Search PubMed⌕ Search

Biomedical subjects

H Matsue

Publications and source records attributed to H Matsue.

At least 37 records · Page 2Linked to original sources
PubMed1996

Identification of mRNA-rich keratinocytes in the basal/suprabasal layers of psoriatic skin.

In this study, we examined the relative amounts of mRNA expressed in normal versus psoriatic epidermis, using in situ hybridization with a biotinylated oligonucleotide poly d(T) probe. The hybridization image was analyzed by Laser Scanning Confocal Microscopy (LSCM). In normal human skin, hybridization signals were detected homogeneously throughout the epidermis, mostly in nucleus. These signals disappeared following RNase T2 or RNase A treatment, indicating that the target for this probe is RNA; by implication, mRNA. In psoriatic lesions, the overall signal intensity was significantly elevated, especially in the basal/suprabasal layers. Moreover, those signals were most prominent in the cytoplasm, not in the nucleus. In contrast, the signals of uninvolved epidermis adjacent to the psoriatic lesions were indistinguishable from those of normal skin in both signal intensity and hybridization profile. Our data are consistent with the notion that one of the characteristic features of psoriasis is an elevated (or uncontrolled) synthesis of mRNA and proteins.

Biopsy, Needle↗
PubMed1996

[Metabolic evaluation of cultured porcine hepatocyte monolayers in human plasma from hepatic failure patients--basic study on development of a bioreactor in hybrid artificial liver].

In order to develop a functional bioreactor for hybrid artificial liver, function of cultured porcine hepatocyte monolayers in human plasma from hepatic failure patients (group III, n = 5) was investigated. Culture media, Leibovitz L-15 medium (group I, n = 7) and normal human plasma (group II, n = 3), were used as controls. Morphologically, no degeneration of porcine hepatocytes in hepatic failure plasma was observed for 5 days in culture. Levels of ureogenesis showed no significant difference against the controls at day 1, 2, 5 in culture, but the level at day 3 was significantly higher than that of group II. Levels of gluconeogenesis showed a close tendency as those of ureogenesis, but the level at day 3 was significantly lower than that of group I. Levels of intracellular DNA contents, showing between 1.85 +/- 0.39 and 1.35 +/- 0.05 microgram/cm2, were compatible with those of controls during first three days in culture, but level of group III at day 5 was significantly higher than that of group I. After incubation of porcine hepatocyte in hepatic failure plasma, the amount of valine, leucine, isoleucine, glutamine, arginine, and citrulline was significantly decreased. Elevated phenylalanine and tyrosine were also decreased, but Fischer's ratio, the ratio of branched chain amino acid against aromatic amino acids, was not significantly increased. Data obtained by this investigation showed that cultured porcine hepatocytes held proper hepatic function in the hepatic failure plasma. It is concluded that culture porcine hepatocyte monolayers were a promising candidate for a bioreactor of a hybrid artificial liver.

Adult↗
PubMed1995

Folate receptors targeted to clathrin-coated pits cannot regulate vitamin uptake.

Potocytosis is an endocytic process that is specialized for the internalization of small molecules. Recent studies on the uptake of 5-methyltetrahydrofolate by the folate receptor have suggested that the glycosyl-phosphatidylinositol anchor on this protein causes it to cluster and be internalized by caveolae instead of coated pits. To test this hypothesis directly, we have constructed a chimeric folate receptor that has the glycosyl-phosphatidylinositol anchor replaced with the transmembrane domain and cytoplasmic tail of the low density lipoprotein receptor. The cells with wild-type receptors delivered 5-methyltetrahydrofolate to the cytoplasm more rapidly than did cells expressing the chimeric receptor. This suggests that efficient delivery to the cytoplasm depends on caveolae. In sharp contrast to cells with wild-type folate receptors, cells internalizing folate by clathrin-coated pits were unable to decrease vitamin uptake when they were either folate replete or confluent.

Animals↗
PubMed1995

Keratinocytes constitutively express the Fas antigen that mediates apoptosis in IFN gamma-treated cultured keratinocytes.

