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Biomedical subjects

Y Mizushima

Publications and source records attributed to Y Mizushima.

At least 19 recordsLinked to original sources
PubMed1996

Characterization of T cell receptor beta chains of accumulating T cells in skin allografts in mice.

The study of T cells involved in the immune reaction that occurs in engrafted organs should provide information that would be helpful in the regulation of allograft rejection in organ transplantation. Toward this end, we focused on detection and characterization of accumulating T cells in mouse skin allografts from B10.A(4R) to C57BL/6 mice in vivo. T cell receptor beta genes were amplified by reverse transcriptase-PCR from mRNA of the skin grafts, and accumulating T cell receptor beta gene clonotypes were identified by their single strand conformation polymorphism. Their joining region usage and the amino acid sequences of the complementarity-determining region-3 were then determined. The results were as follows: (1) Distinct oligoclonal accumulation of T cells was more prevalent in the skin allografts than in the syngenic skin grafts. (2) Although the accumulating T cell clonotypes appeared to use many different variable-region gene families, preferential combinations of variable region-joining region were found. (3) Several homologous amino acid sequences were found in these accumulating TCR beta genes in allografts, suggesting that these T cells are driven by the same or similar antigens. (4) In addition, little T cell accumulation was found in spleens from the mice with allografts or syngenic skin grafts. Taken together, accumulating T cells in the skin allografts were detected in vivo, and some appeared to have characteristics in common. This may lead to T cell clonotype-specific therapy in organ transplantation.

Amino Acid Sequence↗
PubMed1996

Role of gamma delta T cells in pathogenesis and diagnosis of Behcet's disease.

BACKGROUND: Behcet's disease (BD) is a multisystem disorder of unknown pathogenesis. The diagnosis is based on a set of international clinical criteria. Previous investigations have suggested that immunological cross-reactivity between peptides within streptococcal heat-shock proteins and human peptides might be involved in the pathogenesis of BD. We tested four peptides from mycobacterial heat-shock proteins to see if they specifically stimulated gamma delta T cells from BD patients. We then investigated this response to see whether it could be used as a laboratory test to diagnose BD. METHODS: We used a T-cell proliferative test to assay responses to four mycobacterial 65 kDa heat-shock-protein peptides and to four homologous peptides derived from the sequence of the human 60 kDa heat-shock protein. FINDINGS: We elicited significant gamma delta T-cell responses to the mycobacterial peptides in 25 (76%) of 33 patients with BD, compared with 2 (3.6%) of 55 controls with recurrent oral ulcers, systemic disease, or no disorders. The proportion of BD patients who had false-negative results decreased if the test was done during clinical manifestation of disease activity. There was a correlation between disease activity and T-cell responses. Four homologous peptides from human 60 kDa heat-shock protein also specifically stimulated T cells from patients with BD but with lower stimulation indices. INTERPRETATION: Activation of peripheral-blood mononuclear cells with the four heat-shock-protein peptides elicited significant T-cell proliferative responses by the gamma delta subset of T cells, which may regulate alpha beta T cells. Because these peptides have a high specificity for BD, this assay can be used as a laboratory diagnostic test for BD.

Adult↗
PubMed1996

Stable clonal expansion of T cells induced by bone marrow transplantation.

The immune mechanisms of T cells regeneration after bone marrow transplantation (BMT) and the factors maintaining allogeneic marrow graft in the host are still unknown. To pursue this issue, we analyzed T-cell clonality of peripheral blood lymphocytes (PBLs) in BMT recipients, using reverse transcription polymerase chain reaction with T-cell receptor (TCR) V beta gene segment-specific primers and single-strand conformation polymorphism. PBLs from patients and donors showed a heterogeneous T-cell population with oligoclonal accumulations of CD8+ T cells. When PBLs were cultured in HLA-matched mixed lymphocytes reaction in vitro, no distinct clonal expansion was observed. However, after BMT, oligoclonal expansions were induced in the recipients in vivo, without a restriction of TCR V beta gene usage. Although part of the expansion was transient, the majority was repeatedly detected even several months later. Our results suggested that certain in vivo mechanisms maintain a stable clonal expansion of distinct T cells in marrow recipients. We also found in a single patient with graft-versus-host disease a replacement of expanded clones by other clones during follow-up. Diminishing numbers of accumulation clones were found in long-term marrow recipients, indicating a general tendency for clonal expansion to subside progressively. Considered together, our data suggest the involvement of clonally expanded T cells in lymphoid regeneration and in acute and chronic immune responses after BMT.

