Biomedical subjects

S Habu

Publications and source records attributed to S Habu.

At least 19 recordsLinked to original sources

PubMed2003

Increased susceptibility to airway responses in CD40-deficient mice.

The interaction between CD40 and its ligand (CD154) is crucial for IL-12 production and effective humoral immunity such as IgE production. Although the interaction seems to play a crucial role in asthmatic inflammation, previous studies investigating the role of the CD40 and CD154 interaction in experimental animal models of asthma are complicated due to multistep reactions in developing asthma. Here, in order to investigate the role of CD40 in the effector phase in the development of airway responses, we used CD40-deficient mice backcrossed with mice transgenic for an ovalbumin (OVA)-specific TCR (TCRtg). Using intranasal OVA administration followed by aerosol inhalation of OVA, greater airway hyperreactivity and eosinophilia in bronchoalveolar lavage fluid (BALF) were observed in CD40-deficient mice backcrossed with TCRtg mice (CD40-/-/ TCRtg mice), compared with control littermates (CD40+/+/ TCRtg mice). CD4+ helper T cell subset analysis of lung draining lymph nodes revealed that the Th1 component was significantly decreased in CD40-/-/ TCRtg mice. Airway hyperreactivity and airway eosinophilia significantly correlated with the predomination of Th2 cells. Cytokine measurements in BALF also showed decreased IL-12 and the predominance of Th2 cells in CD40-/-/ TCRtg mice. These results suggest that CD40 may play a protective role in developing asthma in the phase after establishing specific memory T cells through the regulation of the balance between Th1 and Th2 cells presumably via induction of IL-12.

Animals↗

PubMed2002

Lactobacillus casei strain Shirota suppresses serum immunoglobulin E and immunoglobulin G1 responses and systemic anaphylaxis in a food allergy model.

BACKGROUND: Our previous study using allergen-sensitized murine splenocyte cultures has shown that Lactobacillus casei strain Shirota (LcS), a lactic acid bacterium widely used as a starter for fermented milk products, suppresses IgE production through promoting a dominant Th1-type response mediated by IL-12 induction. OBJECTIVE: We tried to evaluate the ability of LcS to suppress both IgE response and allergic reactions in vivo using a food allergy model with ovalbumin-specific T cell receptor transgenic (OVA-TCR-Tg) mice. METHODS: The ability of heat-killed LcS to induce IL-12 in serum was tested. OVA-TCR-Tg mice were fed a diet containing OVA for 4 weeks and injected with LcS intraperitoneally three times in the first week of this period. Cytokine and antibody secretion by splenocytes, and serum IgE and IgG1 responses were examined. The inhibitory effect of LcS on systemic anaphylaxis induced by intravenous challenge of OVA-fed OVA-TCR-Tg mice with OVA was also tested. RESULTS: Intraperitoneal injection of LcS induced an IL-12 response in the serum of OVA-TCR-Tg mice. In the food allergy model, LcS administration skewed the pattern of cytokine production by splenocytes toward Th1 dominance, and suppressed IgE and IgG1 secretion by splenocytes. The ability of LcS to modulate cytokine production was blocked by anti-IL-12 antibody treatment. LcS also inhibited serum OVA-specific IgE and IgG1 responses and diminished systemic anaphylaxis. CONCLUSION: LcS administration suppresses IgE and IgG1 responses and systemic allergic reactions in a food allergy model, suggesting a possible use of this lactic acid bacterium in preventing food allergy.

Anaphylaxis↗

PubMed2001

Overexpression of AML1 transcription factor drives thymocytes into the CD8 single-positive lineage.

To understand the gene regulation involved in the development of single-positive (SP) thymocytes, we generated transgenic mice in which the AML1 transcription factor is overexpressed. In these mice the number of CD8 SP thymocytes was greatly increased, and this continued to be true even when MHC class I was absent. This promotion to the CD8 SP lineage was not, however, observed when both class I and class II were absent. Furthermore, even thymocytes carrying MHC class II-restricted TCR differentiated into the CD8 SP lineage when AML1 was overexpressed. The selected CD8 SP cells were, however, unable to mature, as judged by the expression level of heat-stable Ag. Thus, overexpression of AML1 is able to skew class II-restricted thymocytes into the CD8 SP lineage, but not to drive the maturation of resulting selected CD8 SP cells.

Animals↗

PubMed2001

p53 Homologue p63 represses epidermal growth factor receptor expression.