The Fas antigen is a cell surface protein that can mediate apoptosis in many cell types. Although its physiological function is still unclear, recent evidence indicates that this surface molecule is involved in apoptosis in the immune system and the liver. The epidermis is an organ that undergoes terminal differentiation with the eventual death of keratinocytes, and it has been suggested that this is a specialized form of apoptosis. In the present study, we examined whether or not the Fas antigen is involved in keratinocyte apoptosis. Immunoreactivity for the Fas antigen was found throughout the epidermis in normal human skin sections and cultured normal human keratinocytes, and mRNA for the Fas antigen was found to be constitutively expressed in normal epidermis and cultured normal keratinocytes by RT-PCR analysis. To determine whether the Fas antigen in keratinocytes is functional, we used a cytotoxic monoclonal antibody (mAb) against the Fas antigen to induce apoptosis. This antibody did not induce apoptosis of cultured keratinocytes even though they expressed the Fas antigen. We then tested the ability of several cytokines (TGF beta, TNF alpha and IFN gamma) to induce Fas-mediated keratinocyte apoptosis. Only pretreatment with IFN gamma followed by the addition of the anti-Fas mAb induced apoptosis, as assessed by cell viability, morphological changes and ultrastructural characteristics, suggesting that constitutive expression of the Fas antigen is not sufficient to induce apoptosis in keratinocytes and that keratinocyte apoptosis via the Fas antigen-mediated mechanism may require the activation of keratinocytes by IFN gamma, which is thought to be produced by activated T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1995

Chemical structure of the carbohydrate moiety of fucose-rich glycopeptides from human pancreatic juice.

Human pancreatic juice, obtained from nine patients after partial excision of the pancreas for bile duct cancer, was fractionated in order to isolate its glycopeptides. Three glycopeptides were purified employing ion-exchange chromatography and gel filtration. All the glycopeptides were found to be free of sialic acid and galactosamine but to have an unusually high content of L-fucose. The chemical structures of the three glycopeptides were determined using 500-MHz [1H]-NMR spectroscopy. One of them, glycopeptide, GP-4, possessed a biantennary structure with three L-fucose residues. The second glycopeptide, GP-3, had a triantennary structure with four L-fucose residues, and the third one, G-2, had a tetra-antennary structure with five L-fucose residues. The chemical compositions of these glycopeptides, including the absence of sialic acid and the high L-fucose content, indicate that they represent a new class of glycopeptide present in the normal human pancreas.

Carbohydrate Sequence↗
PubMed1995

The effects of cyclosporin A and FK506 on proliferation and IL-8 production of cultured human keratinocytes.

The present study was conducted to determine whether cyclosporin A (CsA) and FK506 could be effective in inhibiting the proliferation and cytokine secretion of normal human epidermal keratinocytes (NHEK). NHEK proliferation in the presence of CsA and FK506 at the concentrations 10(-9) to 10(-5) M at 24 and 48 h time points was measured colorimetrically by the MTS assay. CsA had inhibitory effects from 10(-6) to 10(-5) M, while FK506 had no effect, except for toxicity at the very highest concentrations (5 x 10(-6) M and higher). NHEK cells spontaneously secrete IL-8 (243.4 +/- 55.5 pg/ml), and this baseline level was augmented by TNF-alpha alone, or synergistically by TNF-alpha and IFN-gamma, which are thought to be secreted by T cells. Neither CsA nor FK506 had any significant effect on either spontaneous or cytokine-stimulated keratinocyte IL-8 production. Therefore, it is most likely that the two drugs indirectly inhibit the keratinocyte inflammatory response through their actions on T cells or other immunocompetent cells.

Cell Division↗
PubMed1995

Interleukin-7-dependent interaction of dendritic epidermal T cells with keratinocytes.

Dendritic epidermal T cells (DETC), a member of the epithelial tissue-type gamma delta T-cell family, are characterized by their exclusive residence within mouse epidermis, their dendritic morphology, and their monoclonal nature in the T-cell-receptor configuration. Here we review our recent studies on the interleukin (IL)-7-dependent interaction of DETC with neighboring keratinocytes. Keratinocytes express constitutively the mRNAs for IL-7 and secrete biologically relevant amounts of IL-7. This cytokine, in turn, serves as a growth factor for DETC, as evidenced by the proliferative responses to recombinant or keratinocyte-derived IL-7 of the 7-17 DETC line and of DETC freshly purified from mouse skin. The 7-17 DETC line undergoes apoptotic cell death in response to external stimuli known to deplete DETC in situ (e.g., ultraviolet B radiation or corticosteroid treatment), and IL-7 prevents this apoptosis, thereby promoting long-term survival. These results document the crucial role played by IL-7 in maintaining the survival and growth of DETC in epidermis. IL-7 mRNA expression in keratinocytes is abrogated by ultraviolet B radiation, whereas it is up-regulated by interferon-gamma, which is secreted by DETC upon activation. More specifically, interferon-gamma induces the preferential expression of truncated forms (2.6 and 1.5 kb) of IL-7 transcripts, in addition to the 2.9- and 1.7-kb transcripts that are expressed constitutively, and this regulation occurs through the usage of alternative transcription initiation sites. These results suggest unique pathways through which IL-7 production is regulated in keratinocytes by external stimuli (e.g., ultraviolet B) as well as T-cell-derived cytokines (e.g., interferon-gamma). We propose that keratinocyte-derived IL-7 is an essential component of the epidermal cytokine milieu.

