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Biomedical subjects

K Sugamura

Publications and source records attributed to K Sugamura.

At least 109 records · Page 6Linked to original sources
PubMed1995

Differences in the interleukin-2 (IL-2) receptor system in human and mouse: alpha chain is required for formation of the functional mouse IL-2 receptor.

Reconstitution with mouse interleukin-2 (IL-2) receptor subunits demonstrated that the mouse IL-2 receptor complex was different from the human complex in the alpha chain requirement for the functional mouse receptor complex. The heterotrimeric complex of the mouse exogenous alpha and beta chains and the endogenous gamma chain on mouse lymphoid BW5147 cells showed the ability to bind IL-2 with high affinity, resulting in IL-2-induced tyrosine phosphorylation of a cytosolic tyrosine kinase, JAK3, which is involved in IL-2-dependent signals. Exogenous introduction of the beta chain with the endogenous gamma chain, however, could neither confer appreciable IL-2 binding nor IL-2-induced signal transduction on BW5147 cells, unlike the human beta gamma heterodimer. Mouse spleen CD8+ cells, not having the alpha chain initially, showed IL-2-dependent cell proliferation only when expression of the alpha chain was induced. Collectively, these results illustrate that the functional mouse IL-2 receptor complex necessarily includes the alpha chain, and that the regulation of CD8+ T cell growth during immune reaction depends upon alpha chain expression.

Animals↗
PubMed1995

A study by means of a new assay of the relationship between an outbreak of erythema infectiosum and non-immune hydrops fetalis caused by human parvovirus B19.

An enzyme-linked immunosorbent assay (ELISA), used to detect IgG and IgM antibody specific for human parvovirus B19, was established by use of human parvovirus B19 capsid protein VP-1 expressed in Escherichia coli. Paired samples of serum derived from 44 mothers and single samples derived from 24 babies having unexplained non-immune hydrops fetalis (NIHF), were tested by means of the assay. Five cases (11%) of NIHF were suspected of having been induced by intrauterine human parvovirus B19 infection because the samples of maternal serum were positive for parvovirus B19 IgM antibody. Four of the five cases arose during an outbreak of erythema infectiosum. According to our study and previous reports over 90% of NIHF caused by parvovirus B19 intrauterine infection have become clinically overt in the second trimester of pregnancy during a period ranging from 15 to 27 weeks of gestation.

Blotting, Western↗
PubMed1995

Sharing of the IL-2 receptor gamma chain with the functional IL-9 receptor complex.

The third subunit, the so-called common gamma (gamma c) chain, of the IL-2 receptor is shared among the receptors for IL-2, IL-4, IL-7 and IL-15, and dysfunction of the gamma c chain is thought to cause X-linked severe combined immunodeficiency (XSCID) ascribed to impairment of early T cell development. However, cytokines linked to XSCID are as yet unidentified. A mAb specific for the gamma c chain, TUGm2, profoundly inhibited cell proliferation in response to IL-9. Another mAb, TUGm3, immunoprecipitated [125I]IL-9 cross-linked with either the IL-9 receptor or the gamma c chain. These results demonstrate that the gamma c chain is included in the functional receptor complex for IL-9, which was initially characterized as a T cell growth factor and is essential for IL-9-dependent growth signal transduction.

Animals↗
PubMed1995

Interleukin (IL)-15 promotes the growth of murine epidermal gamma delta T cells by a mechanism involving the beta- and gamma c-chains of the IL-2 receptor.

Dendritic epidermal T cells (DETC) are skin-specific members of the epithelial gamma delta T-cell family in mice. We have reported previously that the growth of DETC is promoted by interleukin (IL)-2 in an autocrine fashion, or by IL-7, which is secreted by neighboring keratinocytes. Here we report that DETC growth is promoted by IL-15, a newly discovered T-cell growth factor that is produced in lymphoid as well as nonlymphoid tissues. Recombinant IL-15 promoted the growth of the 7-17 DETC line in a time- and dose-dependent fashion. Using monoclonal antibodies against alpha-, beta-, or gamma c-chains of the IL-2 receptor complex, we observed that the combination of anti-beta chain and anti-gamma c chain antibodies blocked IL-15 responsiveness completely, whereas anti-alpha chain had no effect. These results indicate that this gamma delta T-cell line uses the beta/gamma c heterodimer for proliferative responses to IL-15. Antibodies against IL-2 or IL-7 did not block IL-15-driven proliferation of 7-17 DETC, indicating that IL-15 promotes their growth in an IL-2- and IL-7-independent manner. Both the surface expression of beta/gamma c heterodimers and the IL-15 responsiveness of 7-17 DETC were highest 1 to 8 days after concanavalin A stimulation, and both declined substantially 21 days after stimulation, illustrating regulation by the state of cell activation. Working with epidermal cells that were freshly procured from CBA mice, we noted that IL-15 promoted conavalin-A-triggered growth of Thy-1+ cells (i.e., DETC), but not of the Thy-1- cells. The gamma c-chain was not expressed by freshly procured DETC, becoming detectable within 48 h after concanavalin A stimulation. We propose that IL-15 facilitates the growth of epithelial gamma delta T cells by a beta/gamma c receptor-dependent mechanism.