Adolescent↗
PubMed1996

High frequencies of identical T cell clonotypes in synovial tissues of rheumatoid arthritis patients suggest the occurrence of common antigen-driven immune responses.

OBJECTIVE: To investigate T cell antigen receptor (TCR) clonotypes in rheumatoid arthritis (RA) lesions. METHODS: Reverse transcriptase-polymerase chain reaction with TCR V beta family-specific primers and subsequent single-strand conformation polymorphism (SSCP) analysis were performed. Direct nucleotide sequencing was also conducted. RESULTS: A distinct clonal expansion of T cells was observed in the synovium. Furthermore, identical bands in samples of different areas of the same lesion were obtained by SSCP analysis. Nucleotide sequencing revealed that T cell clonotypes of identical mobility on SSCP analysis had the same nucleotide sequence and thus were identical clones. In 6 RA patients, 60-100% of the expanded T cell clonotypes had identical migration patterns in 2 different samples, indicating that this percentage represents commonly existing T cell clonotypes in the affected joint. Furthermore, the J beta 2.1 gene segment was used predominantly by the TCR V beta clonotypes that commonly expanded in the different portions of the same joint. CONCLUSION: These results suggest that the immune response in RA is not random, but rather is driven by common stimuli.

Adult↗
PubMed1996

Time course analysis of alpha+ beta+ T cell clones during normal pregnancy.

During normal pregnancy, the fetus continues to mature inside the uterus without rejection. Inherited paternal antigens could be targeted by the maternal immune system. These reactions are believed to play a role in a number of habitual abortions. However, the precise maternal mechanisms preventing fetal tissue rejection are not well understood. Maternal T cells should recognize fetal antigens, so it is conceivable that antigen-specific T cell response to fetal antigens would occur by proliferation and accumulation of certain T cell clones in the pregnant mother. To elucidate the maternal immune response to the fetus we investigated the clonality of expanded T cells in peripheral blood lymphocytes in ten normal pregnant women. We employed reverse transcriptase-polymerase chain reaction for T cell receptor beta chain gene and subsequently analyzed the PCR product by single-strand conformation polymorphism analysis. A large number of distinctly expanded T cell clones were detected during pregnancy. These accumulations were observed as early as the ninth to tenth week post-conception and reached a maximum during the second trimester, suggesting the existence of dynamic antigen-specific T cell responses in the pregnant mother. However, after the 30th week of gestation, nearly all expanded T cell clones disappeared before parturition and the degree of clonality reached almost normal levels. Our results clearly indicate the existence of dynamic maternal T cell responses during pregnancy.

Adult↗
PubMed1996

Antisense phosphorothioate oligonucleotide inhibits interleukin 1 beta production in the human macrophage-like cell line, U937.

To find more efficacious therapeutic possibilities for treatment of inflammatory disease, we studied the effects of antisense oligonucleotides on interleukin 1 beta (IL-1 beta) production of the human macrophage-like U937 cells. U937 cells were incubated with several kinds of oligonucleotides. Total human IL-1 beta production was determined using an enzyme-linked immunosorbent assay. An antisense phosphorothioate oligonucleotide (S-oligo), complementary to the sequence, including initiation codon of the IL-1 beta gene, inhibited IL-1 beta production in a dose-dependent and sequence-specific manner. The effect of the antisense S-oligo was neutralized by mixing with a sense but not with a scramble S-oligo. Cellular uptake of S-oligo scanned with a laser confocal imaging system was time and temperature dependent, and its intracellular distribution was mainly to the cytosols in U937 cells. Human IL-1 beta antisense S-oligo inhibited IL-1 beta production of U937 cells, suggesting a potential to reduce some kinds of inflammatory processes.

Base Sequence↗
PubMed1996

Cellular uptake and biological effects of antisense oligodeoxynucleotide analogs targeted to herpes simplex virus.

In this study, we synthesized antisense oligodeoxynucleotides (ODNs) with phosphodiester, phosphorothioate (S-ODNs), or methylphosphonate linkages complementary to the splicing acceptor site of immediate-early pre-mRNA 5 of herpes simplex virus type 1 (HSV-1). The antiviral activity of each analog on cytopathic effect in cells infected with HSV-1 or HSV-2 was assessed and compared with the cellular uptake of the analog. We found that antisense S-ODNs showed the most potent antiherpetic activity, with 50% inhibitory concentrations of 5 microM for HSV-1 and 0.25 microM for HSV-2. The antiviral effect of antisense S-ODNs was stronger and longer acting than that of acyclovir. Cell association of S-ODNs was the highest and paralleled antiviral activity. Furthermore, some fluorescein isothiocyanate (FITC)-labeled S-ODNs were recognized in the nuclei in HSV-1 infected cells by confocal laser scanning microscopy. S-ODNs located in the nucleus could access the targeted mRNA, which might be responsible for the antiviral activities. Although our study also showed non-sequence-specific activity, which implies that multiple mechanisms are involved, S-ODNs are a promising novel anti-herpetic agent.

