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Biomedical subjects

Y Shinkai

Publications and source records attributed to Y Shinkai.

At least 37 records · Page 2Linked to original sources
PubMed1997

Amyloid beta-protein (A beta) accumulation in the leptomeninges during aging and in Alzheimer disease.

The results of well-characterized two-site enzyme immunoassays showed that the crude leptomeninges (consisting of the pia matter, arachnoid matter, and leptomeningeal vessels [LV]) from aged control brains and brains affected by Alzheimer disease (AD) contain very high levels of amyloid beta-protein (A beta). To learn about the source of A beta, we carefully dissected out both leptomeninges (LM) and LV under a dissecting microscope and determined the levels of soluble A beta in each. The purity of these dissected tissues was confirmed by the absence or presence of alpha-smooth muscle actin representing LV by Western blotting. Surprisingly, the amounts of A beta in each dissected sample were nearly equivalent on a weight basis. In each compartment from aged controls the level of A beta 1-42 was comparable to that of A beta 1-40, while in AD brain A beta 1-40 was a predominant species in both LM and LV. In some cases careful immunocytochemical examination revealed the presence of A beta deposits that were immunolabeled by several A beta monoclonal antibodies in leptomeningeal layers (most often in the arachnoid matter). The extent of A beta deposition in LM appeared to be much less than that explained by the soluble A beta levels, suggesting that immunocytochemically undetectable A beta can accumulate in LM. These observations indicate that leptomeninges are a large reservoir of A beta in normal aged individuals and in AD patients.

Adult↗
PubMed1996

Activated Ras signals differentiation and expansion of CD4+8+ thymocytes.

We describe a novel approach to assay the ability of particular gene products to signal transitions in lymphocyte differentiation in vivo. The method involves transfection of test expression constructs into RAG-1-deficient embryonic stem cells, which are subsequently assayed by the RAG-2-deficient blastocyst complementation approach. We have used this method to demonstrate that expression of activated Ras in CD4-8- (double negative, DN) prothymocytes in vivo induces their differentiation into small CD4+8+ (double positive, DP) cortical thymocytes with accompanying expansion to normal thymocyte numbers. However, activated Ras expression in DP cells does not cause proliferation or maturation to CD4+8- or CD4-8+ (single positive) thymocytes. Therefore, signaling through Ras is sufficient for promoting differentiation of DN to DP cells, but further differentiation requires the activity of additional signaling pathways.

Animals↗
PubMed1995

Expression and induction of nuclear factor-kappa B-related proteins in thymocytes.

Thymocytes mature in response to the cues from the thymic micro-environment, which regulate stage-specific gene expression during development. We find that several proteins that bind the kappa B sequence in vitro are constitutively activated in freshly isolated thymocytes. These include the rel-related p50 homodimers, p50/p65 heterodimers, low levels of c-rel, and two other factors that may be thymus specific. Disruption of the thymic micro-environment resulted in loss of DNA-binding, suggesting that lymphocyte-stromal cell interactions induce and maintain these proteins in a DNA-binding form. Phorbol ester and ionomycin treatment induced p50, p65, and p68 c-rel kappa B DNA-binding activity. Expression of p68 c-rel protein, but not p50 or p65, was suppressed by the immunosuppressive drug FK506. Because FK506 specifically inhibits the appearance of mature single-positive thymocytes, gene expression regulated by p68 c-rel may play a role in selection and maturational signals involved in the double-positive to single-positive transition.

Animals↗
PubMed1995

Amyloid beta-proteins 1-40 and 1-42(43) in the soluble fraction of extra- and intracranial blood vessels.

To investigate the process of amyloid beta-protein (A beta) accumulation in cerebral amyloid angiopathy (CAA), the levels of A beta were determined in the soluble fraction of extra- and intracranial blood vessels and leptomeninges obtained at autopsy. Two enzyme immunoassays were employed that are known to sensitively and specifically quantify two A beta species, A beta 1-40 and 1-42(43). A beta was detectable in the intracranial blood vessels and leptomeninges with the latter containing the highest levels, while it was undetectable in the extracranial blood vessels. Thus the levels of soluble A beta correlated well with the predilection sites for CAA. Among individuals aged 20 to 90, the A beta levels in the leptomeninges increased sharply in those aged 50 to 70 and thereafter tended to decline. However, only slight degrees of CAA were detected by immunocytochemistry, even when those leptomeninges contained high levels of A beta comparable with those in Alzheimer's disease. The level of A beta 1-42 was almost always severalfold that of A beta 1-40 in the soluble fraction of leptomeninges. This is in good agreement with the immunocytochemical result showing the presence of A beta 40-negative, A beta 42(43)-positive meningeal vessels. These results indicate that A beta 1-42 is the initially deposited species in CAA and that the disruption of A beta homeostasis precedes A beta deposition in the meningeal vessels.