Tumor suppressor p53 has been shown to transactivate epidermal growth factor receptor (EGFR) expression through binding to a putative p53 responsive element in the EGFR promoter between nucleotides -265 and -239 (EGFRp53RE). Isotypes of p63 gene products, recently identified as p53 relatives, have a similar function to transactivate several p53 target gene promoters. However, our results indicate that TAp63gamma has a very low ability to bind to the EGFRp53RE and surprisingly represses both basal EGFR promoter activity and endogenous EGFR expression. Transient transfection assays show that the EGFR promoter region between -348 and -293, containing two Sp1 sites, is crucial for the repression of the EGFR expression by TAp63gamma. Mutations in these Sp1 sites in the reporter constructs result in loss of the TAp63gamma repression effect. We further show that TAp63gamma directly interacts with Sp1 by immunoprecipitation analysis and that TAp63gamma impairs Sp1 binding to the target DNA site in electrophoretic mobility shift assays. These results suggest that TAp63gamma is involved in the regulation of the EGFR gene expression through interactions with basal transcription factors.

DNA↗

PubMed2001

Identification of a novel retrovirus long terminal repeat (LTR) that is targeted by p51A (TAp63gamma) and selective dominant-negative activity of p73L (DeltaNp63alpha) toward p53-responsive promoter activities.

The p51/p73L/p63/p40 gene, recently identified as a p53 homolog, encodes two major isoforms, p51A and p73L, which are suggested to have similar functions synonymous with p53 and dominant-negative activity toward both p53 and p51A, respectively. We have cloned a high affinity genomic fragment bound to p51A that was assigned to be a novel retrovirus long terminal repeat. Strikingly, this fragment was found to bind to both p53 and p73L with similar affinity to p51A. Additional demonstration with known p53 response elements suggested that DNA-binding profiles of p51A and p73L were very similar but were distinct from that of p53. Consistent with this novel finding, transient cotransfection experiments in mammalian cells suggested that p73L, when it was expressed at a low level, selectively suppressed p53-dependent transactivation of p21-luc and mdm2-luc but not of cyclinG-luc and bax-luc reporters. These data raise the possibility that p73L differentially modulates the p53 function according to the distinct DNA-binding affinity between these two proteins.

Animals↗

PubMed2001

Surface molecules essential for positive selection are retained but interfered in thymic epithelial cells after monolayer culture.

It has been reported that the three-dimensional structure of thymic epithelial cells (TECs) is responsible for thymic positive selection but that this ability disappears when TECs are cultured in monolayer. These results have supported the hypothesis that certain TEC-specific molecules are extinguished during monolayer culture. In this study, using MHC class II-restricted T-cell receptor transgenic mice, we demonstrated that preselected CD4(+)8(+) (DP) thymocytes were inhibited from developing into CD4(+)8(-) (CD4SP) cells in reaggregate thymus organ culture with monolayer-cultured TECs, but this inhibition was removed when TECs were cultured in monolayer with protein synthesis inhibitor or when the cultured TECs were treated with fixative. These results seem to be inconsistent with the previous hypothesis and indicate that monolayer culture allows TECs to retain the surface molecules necessary for positive selection but interferes with their function, which must be sustained for three dimensional structure.

Animals↗

PubMed2001

Transcriptional dysregulation of the p73L / p63 / p51 / p40 / KET gene in human squamous cell carcinomas: expression of Delta Np73L, a novel dominant-negative isoform, and loss of expression of the potential tumour suppressor p51.

We have recently identified a second p53 -related p73L gene, also referred to as p63 / p51 / p40 / KET gene, which encodes the 2 major isoforms p73L and p51 resulting from different exon usage at their amino terminal regions. Although p73L and p51 are suspected to play oncogenic and tumour suppressive roles in mammalian cells, respectively, no evidence of linkage between the expression of these isoforms and human cancers has been reported so far. In this study, we first investigated the expression profile of p51 and p73L in various human tumour cell lines and found that a novel isoform, termed DeltaNp73L, was predominantly expressed in squamous cell carcinomas. The expression profile of DeltaNp73L/p73L/p51 in primary human skin cancer specimens showed that the expression of p51 was frequently lost (62%) but was detected in all normal skin samples. In p51-expressing skin cancers, DeltaNp73L expression was associated at a high frequency (75%) though it was not detected in normal skin tissues. Transient co-transfection data indicate the possibility that DeltaNp73L can inhibit p53-, and more preferentially, p51-mediated transactivation. These data suggest that the loss of expression of p51 and/or the expression of DeltaNp73L might contribute to the pathogenesis of human squamous cell carcinomas.

Carcinoma, Squamous Cell↗

PubMed2001

Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development.