Animals↗
PubMed1996

[A combination chemotherapy with CDDP and 5-FU effective for pulmonary metastases in a case of parotid gland carcinoma].

The patient was a 40-year-old female (154 cm, 45 kg). Several months after receiving radiotherapy, surgery and chemotherapy (Epi-ADR, CDDP, PEP) for an undifferentiated carcinoma of the left parotid gland, she had multiple pulmonary metastases without local recurrence. Following 2 cycles of combination chemotherapy with CDDP (30 mg/1 hr, days 1 approximately 5) and 5-FU (1,000 mg/24 hrs, days 2 approximately 6), most pulmonary nodules disappeared. For the remaining pulmonary nodules, one cycle of combination chemotherapy with 5-FU (850 mg/24 hrs, days 1 approximately 5), leucovorin (9 mg x 3/day, days 1 approximately 5) and CDDP (110 mg/2 hrs, day 7) was added, but further improvement was not obtained on chest CT. Side effects were tolerable in both regimens. It was suggested that the combination chemotherapy with CDDP and 5-FU might be useful for the treatment of advanced parotid gland carcinomas.

Adult↗
PubMed1996

[Acute lupus pneumonitis presenting as multiple nodular shadows].

A 24-year-old woman consulted a physician because of fever, dry cough, and arthralgia of one month duration. She was transferred to our hospital for more detailed examination of pancytopenia and diffuse infiltrative shadows on a chest X-ray film. On admission she was depressed, and had oral ulcers. Pleural and pericardial effusions were observed. Laboratory studies were positive for anti-nuclear antibody, anti-DNA antibody, and lupus erethymatosus. Casts were found in the urine, and systemic lypus erythematosus was diagnosed. Chest radiography and CT scan showed nodular shadows in the right S8 (2nodules with 1 cm diameter)(1 cm x 2), right S10 (0.5 cm diameter), and left S8 (0.5 cm diameter) regions, as well as diffuse infiltrative shadows in both lower lung fields. A specimen obtained by transbronchial lung biopsy revealed acute interstitial pneumonia with arteritis. After treatment with prednisolone (60 mg/day), the nodular and infiltrative shadows rapidly disappeared. Cases of lupus pneumonitis presenting as nodular shadows are very rare and are valuable in diagnostic imaging.

Acute Disease↗
PubMed1996

Cellular uptake mechanism for oligonucleotides: involvement of endocytosis in the uptake of phosphodiester oligonucleotides by a human colorectal adenocarcinoma cell line, HCT-15.

We used a 20-mer antisense oligonucleotide against human MDR1 mRNA to study the mechanism of uptake of oligonucleotides by a human colorectal adenocarcinoma cell line, HCT-15.5'-end-32P-labeled oligonucleotides were mainly used. The uptake of phosphodiester oligonucleotides (D-oligo) by HCT-15 cells increased with time, but the uptake of phosphorothioate oligonucleotides (S-oligo) showed minimal increases with time. HeLa cells showed a 4-fold greater D-oligo uptake than did HCT-15 cells, and other cell lines exhibited a 25-fold lower uptake than did HCT-15 cells. On the other hand, S-oligo uptake varied only severalfold among the cell lines used. Doligo up-take consisted of a saturable component (Michaelis constant K(m) = 0.16 microM and maximal uptake rate Vmax = 4.8 pmol/min/mg for HCT-15 cells and K(m) = 0.21 microM and Vmax = 35.6 pmol/min/mg for HeLa cells), i.e., the difference between the cell types was mainly in Vmax. The uptake of labeled D-oligo was inhibited by unlabeled S-oligo at a much lower concentration (Ki = 0.01 microM) than by unlabeled D-oligo (K(m) = 0.16 microM), although the uptake rate of D-oligo was greater than that of S-oligo. The D-oligo uptake was highly temperature dependent. Of the sulfhydryl-modifying reagents (p-chloromercuriphenyl-sulfonic acid and p-chloromercuribenzoic acid), only p-chloromercuribenzoic acid inhibited D-oligo uptake, suggesting the involvement of a protein whose sulfhydryl residues inside the cell are critical for D-oligo uptake. Chloroquine and monensin, which are known to alter the intracellular fate of ligands and receptors, did not affect D-oligo uptake, but phenylarsine oxide, an inhibitor of internalization processes, inhibited uptake significantly. Down-regulation and recovery of D-oligo uptake induced by excess unlabeled D-oligo pretreatment occurred, but this was only partial. Studies with a digitonin equilibrium method and confocal microscopy indicated that intracellular localization of D-oligo was mainly in a vesicular compartment. More stable 3'-end-32P-labeled oligonucleotides were also used, to compare their uptake characteristics with those of the 5'-end-labeled oligonucleotides. The uptake characteristics for the two labels were similar in terms of saturability, greater D-oligo uptake, compared with S-oligo, and the inhibitory effect of p-chloromercuribenzoic acid; however, the stable 3'-end label showed greater uptake than the 5'-end label. Our findings suggest that D-oligo uptake by HCT-15 cells is mediated by endocytosis involving binding sites on the plasma membrane.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1995