Adult↗
PubMed1995

CD3 epsilon and CD3 zeta cytoplasmic domains can independently generate signals for T cell development and function.

To determine whether CD3 epsilon and CD3 zeta proteins have unique roles in TCR-dependent functions, chimeric genes encoding the extracellular and transmembrane domains of the human IL-2 receptor alpha chain (Tac) fused to a cytoplasmic domain of either the CD3 epsilon or CD3 zeta chain were introduced as transgenes into both normal and RAG2-deficient (RAG2-/-) mice. Developmental arrest of T lineage cells at the CD4, CD8 double-negative stage in the transgenic RAG2-/- thymus was released to the CD4, CD8 double-positive (DP) stage by in vivo cross-linking of TT epsilon or TT zeta with anti-Tac antibody. In TT epsilon + or TT zeta +, RAG2-/- mice, in vitro cross-linking of TT epsilon and TT zeta induced DP thymocyte cell death and proliferation of mature single-positive T cells. Overall, no qualitative differences were observed between TT epsilon- and TT zeta-mediated functions, suggesting that different CD3 components deliver qualitatively similar signals in inducing TCR-dependent functions.

Animals↗
PubMed1994

Nuclear factors that mediate intrathymic signals are developmentally regulated.

Thymocytes mature through several stages of development, defined by cell surface markers such as CD3, CD4, and CD8, in response to environmental cues. Signal transduction resulting from lymphocyte-stromal cell interactions is likely to activate inducible transcription factors which in turn govern stage-specific gene expression. In this report we show that inducible transcription factors such as AP-1 and NF-AT are constitutively nuclear, in response to intrathymic signals, in freshly isolated thymocytes at all stages of maturation. In CD4+CD8+ double positive (DP), but not in the more immature CD4-CD8- double negative (DN) thymocytes, constant stimulus from the thymic environment is required to maintain nuclear AP-1. Thus, disruption of the thymus and incubation of thymocytes at 37 degrees C downregulates DNA binding by nuclear factors AP-1 and NF-AT. Similar treatment of thymocytes has previously been shown to downregulate CD3 zeta chain phosphorylation and increase T cell receptor CD3 expression on DP thymocytes, which is a feature of repertoire selection. Since mature T cells maintain inducible nuclear factors in an inactive form until an encounter with antigen, we propose that downregulation of nuclear DNA binding proteins may reflect another feature of this stage of T cell maturation.

Aging↗
PubMed1994

Influence of immunoglobulin heavy- and light-chain expression on B-cell differentiation.

To study the influence of immunoglobulin heavy-chain (HC) and light-chain (LC) expression in promoting B-cell differentiation, we have introduced functional immunoglobulin HC and/or LC transgenes into the recombinase activating gene-2-deficient background (RAG-2-/-). RAG-2-/- mice do not undergo endogenous V(D)J rearrangement events and, therefore, are blocked in B- and T-cell development at the early pro-B- and pro-T-cell stages. Introduction of immunoglobulin HC transgenes into the RAG-2-/- background promotes the development of a B-lineage cell population that phenotypically has the characteristics of pre-B cells. We have shown further that this population has altered growth characteristics as measured by interleukin-7 responsiveness in culture. Bone marrow cells from immunoglobulin HC transgenic RAG-2-/- mice have up-regulated expression of germ-line kappa LC gene transcripts and down-regulated expression of lambda 5 surrogate LCs (SLCs). Although mu HC/SLC complexes are detectable intracellularly in HC/RAG-2-/- pre-B-cell populations, HC expression is not readily detectable on the surface of these cells. lambda LC RAG-2-/- mice had a bone marrow B-lineage cell phenotype indistinguishable from that of RAG-2-/- littermates, indicating that LC expression by itself has no influence on pro-B cell differentiation. Strikingly, simultaneous introduction of mu HC and lambda LC transgenes into RAG-2-/- mice led to the generation of a substantial population of "monoclonal" peripheral B-cells that were functional with regard to immunoglobulin secretion, indicating that T cells or diverse immunoglobulin repertoires are not necessary for peripheral B-cell development.