Animals↗
PubMed1994

Novel fucose-rich glycosaminoglycans from squid ink bearing repeating unit of trisaccharide structure (-6GalNAc alpha 1-3GlcA beta 1-3Fuc alpha 1-)n.

Three fucose-rich glycosaminoglycans (GAGs), illexin A, illexin B and illexin C, were isolated from squid ink of Illex argentinus. The GAGs were obtained by the method of Actinase E digestion, DEAE Sephadex A-50 ion-exchange chromatography and Sephacryl S-300 gel filtration. They gave a single band on the electrophoresis using cellulose acetate membrane and contained equimolar ratios of Fuc, GlcA and GalNAc. Molecular weights of illexin A, illexin B, and illexin C were determined by Sephacryl S-300 gel filtration to be 50,000, 50,000-80,000, and 80,000, respectively. These GAGs were not digested with any glycosaminoglycanases. On the basis of one and two dimensional NMR spectra, glycosidase treatment and periodate oxidation of the pyridylaminated trisaccharide obtained by acid hydrolysis of illexin C, the unique repeating structure of the GAGs was determined to be (-6GalNAc alpha 1-3GlcA beta 1-3Fuc alpha 1-)n.

Amino Acids↗
PubMed1994

Contact sensitization induces proliferation of heterogeneous populations of hapten-specific T cells.

To characterize the T cells that are activated during the induction of contact hypersensitivity (CH), two sets of studies were conducted: 1) dinitrophenol (DNP)-specific proliferative responses of T cells in draining lymph nodes of BALB/c mice sensitized epicutaneously to dinitrofluorobenzene (DNFB) were examined, and 2) from these lymph node cells, DNP-specific T cells were cloned by limiting dilution microculture and analyzed by FACS for surface markers, by RT-PCR, HT2 bioassay and ELISA for cytokine expression at mRNA and protein levels respectively, and by proliferation assay for cytokine and antigen-presenting cell (APC) requirements. Our results show that alpha beta TCR-bearing T cells of both the CD4+ and CD8+ subtypes from lymph nodes of DNFB-skin-painted mice proliferate specifically to dinitrobenzene sulfonate (DNBS) in vitro. Four DNP-specific, CD4+ T-cell clones were characterized: clone 5S4 secreted IL-4 and required Il-4 for optimal growth; clone 5S10 secreted IL-2 and required IL-2 for optimal growth; clone 5S2 secreted IL-4 but required IL-2 for optimal growth; and clone 5S8 secreted IL-2 predominantly at 5 months, but switched to production of IL-4 at 7 months. All four clones secreted IL-10, and proliferated to DNBS when Langerhans cell (LC)-enriched epidermal cells were used as APC. These findings indicate that heterogeneous populations of DNP-specific T cells are activated in draining lymph nodes during the induction of CH to DNFB in BALB/c mice.

Administration, Cutaneous↗
PubMed1994

An investigation of the antitumor peptidoglycan fraction from squid ink.

Squid ink, which has little commercial use and is usually discarded, was extracted using a Tris-HCl buffer (pH 6.8). The extract was fractionated using DEAE Sephacel ion-exchange chromatography and Sephacryl S-300 gel filtration to give a peptidoglycan fraction which exhibited strong antitumor activity against Meth-A fibrosarcoma in BALB/c mice following intraperitoneal administration. The fraction was composed of 7.8% peptide, 57% polysaccharide and 30% pigment. The polysaccharide component had a unique structure with equimolar ratios of GlcA, GalNAc and Fuc. Since the fraction has no direct cytotoxic effect on Meth-A cells, inhibition of tumor growth may be due to stimulation of host-mediated responses.

Animals↗
PubMed1993

Keratinocyte-derived IL-7 serves as a growth factor for dendritic epidermal T cells in mice.

