta 5.1, case 5: V beta 2, case 6: V beta 6, case 7: V beta 8, case 8: V beta 7, V beta 20, case 9: V beta 6), whereas no predominant V beta usage was demonstrated in normal controls. It remains to be elucidated whether the BAL lymphocytes expressing predominant V beta genes are involved in the activation of alveolar macrophages, fibroblasts, thereby inducing the production of autoantibodies.
Base Sequence↗