Animals↗
PubMed1995

[Flow cytometric measurement of p53 protein and DNA content in colorectal carcinomas with liver metastasis].

To investigate the possible relation between p53 protein, DNA content and liver metastasis of colorectal cancer, overexpression of p53 and DNA content were measured by flowcytometer in 113 primary lesions, which included 34 cases with simultaneous liver metastasis and 79 cases with curative resection, and 25 metastatic lesions of the liver. Overexpression of p53 and DNA aneuploidy were found in 44 (38.9%) and 77 (68.1%) of 113 primary lesions, respectively. However, p53 protein and DNA aneuploidy were unrelated to the clinicopathological findings, such as liver metastasis, venous invasion and lymph node metastasis. Comparing the overexpression of p53 protein between primary and metastatic lesions, p53 protein was recognized in 18 (72.0%) of 25 metastatic lesions of the liver. Incidence of p53 protein was significantly higher in metastatic lesions of the liver than in primary lesions (p < 0.01). On the other hand, p53 protein was found in 27 (60.0%) of 45 diploid lesions and in 35 (37.6%) of 93 aneuploid lesions. There was a significant difference in p53 protein between diploid and aneuploid tumors (p < 0.05). These results suggest that p53 protein may not correlate with the occurrence of liver metastasis and might be produced in the metastatic lesion of the liver after metastasis.

Colorectal Neoplasms↗
PubMed1995

T cell reconstitution by haploidentical BMT does not restore the diversification of the Ig heavy chain gene in patients with X-linked SCID.

We previously examined the Ig heavy (H) chain gene of pretransplant patients with X-linked SCID (XSCID), having defects in the gene of the IL-2 receptor (R) gamma chain. In the present study, we analyzed two post-transplant XSCID patients, in whom T cell-depleted haploidentical BMT resulted in lymphoid split chimeras, i.e., donor functional T cells coexisting with recipient B cells. Although the recipient B cells produced IgM, no isohemagglutinin or Ag-specific Ab was detected. To investigate the cause of failure to produce Ab in the patients, we sequenced the complementarity determining region 3 (CDR3) and adjacent region of Ig H chain gene, which govern Ab specificity. Among the 64 post-transplant CDR3 junctional sequences, combinatorial and junctional diversity were normal compared with those in age-matched controls. All of the post-transplant joining regions except one clone were equal to germline and the frequency of somatic mutation was significantly lower than that in age-matched controls. The results indicated that T cell reconstitution by BMT does not restore diversification of the Ig gene in the IL-2R gamma chain-deficient B cells, which might be associated with the defect in the Ag-specific Ab production.

Base Sequence↗
PubMed1994

IL-2 can support growth of CD8+ T cells but not CD4+ T cells of human IL-2 receptor beta-chain transgenic mice.

We have generated transgenic mice expressing the human (h) IL-2R beta-chain on lymphoid cells under the control of the mouse H-2Kd promoter. Spleen cells and thymocytes of the transgenic mice were cultured in the presence of 5 nM hIL-2. After a 10-day culture, the expanded populations were analyzed by flow cytometry and shown to be composed of CD8+ T cells and gamma delta T cells. Surprisingly, CD4+ T cells of the transgenic mice did not proliferate in response to hIL-2, although the CD4+ T cells expressed the transgenic hIL-2R beta-chain as well as the endogenous gamma-chain on their surface and bound 125I-labeled IL-2. When CD4+ T cells of the transgenic mice were stimulated with anti-CD3 mAb, the CD4+ T cells proliferated in response to hIL-2. These findings suggest that CD4+ T cells may require another triggering signal to respond to IL-2 even when IL-2Rs are expressed. By contrast, CD8+ T cells and gamma delta T cells respond to IL-2 as long as IL-2Rs are expressed.

Animals↗
PubMed1994

Interleukin 2-induced activation of JAK3: possible involvement in signal transduction for c-myc induction and cell proliferation.

We have investigated the role of JAK3 in interleukin 2 (IL-2)-induced signal transduction with a human T cell line, ED40515(-), lacking expression of the IL-2 receptor gamma chain and its sublines transfected with wild-type or mutant cDNAs of the IL-2 receptor gamma chain. Our results demonstrated that the membrane-proximal cytoplasmic region, encompassing the src homology region 2 (SH2)-like subdomain, of the gamma chain is essential for association and activation of JAK3. Furthermore, IL-2-induced activation of JAK3 paralleled induction of the c-myc gene and DNA synthesis but not induction of the c-fos and c-jun genes. These results support the hypothesis that JAK3 plays a pivotal role in the IL-2 receptor-mediated signals for cell growth.

