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Tetsuro Sasada

Publications and source records attributed to Tetsuro Sasada.

13 recordsLinked to original sources
PubMed2006

The TCR C beta FG loop regulates alpha beta T cell development.

The TCRbeta chain constant domain contains an unusually elongated, solvent-exposed FG loop. This structural element forms one component of an alphabeta TCR cavity against which CD3epsilongamma may abut to facilitate Ag-specific signaling. Consistent with this notion, in the present study we show that N15alphabeta TCR transfectants expressing a FG loop-deleted chain (betaDeltaFG) stimulate less tyrosine protein phosphorylation than those bearing a wild-type beta-chain (betawt) upon TCR cross-linking. Furthermore, coimmunoprecipitation studies suggest a weakened association between the CD3epsilongamma heterodimer and the beta-chain in TCR complexes containing the betaDeltaFG variant. To further investigate the biologic role of the Cbeta FG loop in development, we competitively reconstituted the thymus of Ly5 congenic or RAG-2-/- mice using bone marrow cells from betawt or betaDeltaFG transgenic C57BL/6 (B6) mice. Both betawt and betaDeltaFG precursor cells generate thymocytes representative of all maturational stages. However, betaDeltaFG-expressing thymocytes dominate during subsequent development, resulting in an excess of betaDeltaFG-expressing peripheral T cells with reduced proliferative and cytokine production abilities upon TCR stimulation. Collectively, our results show that the unique Cbeta FG loop appendage primarily controls alphabeta T cell development through selection processes.

Animals↗
PubMed2004

Ran, a small GTPase gene, encodes cytotoxic T lymphocyte (CTL) epitopes capable of inducing HLA-A33-restricted and tumor-reactive CTLs in cancer patients.

PURPOSE: The purpose is to identify a gene coding for tumor-associated antigen and peptide capable of inducing CTLs reactive to tumor cells with a HLA-A33-restricted fashion to provide scientific basis for specific immunotherapy to HLA-A33+ cancer patients. EXPERIMENTAL DESIGN: An expression gene-cloning method was used to identify the tumor-associated antigen gene. Northern blot analysis and immunohistochemistry were used to examine the mRNA and protein expression levels in various cells and tissues, respectively. Synthetic peptides were examined for their ability to induce HLA-A33+ tumor-reactive CTLs in peripheral blood mononuclear cells from cancer patients. RESULT: A gene of small GTPase, Ran, which controls the cell cycle through the regulation of nucleocytoplasmic transport, mitotic spindle organization, and nuclear envelope formation, was found to encode epitopes recognized by the HLA-A33-restricted CTLs established from T cells infiltrating into gastric adenocarcinoma. The expression of the Ran gene was increased in most cancer cell lines and cancer tissues at both the mRNA and protein levels. However, it was not enhanced in the surrounding normal cells or tissues. It was also undetectable in normal tissues as far as tested. Ran-derived peptides at positions 48-56 and 87-95 could induce CD8+ peptide-specific CTLs reactive to tumor cells from HLA-A33+ epithelial cancer patients in a HLA class I-restricted manner. CONCLUSIONS: Because of its increased expression in cancer cells and involvement in malignant transformation and/or the enhanced proliferation of cancer cells, the two Ran-directed peptides could be potent candidates in use for specific immunotherapy against HLA-A33+ epithelial cancers.

Adenocarcinoma↗
PubMed2004

Usefulness of 99mTc-sestamibi scintigraphy in suggesting the therapeutic effect of chemotherapy against gastric cancer.