Mutations in the CD40 ligand (CD40L) gene lead to X-linked immunodeficiency with hyper-IgM, which is often associated with autoimmune diseases. To determine the contribution of defective CD40-CD40L interactions to T cell autoreactivity, we reconstituted CD40-CD40L interactions by transferring T cells from CD40-deficient mice to syngenic athymic nude mice and assessed autoimmunity. T cells from CD40-deficient mice triggered autoimmune diseases accompanied with elevations of various autoantibodies, while those from wild-type mice did not. In CD40-deficient mice, the CD25(+) CD45RB(low) CD4(+) subpopulation which regulates T cell autoreactivity was markedly reduced. CD40-deficient APCs failed to induce T regulatory cells 1 producing high levels of an inhibitory cytokine, IL-10 in vitro. Furthermore, autoimmune development was inhibited when T cells from CD40-deficient mice were cotransferred with CD45RB(low) CD4(+) T cells from wild-type mice or with T regulatory cells 1 induced on CD40-expressing APCs. Collectively, our results indicate that CD40-CD40L interactions contribute to negative regulation of T cell autoreactivity and that defective interactions can lead to autoimmunity.

Animals↗

PubMed2001

Limited effect of chromatin remodeling on D(beta)-to-J(beta) recombination in CD4+CD8+ thymocyte: implications for a new aspect in the regulation of TCR beta gene recombination.

We have generated mutant mice in which TCR beta chain enhancer (E(beta)) was replaced with the TCR alpha chain enhancer (E(alpha)). Using this mouse model, we analyzed (i) recombination status of the TCR beta chain genes after functional V(D)J rearrangements occurred in the first allele during double-negative (DN)-to-double-positive (DP) transition and (ii) involvement of E(beta) for the expression of rearranged TCR beta chain genes. Our data show that E(alpha) substituted for E(beta) function to express a similar extent of TCR beta chains exactly at the same time as did E(beta) (CD25+CD44- DN stage), although the proportion of TCR beta+ cells at this stage was low in mutant mice. At the DP stage, germline transcription and histone acetylation of D(beta)-J(beta) loci were detectable at a high degree in both mutant and wild-type mice. However, DP cells in mutant mice retained the germline D(beta)-J(beta) configuration at a higher frequency than that of wild-type mice, whereas both DP cells expressed TCR beta chains to a similar extent. These data suggest that chromatin opening has a limited impact on D(beta)-to-J(beta) recombination at the DP stage and that E(alpha) is functionally equivalent to E(beta) in promoting expression of functionally rearranged TCR beta chain genes through DN-to-DP transition.

Animals↗

PubMed2001

CD4 T cells monospecific to ovalbumin produced by Escherichia coli can induce colitis upon transfer to BALB/c and SCID mice.

Although some animal models suggest an involvement of CD4 T cells reactive to luminal microbial antigen(s) for the pathogenesis of inflammatory bowel diseases (IBD), direct linkage between microflora-driven clonal expansion of CD4 T cells and the development of colitis has not been well studied. Here, BALB/c and SCID mice were given CD4 T cells purified from Rag-2(-/-) mice crossed to transgenic mice expressing TCR specific to ovalbumin (OVA) then administered with antibiotic-resistant Escherichia coli producing OVA (ECOVA) or LacZ (ECLacZ) via the rectum. The ECOVA-inoculated BALB/c and SCID mice developed a subacute colitis with microscopic features of distortion of crypt architecture, loss of goblet cells, and focal infiltration by mononuclear cells in the lamina propria (LP) and submucosa. Expanding OVA-specific CD4 T cells were detected in colonic follicles of mice with ECOVA. Early in colitis, OVA-specific CD4 T cells producing IFN-gamma predominate in the LP of the colon, which was followed by an emergence of OVA-specific CD4 T cells producing IL-4 and IL-10 at a later time point. Co-transfer of an IL-10-secreting OVA-specific CD4 T cell line prevented colitis. Thus, an expansion of CD4 T cells monospecific to OVA, an antigen non-cross-reactive to colonic tissue, can mediate both induction and inhibition of the colitis which was associated with hyperplasia of lymph follicles.

Administration, Rectal↗

PubMed2001

Ciliary neurotrophic factor, a gp130 cytokine, regulates preovulatory surges of luteinizing hormone and prolactin in the rat.