Cell Division↗
PubMed1994

Impairment of ligand binding and growth signaling of mutant IL-2 receptor gamma-chains in patients with X-linked severe combined immunodeficiency.

The IL-2R gamma-chain is an indispensable subunit for the functional IL-2R. Recently, mutations of the gamma-chain have been reported to be closely associated with X-linked severe combined immunodeficiency (XSCID). The present study reveals that three patients with XSCID have three different mutations in the gamma-chain; a point mutation, a two consecutive-base deletion, and lack of the second exon in mRNA. The point mutation that we have detected is C to T, which results in one amino acid substitution of valine for alanine in the extracellular domain of the IL-2R gamma-chain (named AV mutant). The two-base deletion detected causes a frame shift of the coding region in the SH2 subdomain in the cytoplasmic domain (named tSH mutant). Transfection studies performed with the mutant gamma-chains demonstrated that the AV mutant and tSH mutant failed to bind to IL-2 and to transduce growth signals, respectively. These findings indicate that the gamma-chain gene mutations that accompany XSCID induce loss of the gamma-chain function, possibly resulting in stagnation of the differentiation and development of T cells.

Base Sequence↗
PubMed1994

Physical association of JAK1 and JAK2 tyrosine kinases with the interleukin 2 receptor beta and gamma chains.

The functional interleukin 2 (IL-2) receptors contain the beta and gamma chains which are necessary for the transduction of cell growth signals. Monoclonal antibodies specific for the beta chain and gamma chain coimmunoprecipitated JAK1 and 114-kDa JAK2 tyrosine kinases, respectively. Tyrosine phosphorylation of JAK1 and JAK2 was induced upon IL-2 stimulation, and IL-2 activated the JAK2 kinase. These results demonstrate that the JAK1 and JAK2 tyrosine kinases are physically associated with the beta chain and gamma chain, respectively, and suggest that regulation of the kinases may be linked to IL-2-induced signal transduction.

Base Sequence↗
PubMed1994

Regulation by interleukin-2 (IL-2) and interferon gamma of IL-2 receptor gamma chain gene expression in human monocytes.

The interleukin-2 receptor gamma chain (IL-2R gamma) gene codes for a subunit of the IL-2R and is expressed in human lymphoid cells. The present study was undertaken to determine whether human monocytes expressed the IL-2R gamma gene constitutively or after activation by IL-2 or interferon gamma (IFN gamma). Fresh human monocytes constitutively expressed low but significant levels of IL-2R gamma mRNA, and nuclear run-on experiments showed that IL-2R gamma gene was transcriptionally active. Stimulation with IL-2 or IFN gamma induced a major increase of IL-2R gamma mRNA in a time- and a dose-dependent manner. However, neither cytokine increased the transcriptional activity of the gene. The enhancement of IL-2R gamma mRNA expression by either IL-2 or IFN gamma was concomitant with the stabilization of the mRNA, suggesting a postranscriptional level of control. Finally, the augmented expression of IL-2R gamma in IL-2- and IFN gamma-treated monocytes was associated with an increased IL-2-binding activity, compared with that of unstimulated cells. These results provide the first evidence of the expression of the IL-2R gamma gene in nonlymphoid cells and of its modulation by IL-2 and IFN gamma through posttranscriptional mechanisms.

Flow Cytometry↗
PubMed1994

Cooperative interactions between the interleukin 2 receptor alpha and beta chains alter the interleukin 2-binding affinity of the receptor subunits.

The interleukin 2 (IL-2) receptor (IL-2R) is a multisubunit receptor that includes three major IL-2 binding subunits, the IL-2R alpha, beta, and gamma chains. We have detected and analyzed cooperative interactions between the IL-2R alpha and beta chains (IL-2R alpha and IL-2R beta, respectively) in COS cells transfected with cDNAs encoding the IL-2R alpha, the IL-2R beta, or both cDNAs. We demonstrated that IL-2 F42A, an analog that fails to bind to the isolated IL-2R alpha subunit and would be predicted by the hierarchical affinity-conversion model to have impaired binding to cells expressing both chains, instead readily binds to the IL-2R alpha/beta heterodimer in COS cells. Furthermore, this binding is abolished by the antibody HIEI that separates the two IL-2R subunits. The monoclonal antibodies anti-Tac and Mik-beta 1 directed at the IL-2-binding sites on IL-2R alpha and IL-2R beta, respectively, block ligand binding to the heterodimer. This binding pattern is inconsistent with the strict hierarchical affinity-conversion model that mandates an initial binding of IL-2 to IL-2R alpha followed by binding of the IL-2/IL-2R alpha complex to IL-2R beta. Instead, our results support an alternative model of preformed complexes of IL-2R beta with other IL-2R subunits. In this alternative model, IL-2R alpha and -beta exist in part as preformed complexes in which the affinity of IL-2R beta for IL-2 is altered by the proximity of IL-2R alpha, through mechanisms that do not require the prior binding of IL-2 to IL-2R alpha.