PURPOSE: Imaging with (99m)Tc-sestamibi ((99m)Tc-MIBI) has been used to assess 170-kDa P-glycoprotein (P-gp) expression and predict chemotherapy responses in several types of malignancy, such as breast and lung cancers. The purpose of this study was to evaluate the relationship between (99m)Tc-MIBI accumulation in tumors and sensitivity to chemotherapy in gastric cancer patients. EXPERIMENTAL DESIGN: Thirty-six patients with advanced gastric cancer underwent (99m)Tc-MIBI scintigraphy before chemotherapy. Patients also underwent endoscopic biopsy, and the expression of P-gp or multidrug resistance-associated protein was analyzed by immunohistochemical staining. The relationship between the accumulation of (99m)Tc-MIBI in tumors and responses to chemotherapy with 5-fluorouracil/cis-diamminedichloroplatinum(II) or epirubicin was examined. RESULTS: Higher accumulation of (9m)Tc-MIBI in tumors was observed in 25 and 23 of 36 gastric cancer patients at the early (30 min) and delayed (120 min) images, respectively. Accelerated accumulation of (99m)Tc-MIBI negatively correlates with increased expression of P-gp, but not of multidrug resistance-associated protein, as determined by immunohistochemistry in gastric cancer tissues. The response rate to 5-fluorouracil/cis-diamminedichloroplatinum(II) chemotherapy in patients with high (99m)Tc-MIBI accumulation (15.4%) was much lower than that in patients with low (99m)Tc-MIBI accumulation (54.5%). In contrast, patients with high (99m)Tc-MIBI accumulation show a higher response rate (41.7%) to chemotherapy with epirubicin, which is known to be a substrate of P-gp transporter. CONCLUSIONS: (99m)Tc-MIBI scintigraphy is useful to suggest the responses to chemotherapy of patients with advanced gastric cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Immediate early response gene X-1, a stress-inducible antiapoptotic gene, encodes cytotoxic T-lymphocyte (CTL) epitopes capable of inducing human leukocyte antigen-A33-restricted and tumor-reactive CTLs in gastric cancer patients.

Peptide-based vaccine therapy, which is designed to elicit T-cell immunity against tumors, is an attractive approach for the treatment of cancer patients. To provide a scientific basis for peptide therapy, an increasing number of CTL-directed peptides have been identified, and some of them have been tried as antigen-specific immunotherapy in the past decade. Only a few studies, however, have been performed on such peptides restricted with alleles other than HLA-A2 and -A24. In the present study, we show that immediate early response gene X-1 (IEX-1), a stress-inducible protein associated with the regulation of cell proliferation and apoptosis, produces antigenic epitopes recognized by 850B-CTLs, HLA-A33-restricted CTLs newly established from T cells infiltrating into gastric adenocarcinoma. The IEX-1 gene was highly expressed in most cell lines and tissues from various types of cancer at both the mRNA and protein levels. However, it was not expressed at the protein level in any normal epithelium or connective tissues tested. Three IEX-1-derived peptides at positions 47-56, 61-69, and 65-73, which were recognized by the 850B-CTLs, could induce CD8(+) peptide-specific CTL reaction to tumor cells from HLA-A33(+) gastric cancer patients and other epithelial cancer patients, but not from healthy donors, in an HLA class I-restricted manner. Because increased expression of IEX-1 is suggested to be involved in the resistance to apoptosis and in the proliferation of cancer cells, these antigenic peptides could be potent candidates for peptide-based specific immunotherapy against HLA-A33(+) gastric cancer and other epithelial cancers.

Adenocarcinoma↗
PubMed2003

CD4+CD25+ regulatory T cells in patients with gastrointestinal malignancies: possible involvement of regulatory T cells in disease progression.