Ciliary neurotropic factor (CNTF) is a neuroregulatory cytokine belonging to the interleukin-6 type cytokine superfamily. Although a few studies have reported a facilitatory action of CNTF on the reproductive axis in rodents, information along this line is still very limited. In this study, we examined a possible role of CNTF in the generation of ovarian steroid-induced luteinizing hormone (LH) and prolactin (PRL) surges in the rat, a crucial physiological event in mammalian reproduction. Experiments were performed on both normally-fed and 3-day-fasted rats, ovariectomized and primed with estradiol and progesterone. Blood was collected every 30 min between 11:00 and 18:00 h, to measure LH and PRL. Drugs were given intracerebroventricularly at 11:00 h. Compared to control serum, undiluted as well as threefold dilutions of anti-CNTF serum caused partial but significant suppression of LH surges. Both concentrations of the antibody also delayed the onset of PRL surge to a comparable degree. Fasted rats did not exhibit significant surges of the hormones, while 0.3 and 1.0 nmol, but not 0.1 nmol, recombinant human CNTF led to a dose-dependent recovery of both LH and PRL surges. These results demonstrate for the first time a significant role of CNTF in the generation of preovulatory LH and PRL surges in the rat. CNTF may thus be another humoral signal linking nutrition and reproductive function.

Animals↗

PubMed2001

Radioimmunoassay for somatostatin receptor type 2.

OBJECTIVE: To develop radioimmunoassay for somatostatin receptor type 2 (SSTR2) and search for its presence in certain rat tissues. METHODS: Anti-SSTR2 antiserum has been raised in New Zealand white rabbits immunized with a conjugate of synthetic SSTR2 with bovine serum albumin. Radioiodination of SSTR2 was performed by chloramin T method followed by purification of radioiodinated material on Sephadex G-25 column. RESULTS: The obtained antibody did not crossreact with SSTR1, SSTR3, SSTR4, SSTR5, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. The assay was performed with a double antibody system. SSTR2 was extracted from the tissues with acid acetone. The dilution curve of acid acetone-extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue SSTR2 was about 89 %, and the intra-assay and inter-assay variations were 4.9 % and 7.8 %, respectively. SSTR2 was found in the hypothalamus, cerebrum, cerebellum, pituitary, stomach and testis. CONCLUSIONS: These data suggest that this assay system is suitable for the estimation of SSTR2 in the tissues.

Acetone↗

PubMed2001

[Implication on thyroid function tests].

We tried to investigate the present problems, concerning the reference individual and interval in thyroid function tests and find the solutions for them. We are now using healthy adults for the reference individual and interval. Recently, we found the sex-difference and age-related changes for reference individual and interval in free T3 measurement. We raised the questions on whether there are any sex-differences and/or age-related changes or not. Surprisingly, there were almost no detailed data about them especially in Japan. Therefore, we examined the Europe data, and found out some kind of sex-differences and age-related changes. We propose the following examinations using many Japanese population in order to provide a precise and proper reference individual and interval: 1. Whether there are any sex-difference in thyroid function tests? 2. Whether there are age-related change in thyroid function tests, for instance, simply dividing population into the immature, adult and the aged?

Adult↗

PubMed2000

Diminution of the AML1 transcription factor function causes differential effects on the fates of CD4 and CD8 single-positive T cells.

In the thymic cortex, T lymphocytes are positively selected to survive and committed either to the CD4 single-positive (SP) or the CD8 SP lineage. The SP cells then pass through a step of maturation in the medulla and are delivered to peripheral lymphoid tissues. We examined the role of AML1, the gene encoding a transcription factor, in the above processes by using the transgenic mice expressing a dominant interfering form of AML1 as well as mice targeted heterozygously for AML1. One phenotypic change seen in the AML1-diminished mice was the reduction in the numbers of both CD4 SP and CD8 SP thymocytes, reflecting the partial impairment of the transition from the double-positive to SP stage. In addition, distinct from the above abnormality, perturbed were several aspects of SP cells, including the maturation of SP thymocytes, the recent thymic emigration, and the proliferative responsiveness of peripheral T cells to TCR stimulation. Interestingly, the AML1 diminution caused inhibitory and enhancing effects on the CD4 SP and CD8 SP cells, respectively. These differential effects are most likely related to the reduction in the peripheral CD4 SP/CD8 SP ratio observed in the AML1-diminished mice. The AML1 transcription factor thus maintains the homeostasis of each SP subset by functioning at the later stages of T lymphocyte differentiation.

Animals↗

PubMed2000

Oral immunization with size-purified microsphere beads as a vehicle selectively induces systemic tolerance and sensitization.

