Yukio Koide

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.

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.

Protective CTL response is induced in the absence of CD4+ T cells and IFN-γ 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.

Induction of protective immunity to Listeria monocytogenes by immunization with plasmid DNA expressing a helper T-cell epitope that replaces the class II-associated invariant chain peptide of the invariant chain.

ABSTRACT Listeria epitope-specific helper T (Th) cells were able to be primed and induced in vivo by immunization with a plasmid carrying an invariant chain (Ii) gene whose class II-associated invariant chain peptide (CLIP) region was replaced by a Listeria Th epitope. Immunization of C3H/He mice with an Ii-LLO 215-226 plasmid induced specific interferon-γ- and interleukin 2-producing Th cells and conferred significant protective immunity against listerial infection.

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.

Induction of protective immunity to Listeria monocytogenes with dendritic cells retrovirally transduced with a cytotoxic T lymphocyte epitope minigene

ABSTRACT In the present study, we developed a cytotoxic T lymphocyte (CTL) epitope minigene-transduced dendritic cell (DC)-based vaccine against Listeria monocytogenes. Murine bone marrow-derived DCs were retrovirally transduced with a minigene for listeriolysin O (LLO) 91-99, a dominant CTL epitope of L. monocytogenes, and were injected into BALB/c mice intravenously. We found that the DC vaccine was capable of generating peptide-specific CD8+ T cells exhibiting LLO 91-99-specific cytotoxic activity and gamma interferon production, leading to induction of protective immunity to the bacterium. Furthermore, we demonstrated that the retrovirally transduced DC vaccine was more effective than a CTL epitope peptide-pulsed DC vaccine and a minigene DNA vaccine for eliciting antilisterial immunity. These results provide an alternative strategy in which retrovirally transduced DCs are used to design vaccines against intracellular pathogens.

Retrovirus-mediated gene transfer of granulocyte colony-stimulating factor receptor (G-CSFR) cDNA into MDS cells and induction of their differentiation by G-CSF

Myelodysplastic syndromes (MDS) are clonal disorders in which the proper differentiation of hematopoietic stem cells is impaired. There is no effective treatment for this stem cell disorder at present. In an attempt to find a new strategy that promotes the differentiation of MDS blast cells, we tried retroviral transduction of granulocyte colony-stimulating factor receptor (G-CSFR) into an interleukin-3-dependent MDS cell line, MDS-L, since expression of G-CSFR is known to be essential for the differentiation of myeloid progenitor cells and this expression is impaired in most MDS cells. Ectopic expression of human G-CSFR cDNA in MDS-L cells gave rise to granulocytic differentiation by G-CSF stimulation. G-CSF caused the transformants expressing G-CSFR to display a morphological characteristic of mature granulocytes, upregulated CD11b on the cell surface, and improved NBT reduction activity. These results demonstrate that MDS-L cells ecopically expressing G-CSFR are induced to granulocytic differentiation upon exposure to G-CSF, and shed light on the molecular mechanisms of maturation arrest in MDS cells.

Protective Cytotoxic T Lymphocyte Responses Induced by DNA Immunization against Immunodominant and Subdominant Epitopes of Listeria monocytogenes Are Noncompetitive

ABSTRACT 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.

Biased T cell receptor Vbeta gene expression in bronchoalveolar lavage fluid from Japanese patients with sarcoidosis.

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.