Animals↗
PubMed1994

The expression of Vpre-B/lambda 5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice.

Precursor B (pre-B) cells in bone marrow of normal and B cell-deficient mutant mice were analyzed for the expression of Vpre-B/lambda 5 surrogate light chain (SL). The surface expression of SL is confined to the early stages (pro-B and pre-B-I) of pre-B cell development and becomes undetectable once mu heavy chain (microH) is produced. The cell-cycle analysis revealed that cytoplasmic microH+ large cells (large pre-B-II), approximately 30% of which coexpressed SL in the cytoplasm, were most actively cycling, whereas cytoplasmic microH+ small cells (small pre-B-II) were SL- and not in cycle. The analysis of pre-B cells in B cell-deficient mice suggests that the large pre-B-II stage is a critical step for the selection and amplification of cells carrying functionally rearranged microH genes.

Animals↗
PubMed1994

Probing immune functions in RAG-deficient mice.

Inactivation of recombination activating gene (RAG)-1 or RAG-2 in mice results in the inability of developing lymphocytes to initiate V(D)J recombination, leading to the arrest of lymphocyte differentiation at a very early stage. Introduction of functionally assembled antigen-receptor genes or other potentially relevant genes into the RAG-deficient background can bypass the V(D)J recombination block and promote differentiation of the lymphocytes of RAG-deficient mice to various stages. This approach offers new means for analyzing the control of lymphocyte differentiation. In addition, generation of somatic chimeric mice by injecting mutant embryonic stem cells into the RAG-2-deficient blastocysts has also provided a powerful new method for assaying the potential roles of genes or regulatory elements in lymphocyte development or function.

Animals↗
PubMed1994

CD3 epsilon-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2-/- mice in the absence of TCR beta chain expression.

Recent studies have shown that TCR beta chain expression can effect the differentiation of CD4-CD8- double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) thymocytes. The TCR beta chain is expressed on the surface of DP thymocytes in association with CD3 gamma, delta and epsilon chains, suggesting a potential role for CD3 components in this signaling process. We now report detection of a very low level of surface expression of CD3 epsilon on adult DN RAG-2-/- thymocytes. This surface CD3 epsilon was associated with CD3 gamma and delta chains, as detected by anti-CD3 epsilon immunoprecipitation analyses. Significantly, injection of anti-CD3 epsilon mAb into RAG-2-/- mice led to the accumulation of an IL-2R alpha- CD2+ DP cell population and a nearly 100-fold increase in thymic cellularity to essentially normal levels. Together, these data strongly indicate that TCR beta chain-mediated developmental signals are transduced by CD3 components and provide potential insights into mechanisms by which TCR beta chain expression may effect this process.

Animals↗
PubMed1994

Perforin-secreting killer cell infiltration and expression of a 65-kD heat-shock protein in aortic tissue of patients with Takayasu's arteritis.

Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of vascular cell injury in Takayasu's arteritis. To clarify the immunological mechanisms involved, we examined the expression of a cytolytic factor, perforin in infiltrating cells of aortic tissue samples from seven patients with Takayasu's arteritis. We also examined the expression of a 65-kD heat-shock protein (HSP-65), human leukocyte antigen classes I and II, and intercellular adhesion molecule-1 in the aortic tissue. Immunohistochemical studies showed that the infiltrating cells mainly consisted of gamma delta T lymphocytes, natural killer cells, macrophages, cytotoxic T lymphocytes and T helper cells, and that perforin was expressed in gamma delta T lymphocytes, natural killer cells, and cytotoxic T lymphocytes. In situ hybridization analysis also revealed expression of perforin mRNA in the infiltrating cells. Immunoelectron microscopic studies demonstrated that the infiltrating cells released massive amounts of perforin directly onto the surface of arterial vascular cells. We also found that expression of HSP-65, human leukocyte antigen classes I and II, and intercellular adhesion molecule-1 was strongly induced in the aortic tissue and might facilitate the recognition, adhesion and cytotoxicity of the infiltrating killer lymphocytes. These findings provide the first direct evidence that the infiltrating cells in the aortic tissue mainly consist of killer cells, and strongly suggest that these killer cells, especially gamma delta T lymphocytes, may recognize HSP-65 and play a critical role in the vascular cell injury of Takayasu's arteritis by releasing perforin.