Oral administration of antigens has long been recognized as a method to prevent or delay the onset of diseases associated with untoward immune responses to self and non-self antigens. Although oral administration of antigens offers a convenient way to induce systemic tolerance, its therapeutic potential has been seriously limited by the fact that it requires repeated feeding of a large amount of antigens and that it may deteriorate ongoing autoimmune diseases when autoantigens are employed. We have previously shown that orally administered poly-D,L-lactic acid (PDLLA) microspheres containing an antigen were selectively distributed to Peyer's patches (PP) and systemic lymphoid tissues according to their diameter and then released the antigen over a long period of time. We now report that a single dose of intragastric immunization with a PDLLA microsphere 7-10 micrometer in diameter and containing 2 mg of OVA was as effective as 100 mg of water soluble OVA to suppress OVA-specific IgG and DTH response. This was associated with a large increase of Interferon-gamma production by PPT cells stimulated with an antigen and a small increase in secretory IgA specific to OVA. In contrast, administration of an antigen encapsulated in microspheres 3-4 microm in diameter led to an enhanced OVA-specific IgG response and no significant increase in OVA-specific secretory IgA. Thus, by utilizing microspheres of an appropriate diameter as a vaccination vehicle, we were able to selectively induce both systemic tolerance and sensitization by oral ingestion of single low dose of an antigen.

Administration, Oral↗

PubMed2000

Interleukin-4-dependent induction of preproenkephalin in antigen-specific T helper-type 2 (Th2) cells.

Naive Th cells obtained from OVA(323-339)-specific DO11.10 TCR-Tg mice did not express preproenkephalin (PPE) mRNA. However, culture of naive Th cells with OVA(323-339) peptide (OVA-pep) plus IL-2 under Th2-inducing conditions for 7 days resulted in an induction of PPE mRNA. The PPE mRNA was also induced by culturing with OVA-pep plus IL-2 (neutral condition). However, PPE mRNA induction under neutral conditions was totally abrogated by addition of anti-IL-4 mAb. The existence of methionine-enkephalin was also demonstrated in peptidase-digested peptides derived from Th2 cell lysate. These results demonstrate that IL-4 is a critical factor for the induction of PPE mRNA in freshly expanded antigen-specific Th2 cells.

Animals↗

PubMed2000

Molecular basis for functional maturation of thymocytes: increase in c-fos translation with positive selection.

In the process of positive selection, immature CD4+8+ double positive (DP) thymocytes expressing TCR reactive to self-MHC by appropriate avidity develop into mature thymocytes. Positive selection involves not only down-regulation of either CD4 or CD8 but also acquisition of immunocompetent potential such as cell proliferation and cytokine production. To understand the molecular basis for such functional maturation during the positive selection process, we examined whether nonselected DP, selected DP, and CD4+8- single positive thymocytes possess the activation potential for signaling pathways from mitogen-activated protein kinases (extracellular signal-regulated kinase and c-Jun N-terminal kinase) to AP-1. In response to stimulation, a marked induction of c-Fos protein expression as well as cell proliferation is detected only in CD4+8- single positive cells but not in selected and nonselected DP cells, though mitogen-activated protein kinase activities and c-fos transcripts are equally induced. In the presence of proteasome inhibitors, c-Fos protein became detectable in selected DP cells but still not in nonselected DP cells, suggesting that DP cells receiving positive selection signals acquire the capacity to translate the c-fos gene, but it may not be sufficiently high to overcome the degradation of c-Fos protein. These data indicate that the translating ability of the c-fos gene is up-regulated in the thymic positive selection process, from nonselected DP to CD4+8- single positive cells through positively selected DP cells. The distinguished responsiveness to stimulation in thymocytes with and without positive selection may be a result in part of the distinct regulation of the c-fos gene at the translational level.

Animals↗

PubMed2000

Establishment of efficient reaggregation culture system for gene transfection into immature T cells by retroviral vectors.

To overcome low efficiency of retroviral infection into immature T cells, we modified reaggregation fetal thymus organ culture by closely packed co-culture with virus-producing cells (VPC). The viral vector was constructed in chimeric vector, pMX, with IRES and tailless-rat CD2 as a surface marker of infected cells. A rearranged TCR beta gene (Vbeta8.2) was further inserted into the construct for investigating effect of the introduced gene in T cell development. Using this system, we succeeded to transfer the viral vector into immature thymocytes at a remarkably higher efficiency compared to conventional methods using medium containing retrovirus. Moreover, the introduced TCR beta gene was expressed on thymocytes of RAG2-deficient mice to induce in the transition of CD4-CD8- double-negative (DN) into CD4+CD8+ double-positive (DP) cells by transducing beta-selection signaling. Thus, our modified reaggregation culture system is useful for studying the molecular mechanism of T cell development due to a highly efficient gene transfer into immature T cells.

Animals↗