BACKGROUND: Active suppression by CD4+CD25+ regulatory T cells plays an important role in the down-regulation of the response of T cells to foreign and self antigens. Experimental tumor models in mice revealed that regulatory T cells inhibit antitumor immune responses. The purpose of the current study was to demonstrate the possible involvement of CD4+CD25+ regulatory T cells in immune system impairment in patients with gastrointestinal malignancies. METHODS: The phenotypes of lymphocytes, particularly those of CD4+CD25+ T cells, were analyzed in peripheral blood in 149 patients with gastrointestinal malignancies and in ascites in 7 patients with peritoneal dissemination. In addition, cytokine production after in vitro stimulation was examined in CD4+CD25+ and CD4+CD25- T cells isolated from patients with malignant disease. RESULTS: Compared with healthy volunteers, patients with gastrointestinal malignancies had a higher proportion of CD4+CD25+ T cells in peripheral blood, due to the presence of a drastically smaller number of CD4+CD25- T cells. Among patients with gastric carcinoma, those with higher percentages of CD4+CD25+ T cells had a poorer prognosis than did those with lower percentages. CD4+CD25+ T cells also were present in greater proportions in ascites from patients who had advanced-stage disease with peritoneal dissemination. Isolated CD4+CD25+ T cells from patients with malignant disease produced interleukin (IL)-4 and IL-10 but not IL-2 or interferon-gamma; these cells also inhibited cytokine production by CD4+CD25- T cells after in vitro stimulation. CONCLUSIONS: The relative increase in CD4+CD25+ regulatory T cells may be related to immunosuppression and tumor progression in patients with gastrointestinal malignancies. This finding suggests that the use of immunomodulatory therapy to treat patients with gastrointestinal malignancies may be an effective strategy.

Adult↗
PubMed2003

Change in mitochondrial membrane potential in peripheral blood lymphocytes, especially in natural killer cells, is a possible marker for surgical stress on the immune system.

There is accumulating evidence that surgical stresses cause impairment of systemic immune responses, which may promote susceptibility to infection as well as growth of remnant cancer cells in cancer patients. Although alterations in numbers, populations, and functions of lymphocytes have been extensively studied to assess modulation of the immune system, the precise mechanisms of immunosuppression caused by surgical stresses have not been identified, nor have methods been developed to estimate the magnitude of surgical stresses on the immune system. In the present study, to evaluate the effects of surgical procedures on the immune system, the mitochondrial membrane potential (Delta Psi(m)) of peripheral blood lymphocytes (PBL) from 25 patients who underwent various types of operation was measured by flow cytometry using 3,3'-dihexiloxacarbocyanine iodide (DiOC(6)(3)) on the day before operation and on postoperative day (POD) 1, POD 3, and POD 7. The Delta Psi(m) in PBL, especially in natural killer (NK) cell population, was reduced after major surgery. In particular, the reduction of Psi Delta(m) in NK cells appeared to be proportional to the severity of the surgical procedures and reflected the impairment of cellular function. Interestingly, the Delta Psi(m) in NK cells was also negatively correlated with the level of plasma noradrenaline after major surgery, suggesting that the reduction of Delta Psi(m) in NK cells induced by surgical stresses may be mediated, at least in part, by the accompanying increase in plasma noradrenaline. Monitoring of Delta Psi(m) in PBL after operation may be one of the useful markers for estimating the magnitude of surgical stresses on the immune system.

Catecholamines↗
PubMed2003

Disparate peptide-dependent thymic selection outcomes in beta2M-deficient mice versus TAP-1-deficient mice: implications for repertoire formation.

Fetal thymic organ cultures of N15-transgenic RAG-2-/- H-2b mice on normal, beta-2 microglobulin (beta2M)-/- or transporter associated with antigen processing (rAP-1)-/- MHCl-deficient backgrounds were used to examine differentiation of thymocytes bearing a TCR specific for a viral peptide bound to H-2Kb. Strong agonists mediate negative selection in all mice whereas weak agonists are positively selecting in beta2MW-/- mice but negatively selecting on TAP-1-/- or normal backgrounds. Very weak agonists and very weak antagonists are generally without effect in beta2M-/- mice yet foster differentiation in TAP-1-/- animals. The 20-40-fold reduction in beta2M4-/- thymic H-2Kb surface expression suggests that the avidity of the TCR for peptide-MHCI accounts for these differences, consistent with effects of TCR density and individual thymic-peptide abundance in peptide-MHC complexes. TCR-self-MHC interaction dominates Kb-based selection, subtly modulated by peptides as revealed by X-ray crystallography.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Involvement of the TCR Cbeta FG loop in thymic selection and T cell function.