Adult↗
PubMed1993

Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice.

The bcl-2 proto-oncogene can prevent the death of many cell types. Mice were generated that were chimeric for the homozygous inactivation of bcl-2. Lymphocytes without Bcl-2 differentiated into phenotypically mature cells. However, in vitro, the mature T cells that lacked Bcl-2 had shorter life-spans and increased sensitivity to glucocorticoids and gamma-irradiation. In contrast, stimulation of CD3 inhibited the death of these cells. T and B cells with no Bcl-2 disappeared from the bone marrow, thymus, and periphery by 4 weeks of age. Thus, Bcl-2 was dispensable for lymphocyte maturation, but was required for a stable immune system after birth.

Animals↗
PubMed1993

Fc gamma RII/III and CD2 expression mark distinct subpopulations of immature CD4-CD8- murine thymocytes: in vivo developmental kinetics and T cell receptor beta chain rearrangement status.

We have recently identified a dominant wave of CD4-CD8- (double-negative [DN]) thymocytes in early murine fetal development that express low affinity Fc gamma receptors (Fc gamma RII/III) and contain precursors for Ti alpha/beta lineage T cells. Here we show that Fc gamma RII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that Fc gamma RII/III expression is downregulated within the DN subpopulation and before the CD3-CD8low SP stage in T cell receptor (TCR)-alpha/beta lineage-committed thymocytes. DN Fc gamma RII/III+ thymocytes also contain a small fraction of TCR-gamma/delta lineage cells in addition to TCR-alpha/beta progenitors. Fetal day 15.5 DN TCR-alpha/beta lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of Fc gamma RII/III vs. CD2 (Fc gamma RII/III+CD2-, Fc gamma RII/III+CD2+, Fc gamma RII/III-CD2+). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57B1/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (Fc gamma RII/III+CD2(-)-->Fc gamma RII/III+CD2(+)-->Fc gamma RII/III-CD2+) that precedes the entry of DN thymocytes into the CD4+CD8+ double-positive (DP) TCRlow/- stage. Unseparated day 15.5 fetal thymocytes develop into DP thymocytes within 2.5 d and remain at the DP stage for > 48 h before being selected into either CD4+ or CD8+ SP thymocytes. In contrast, Fc gamma RII/III+CD2- DN thymocytes follow this same developmental pathway but are delayed by approximately 24 h before entering the DP compartment, while Fc gamma RII/III-CD2+ display accelerated development by approximately 24 h compared with total day 15.5 thymocytes. Fc gamma RII/III-CD2+ are also more developmentally advanced than Fc gamma RII/III+CD2- fetal thymocytes with respect to their TCR beta chain V(D)J rearrangement. At day 15.5 in gestation, beta chain V(D)J rearrangement is mostly, if not entirely, restricted to the Fc gamma RII/III-CD2+ subset of DN fetal thymocytes. Consistent with this analysis in fetal thymocytes, > 90% of adult thymocytes derived from mice carrying a disrupting mutation at the recombination-activating gene 2 locus (RAG-2-/-) on both alleles are developmentally arrested at the DN CD2- stage. In addition, there is a fivefold increase in the relative percentage of thymocytes expressing Fc gamma RII/III in TCR and immunoglobulin gene rearrangement-incompetent homozygous RAG-2-/- mice (15% Fc gamma RII/III+) versus rearrangement-competent heterozygous RAG-2+/- mice (< 3% Fc gamma RII/III+). Thus, Fc gamma RII/III expression defines an early DN stage preceding V beta(D beta)I beta rearrangement, which in turn is followed by surface expression of CD2. Loss of Fc gamma RII/III and acquisition of CD2 expression characterize a late DN stage immediately before the conversion into DP thymocytes.

Aging↗
PubMed1993

Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes.