Accumulation of distinct T cell clonotypes in human solid tumors.

Tumor-infiltrating lymphocytes (TIL) have been described in a variety of human solid tumors. It is unknown whether such T cells are nonspecific inflammatory cells or a subset of specific host immune responses. To examine this question, we have analyzed the clonotypes of TCR beta-chain messages expressed in TIL, draining lymph nodes, and PBL of 10 patients with uterine or ovarian tumors. We report here that TIL bears distinct T cell clonotype accumulations only in patients without obvious metastasis. In contrast, accumulations of clonally expanded T cells were also found in lymph nodes and PBL of patients with metastatic cancer. The numbers and locations of the accumulated T cell clonotypes seemed to correlate with the stage of tumor invasion and the degree of metastasis. These data support the existence of Ag-driven immune responses to solid tumors in vivo.

Adenocarcinoma↗
PubMed1995

Excessive function of peripheral blood neutrophils from patients with Behçet's disease and from HLA-B51 transgenic mice.

OBJECTIVE: To elucidate the role played by HLA-B51 in the neutrophil hyperfunction of Behçet's disease, we determined the superoxide production by purified peripheral blood neutrophils from Behçet's disease patients, from HLA-B51 positive healthy individuals, and from HLA-B51 transgenic mice. METHODS: Neutrophil function was evaluated by flow cytometric analysis, detecting the conversion of 2',7'-dichlorofluorescin diacetate into dichloroflurescein, induced by superoxide in the neutrophils. RESULTS: A significant correlation between the neutrophil hyperfunction and the possession of HLA-B51 phenotype, regardless of the presence of the disease, was observed in humans. FMLP-stimulated neutrophils (without in vitro priming) from HLA-B51 transgenic mice, but not those from HLA-B35 transgenic mice or from nontransgenic mice, produced substantial amounts of superoxide. CONCLUSION: The HLA-B51 molecule itself may be responsible, at least in part, for neutrophil hyperfunction in Behçet's disease.

Animals↗
PubMed1995

Streptococcus oralis previously identified as uncommon 'Streptococcus sanguis' in Behçet's disease.

The relation between the biochemical and serological properties of 'Streptococcus sanguis' was studied to characterize the strains isolated from dental plaque of patients with Behçet's disease and controls. Seven reference and 100 clinical strains preserved by the Behçet's Disease Research Committee of Japan were identified using established criteria and differentiated with antisera against Strep. oralis ATCC 10557, Strep. sanguis ATCC 10556 and 'Strep. sanguis' ST7, compatible with the criteria. Uncommon serovars (serotypes) KTH-1 (= ATCC 49298), KTH-2 (= ATCC 49296), KTH-3 (= ATCC KTH-4 (= ATCC 49297) and B220 (serovar KTH-1) with both IgA1 protease and neuraminidase (sialidase) were identified as Strep. oralis, whereas common serovars ST3 with IgA1 protease alone and ST7 without both enzymes were identified as Strep. sanguis and Strep. gordonii, respectively. Isolates previously ranked as uncommon serovars were identified as Strep. oralis, whereas the rest ranked as common serovars were identified as the same species as those of the grouping strains. A soft-agar technique was available for species identification except for Strep. oralis serovar KTH-1 reacting with the antiserum against Strep. gordonii ST7. The frequency of isolation of Strep. oralis was higher in Behçet's disease (52%) than in controls (38%), but no difference was observed between the properties of the two groups of isolates. Strep. oralis virulence factors may be involved in breach the mucosal barrier in patients with specific reactivity to these antigens and inducing Behçet's disease.