The asymmetric disposition of T cell receptor (TCR) Cbeta and Calpha ectodomains creates a cavity with a side-wall formed by the rigid Cbeta FG loop. To investigate the significance of this conserved structure, we generated loop deletion (betaDeltaFG) and betawt transgenic (tg) mice using the TCR beta subunit of the N15 CTL. N15betawt and N15betaDeltaFG H-2(b) animals have comparable numbers of thymocytes in S phase and manifest developmental progression through the CD4(-)CD8(-) double-negative (DN) compartment. N15betaDeltaFG facilitates transition from DN to CD4(+)8(+) double-positive (DP) thymocytes in recombinase activating gene (RAG)-2(-/-) mice, showing that pre-TCR function remains. N15betaDeltaFG animals possess approximately twofold more CD8(+) single-positive (SP) thymocytes and lymph node T cells, consistent with enhanced positive selection. As an altered Valpha repertoire observed in N15betaDeltaFG mice may confound the deletion's effect, we crossed N15alphabeta TCR tg RAG-2(-/-) with N15betaDeltaFG tg RAG-2(-/-) H-2(b) mice to generate N15alphabeta RAG-2(-/-) and N15alphabeta.betaDeltaFG RAG-2(-/-) littermates. N15alphabeta.betaDeltaFG RAG-2(-/-) mice show an 8-10-fold increase in DP thymocytes due to reduced negative selection, as evidenced by diminished constitutive and cognate peptide-induced apoptosis. Compared with N15alphabeta, N15alphabeta.betaDeltaFG T cells respond poorly to cognate antigens and weak agonists. Thus, the Cbeta FG loop facilitates negative selection of thymocytes and activation of T cells.

Amino Acid Sequence↗
PubMed2002

CD2 facilitates differentiation of CD4 Th cells without affecting Th1/Th2 polarization.

The role of CD2 in murine CD4 helper T cell differentiation and polarization was examined using TCR-Cyt-5CC7-I transgenic recombination activating gene-2-/- H-2(a) mice on CD2+/+ or CD2-/- backgrounds. In the absence of CD2, thymic development was abnormal as judged by reduction in the steady state number of total, double-positive, and CD4 single-positive (SP) thymocytes, as well as a defect in their restorative dynamics after peptide-induced negative selection in vivo. In addition, in CD2-/- animals, lymph node CD4 SP T cells manifest a 10- to 100-fold attenuated activation response to cytochrome c (CytC) agonist peptides as judged by induction of CD25 and CD69 cell surface expression or [(3)H]TdR incorporation; differences in the magnitude of responsiveness and requisite molar peptide concentrations were even greater for altered peptide ligands. Although the presence or absence of CD2 did not impact the final Th1 or Th2 polarization outcome, CD2 expression reduced the CytC peptide concentration threshold necessary to facilitate both Th1 and Th2 differentiation. In vivo administration of CytC peptide to CD2-/- animals yielded an impaired CD4 SP T cell effector/memory phenotype compared with similarly treated CD2+/+ mice. Analysis of TCR-Cyt-5CC7-I human CD2 double-transgenic mice similarly failed to reveal a preferential Th1 vs Th2 polarization. Collectively, these results indicate that CD2 is important for the efficient development of CD4 SP thymocytes and TCR-dependent activation of mature CD4 lymph node T cells, but does not direct a particular helper T cell subset polarity.

Amino Acid Sequence↗
PubMed2002

Thioredoxin-mediated redox control of human T cell lymphotropic virus type I (HTLV-I) gene expression.