Introduction of TCR alpha transgene, TCR beta transgene, or both into RAG-2-/-mice differentially rescues T cell development. RAG-2-/- mice have small numbers of TCR-CD4-CD8-(double negative, DN) thymocytes that express CD3 gamma delta epsilon and zeta proteins intracellularly. Introduction of a TCR beta transgene, but not a TCR alpha transgene, into the RAG-2-/- background restored normal numbers of thymocytes. These cells were CD4+CD8+ (double positive, DP) and expressed small amounts of surface TCR beta chain dimers in association with CD3 gamma delta epsilon but not zeta. RAG-2-/- mice that expressed alpha and beta TCR transgenes developed both DP and single positive thymocytes. Thus, the TCR beta subunit, possibly in association with a novel CD3 complex, participates in the DN to the DP transition.

Animals↗
PubMed1993

Evidence of perforin-mediated cardiac myocyte injury in acute murine myocarditis caused by Coxsackie virus B3.

We have recently demonstrated that killer cells expressing a cytolytic factor, perforin, infiltrate the hearts of mice with acute viral myocarditis and may play an important role in the mechanism of myocardial damage. To clarify the mechanism of in vivo cardiac myocyte injury mediated by perforin, we investigated the release of perforin molecules from killer cells by immunoelectron microscopy and examined the circular lesions formed by perforin on the membrane of cardiac myocytes. We found that there was massive release of perforin molecules from the killer cells directly onto the surface of the cardiac myocytes. Furthermore, electron microscopy of ultrathin ventricular sections treated with trypsin revealed numerous circular lesions with the characteristics of perforin pores, in the membranes of cardiac myocytes. These findings provide the first direct evidence that killer cells injure cardiac myocytes by releasing perforin and may play a critical role in the myocardial damage occurring in acute viral myocarditis.

Acute Disease↗
PubMed1992

Function and control of recombination-activating gene activity.

The RAG-1 and RAG-2 genes synergistically confer VDJ recombinase activity to nonlymphoid cell lines. To unequivocally test RAG gene function, we created lines of mice that lack functional copies of these genes. Consistent with the possibility that RAG gene encode the tissue-specific components of VDJ recombinase, RAG-2-deficient mice are viable but have a severe combined immune deficiency due to inability to initiate VDJ recombination and thereby generate mature lymphocytes. RAG-2-deficient mice have no obvious defect in any tissue or lineage other than lymphocytes, indicating that VDJ recombinase activity and RAG-2-gene function is required only for lymphocyte development. Levels of RAG-1 and RAG-2 expression in primary murine lymphoid tissues and lymphoid bone marrow cultures generally are much higher than those of transformed precursor B-cell lines. Low-level RAG gene expression in permanent cell lines results from a decline during propagation due to outgrowth of cells with lower RAG expression levels. The low and variable level of RAG gene expression in transformed pre-B cell lines correlates with low and variable rates of endogenous VDJ recombination; therefore, such lines are not reliable models for experiments aimed at studying mechanisms that target this activity to particular variable region gene segments. To generate such a system, we introduced RAG genes into B-lineage lines under the control of a heat shock-inducible promoter; heat-shock treatment induces extremely high-level but transient RAG expression accompanied by parallel induction of VDJ recombinase activity. Such cells efficiently rearrange transfected VDJ recombination substrates in a regulated manner that is dependent on the activity of transcriptional control elements associated with the target V gene segments.

Animals↗
PubMed1992

RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement.

We have generated mice that carry a germline mutation in which a large portion of the RAG-2 coding region is deleted. Homozygous mutants are viable but fail to produce mature B or T lymphocytes. Very immature lymphoid cells were present in primary lymphoid organs of mutant animals as defined by surface marker analyses and Abelson murine leukemia virus (A-MuLV) transformation assays. However, these cells did not rearrange their immunoglobulin or T cell receptor loci. Lack of V(D)J recombination activity in mutant pre-B cell lines could be restored by introduction of a functional RAG-2 expression vector. Therefore, loss of RAG-2 function in vivo results in total inability to initiate V(D)J rearrangement, leading to a novel severe combined immune deficient (SCID) phenotype. Because the SCID phenotype was the only obvious abnormality detected in RAG-2 mutant mice, RAG-2 function and V(D)J recombinase activity, per se, are not required for development of cells other than lymphocytes.

Animals↗