Behcet Syndrome↗
PubMed1995

An interleukin-1 binding region oligopeptide from the human type I IL-1 receptor reduces acute inflammation, delayed hypersensitivity reaction, and lethal endotoxemia in animals.

The effect of an interleukin-1 binding region oligopeptide from the interleukin-1 receptor on various inflammatory responses was investigated in animal models. A synthetic peptide (KICIRIQIS) corresponding to 86-93 of the extracellular domain of the human type I interleukin-1 receptor was used. Carrageenan-induced rat paw edema, a model of acute inflammation, was dose dependently suppressed by intraperitoneal administration of the peptide. The delayed hypersensitivity reaction to sheep red cells was diminished by pretreatment of mice with the peptide at a relatively high dose. In a murine lethal endotoxemia model, animals treated with the interleukin-1 receptor peptide (10 mg/kg x 4) showed significantly better survival than vehicle-treated animals when the peptide was administered from 20 minutes after lipopolysaccharide injection. Improved survival was accompanied by suppression of lipopolysaccharide-induced production of colony-stimulating factor, although the peptide did not improve hypoglycemia. These findings suggest that the interleukin-1 receptor peptide may be a potential treatment for various inflammatory processes.

Acute Disease↗
PubMed1995

Induction of differentiation into monocyte/macrophage cell lineage of a human eosinophilic leukaemia cell line EoL-1 by simultaneous stimulation with tumour necrosis factor-alpha and interferon-gamma.

Human myeloid leukaemia cell lines have been shown to differentiate into distinct cell lineages in vitro in response to several differentiation-inducing agents. A human eosinophilic leukaemia cell line, EoL-1, has been shown to differentiate into mature eosinophilic granulocytes by treatment with the culture supernatant of a human T-cell line, HIL-3. In this study we have studied whether the EoL-1 cell line has potential to differentiate into cell lineage other than eosinophils. We found that EoL-1 cells cultured in the presence of tumour necrosis factor (TNF)-alpha (10 u/ml) and interferon (IFN)-gamma (1000 u/ml) for 2-4 d differentiated into macrophage-like cells in morphology, and expressed CD14 antigen on their cell surface. It is possible that the small subpopulation of EoL-1 cells which contains non-specific esterase (NSE) activity may be preferentially differentiated by TNF-alpha and IFN-gamma. To clarify this issue, we have cloned the EoL-1 cell line and obtained NSE negative and positive sublines. Both EoL-1 sublines differentiated into monocyte/macrophage-like cells, because: (a) EoL-1 sublines were induced to express CD14 antigen, and (b) they attached firmly to the plastic wells; (c) after differentiation they became strongly positive for NSE staining, and secreted TNF-alpha in response to the stimulation with lipopolysaccharide; and (d) they exhibited potent phagocytic activity. Therefore, we found that the EoL-1 cell line has the ability to differentiate not only into mature eosinophilic cells but also into monocyte/macrophage cell lineage, suggesting that EoL-1 cells represent immature cells with ability to differentiate into multiple cell lineages.

CD11 Antigens↗
PubMed1995

Low serum levels of dehydroepiandrosterone may cause deficient IL-2 production by lymphocytes in patients with systemic lupus erythematosus (SLE).

The principal cause of IL-2 deficiency, a common feature of both murine lupus and human SLE, remains obscure. Recent studies of our own as well as others have shown that dehydroepiandrosterone (DHEA), an intermediate compound in testosterone synthesis, significantly up-regulates IL-2 production of T cells, and that administration of exogenous DHEA or IL-2 via a vaccinia construct to murine lupus dramatically reverses their clinical autoimmune diseases. Thus, we have examined serum levels of DHEA in patients with SLE to test whether abnormal DHEA activity is associated with IL-2 deficiency of the patients. We found that nearly all of the patients examined have very low levels of serum DHEA. The decreased DHEA levels were not simply a reflection of a long term corticosteroid treatment which may cause adrenal atrophy, since serum samples drawn at the onset of disease, which are devoid of corticosteroid treatment, also contained low levels of DHEA. In addition, exogenous DHEA restored impaired IL-2 production of T cells from patients with SLE in vitro. These results indicate that defects of IL-2 synthesis of patients with SLE are at least in part due to the low DHEA activity in the serum.

Adult↗