Cell Line↗
PubMed1994

Functional participation of the IL-2 receptor gamma chain in IL-7 receptor complexes.

The gamma chain of the interleukin-2 (IL-2) receptor is shared with the functional IL-4 receptor and is causatively related to X-linked severe combined immunodeficiency (XSCID), which is ascribed to a profound T cell defect. Studies with monoclonal antibodies specific for the IL-2 receptor gamma chain showed that the gamma chain participates in the functional high-affinity receptor complexes for IL-7 that are involved in the differentiation of T and B cells. Participation of the gamma subunit in more than one receptor may enable the elucidation of the mechanisms of XSCID development and lymphocyte differentiation.

Animals↗
PubMed1994

Expression of the mouse interleukin-2 receptor gamma chain in various cell populations of the thymus and spleen.

Expression of the gamma chain, which is shared among functional receptor complexes for interleukin (IL)-2, IL-4 and IL-7, was examined with hematopoietic cells in mouse thymus and spleen by flow cytometry. The gamma chain was expressed in cell populations from the spleen. Stimulation with concanavalin A and lipopolysaccharide caused fluctuation in expression of the gamma chain in T and B cells, respectively. T lineage cells developing in the adult thymus expressed the gamma chain. Fetal thymus at day 15 contained mostly immature thymocytes, which also expressed the gamma chain. These results demonstrate that the gamma chains is widely expressed in T lineage cells, probably indicating that the gamma chain plays a role not only in the proliferation of mature hematopoietic cells but also in the development of immature cells through signal transduction as a common receptor subunit for multiple cytokines.

Animals↗
PubMed1994

The interleukin-2 receptor subunit expression and function on peripheral blood lymphocytes from HIV-infected and control persons.

The expression of interleukin-2 receptor (IL2R) was studied on circulating lymphocytes from HIV-infected (HIV+) and control subjects, using chain-specific monoclonal antibodies and indirect immunofluorescence flow cytometry. The IL2R alpha chain expression was decreased on CD4 and CD8 T cells from HIV+ persons compared to controls. Conversely, beta chain expression was enhanced on both T cell subsets from the patients. IL2R-subunit levels were similar on natural killer cells from patients and controls. To evaluate the function of IL2R, we investigated to what extent IL2 could induce CD69, an early activation marker of lymphocytes. A dose-dependent increase of CD69 expression was observed on T and NK cells from all subjects. The upregulation of CD69 was similar on CD4 T and NK cells from patients and controls, but was more pronounced on CD8 T cells from HIV+ compared to HIV- subjects. Based on inhibition studies, both the alpha and the beta chain contributed to the IL-2-induced CD69 expression on CD4 T cells, pointing to involvement of the high-affinity receptor. The early activation of CD8 T cells and NK cells was mainly dependent on the intermediate-affinity receptor. We conclude that significant changes in IL2R alpha and beta chain expression on circulating T cells occur after HIV infection, but that early activation through IL2 is preserved or enhanced, even in advanced stages.

Adult↗
PubMed1994

Preferential utilization of the immature JH segment and absence of somatic mutation in the CDR3 junction of the Ig H chain gene in three X-linked severe combined immunodeficiency patients.

Human severe combined immunodeficiency (SCID) includes an X-linked SCID (XSCID) characterized by a complete absence of mature T cells, hypogammaglobulinemia and a normal or elevated number of B cells. XSCID results from mutation in the IL-2 receptor (IL-2R) gamma chain gene, which is thought to be involved in not only IL-2R but also IL-4R and IL-7R mediated signals. To investigate the VDJ recombination and Ig repertoire development in the absence of the IL-2R gamma chain, we intended to study the CDR3 junction in peripheral blood B cells of three XSCID patients. A total of 101 different CDR3 junctions were cloned following polymerase chain reaction amplification of polyclonal peripheral blood lymphocyte DNA. Sequence analysis of CDR3 junctions revealed that the primary antibody repertoire of the Ig H chain gene was assembled in a normal fashion. Among the JH segments, overexpression of JH3 segments was significant in XSCID patients compared with age-matched controls. D segment usage in XSCID was very similar to that in age-matched controls. All of the XSCID JH regions except for two clones were equal to germline JH genes, showing little or no evidence of somatic mutation. The results indicate that the immature JH segment is preferentially utilized and somatic mutation is absent in the CDR3 junction of the Ig H chain gene of XSCID patients.

B-Lymphocytes↗