Thioredoxin (TRX) is a small ubiquitous protein with multiple biological functions, including the thiol-mediated redox-regulation of gene expression. We have previously demonstrated that human TRX is overexpressed as a major protein oxidoreductase in human T cell lymphotropic virus type I (HTLV-I)-infected cells. In the present study, we investigated the relationship between TRX and viral gene expression in HTLV-I infection. To study the mechanism that causes overexpression of TRX in HTLV-I-infected cells, we first examined the effect of the HTLV-I transactivator, Tax, on TRX expression. Induction of HTLV-I Tax protein increased the expression of TRX protein in a Tax-transfected Jurkat cell line, JPX-9. Moreover, chloramphenicol acetyltransferase (CAT) analysis with a reporter gene containing the TRX promoter revealed that Tax activates the transcription of TRX gene. To study the role of overexpressed TRX in HTLV-I infection, we next examined the effect of TRX on HTLV-I long terminal repeat (LTR)-mediated transcription using CAT analysis. In an HTLV-I-infected human T cell line MT-2, the HTLV-I LTR transactivation was suppressed by the overexpression of wild-type TRX, but activated by the introduction of inactive mutant TRX. Moreover, in HTLV-I negative Jurkat T cells, the HTLV-I LTR transactivation induced by Tax was also repressed by overexpression of wild-type TRX. Because cellular redox changes were shown to affect the HTLV-I gene expression, it is likely that TRX modulates the HTLV-I gene expression by regulating cellular redox state. Taken together, these findings suggest that overexpressed TRX, which is induced by HTLV-I Tax, may play an important role in HTLV-I infection through the negative regulation of viral gene expression.

Gene Expression Regulation, Viral↗
PubMed2002

Baicalin induces apoptosis via mitochondrial pathway as prooxidant.

Baicalin is a flavonoid and a major component of a herbal medicine, Sho-saiko-to, which is commonly used for treatment of chronic hepatitis in Japan and China. Flavonoids including baicalin have been reported to not only function as anti-oxidants but also cause cytotoxic effect. We investigated the mechanism of baicalin-induced cytotoxicity in leukemia-derived T cell line, Jurkat cells. When cells were cultured with 50-200 microg/ml baicalin for 6h, caspase-3 was activated and then cells fell into apoptosis. Induction of apoptosis by baicalin was accompanied with the marginal generation of intracellular reactive oxygen species (ROS), the increase of the cytosolic fractions of cytochrome c, and the disruption of mitochondrial transmembrane potential (DeltaPsi(m)) prior to the activation of caspase-3. The pre-culture with 5 mM of buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, facilitated baicalin-induced disruption of DeltaPsi(m) and induction of apoptosis. The pre-culture with N-benzyloxycarbonyl-valyl-alanyl-aspartyl fluoromethylketone (Z-VAD-fmk), a pan-caspase inhibitor, partially suppressed the induction of apoptosis. On the other hand, baicalin showed little toxic effect on peripheral blood mononuclear cells (PBMCs) from healthy volunteers. These results indicate that baicalin acts as a prooxidant and induces caspase-3 activation and apoptosis via mitochondrial pathway.

Antineoplastic Agents, Phytogenic↗
PubMed2002

Peptide-induced negative selection of thymocytes activates transcription of an NF-kappa B inhibitor.

Negative selection eliminates thymocytes bearing autoreactive T cell receptors (TCR) via an apoptotic mechanism. We have cloned an inhibitor of NF-kappa B, I kappa BNS, which is rapidly expressed upon TCR-triggered but not dexamethasone- or gamma irradiation-stimulated thymocyte death. The predicted protein contains seven ankyrin repeats and is homologous to I kappa B family members. In class I and class II MHC-restricted TCR transgenic mice, transcription of I kappa BNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides. I kappa BNS blocks transcription from NF-kappa B reporters, alters NF-kappa B electrophoretic mobility shifts, and interacts with NF-kappa B proteins in thymic nuclear lysates following TCR stimulation. Retroviral transduction of I kappa BNS in fetal thymic organ culture enhances TCR-triggered cell death consistent with its function in selection.

Amino Acid Sequence↗