Dendritic epidermal T cells (DETC) are CD3+, CD45+, CD4-, CD8-, TCR-V gamma 3/V delta 1+ T lymphocytes that reside in symbiosis with keratinocytes in mouse epidermis. To address mechanisms by which these cells survive and proliferate within the epidermal environment, we have tested the hypothesis that cytokines secreted by neighboring keratinocytes play relevant roles. The present study was conducted to determine whether keratinocytes produce biologically relevant amounts of IL-7, and, if so, to study its effects on DETC. The long term cultured DETC line, 7-17, and freshly isolated DETC exhibited dose- and time-dependent proliferative responses to rIL-7. These responses were blocked completely by anti-IL-7 antibodies, whereas anti-IL-2 had no effect, indicating that DETC respond to IL-7 by an IL-2-independent mechanism. Proliferative responses depended on the state of cell activation; DETC stimulated 2 to 5 days earlier with Con A proliferated optimally to added IL-7, whereas cells stimulated 10 days earlier did not proliferate. DETC that failed to proliferate responded to IL-7 by showing prolonged survival; cells maintained for 7 days with IL-7 alone retained their capacity to proliferate in response to Con A. Mouse epidermal cells and Pam 212 keratinocyte line both expressed IL-7 mRNA constitutively, as demonstrated by reverse transcription-polymerase chain reaction analyses. The production of IL-7 by mouse keratinocytes was also confirmed; Pam 212 culture supernatants supported DETC proliferation, and this activity was diminished by 50% with added anti-IL-7 antibodies. These results indicate that keratinocytes produce IL-7 in biologically relevant amounts, which, in turn, serve to promote the survival and growth of DETC. IL-7-mediated communication between epithelial cells and gamma delta T cells may represent one mechanism to sustain the indefinite residence of gamma delta T cells in epithelial tissues of mice.

Animals↗
PubMed1993

Profiles of cytokine mRNA expressed by dendritic epidermal T cells in mice.

The epidermis of mice contains, in addition to Langerhans cells, a second dendritic population that is Thy-1+/CD3+/CD4-/CD8-/T-cell receptor-V gamma 3/V delta 1+. These dendritic epidermal T cells (DETC) are now thought to comprise one element in the family of epithelial tissue-resident gamma delta T cells. In the present study, DETCs were examined for their expression of mRNA for cytokines, using a reverse transcription-polymerase chain reaction. Freshly isolated Thy-1+ epidermal cells constitutively expressed mRNA for gamma-interferon, but not IL-2. Within 24 h after stimulation with Con A, these cells then expressed mRNA for gamma-interferon and IL-2, but not IL-4. The rapid onset of expression of mRNA for IL-2 occurred exclusively within the Thy-1+ population, and in a Con A-dependent fashion. When freshly isolated epidermal cells were first stimulated with Con A and then expanded in bulk with rIL-2 for 20-24 d, cells expressing IL-4 mRNA then emerged, upon secondary stimulation with Con A. These "short-term" DETC lines also expressed mRNA for IL-2, interferon-gamma, IL-1 alpha, IL-3, IL-6, IL-7, tumor necrosis factor alpha and beta, and granulocyte macrophage-colony stimulating factor. Interestingly, mRNA for IL-4 and IL-6 was no longer detected in long-term (> 1 year) DETC lines 7-17 and 12-12. In addition, one line (7-17) maintained IL-3 mRNA expression, whereas another (12-12) had lost this capacity. These results emphasize the concept that, as resident cells in epidermis, DETCs exhibit several different immunorelevant activities, and the heterogeneity in cytokine mRNA profiles suggests that DETCs may divide into functional subsets.

Animals↗
PubMed1993

Reciprocal cytokine-mediated cellular interactions in mouse epidermis: promotion of gamma delta T-cell growth by IL-7 and TNF alpha and inhibition of keratinocyte growth by gamma IFN.

A unique subset of gamma delta T cells, termed dendritic epidermal T cells (DETC), resides in symbiosis with keratinocytes in mouse epidermis. We have shown previously that interleukin 7 (IL-7) which is produced by keratinocytes, promotes growth and prevents apoptosis in DETC. To extend this observation, we examined 12 cytokines, each of which is expressed by epidermal cells at mRNA and/or protein levels, for their capacities to modulate the growth of DETC. Cytokines examined included IL-1 alpha, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), granulocyte/macrophage-colony stimulating factor (GM-CSF), and macrophage inflammatory protein-1 alpha (MIP-1 alpha). When tested individually, IL-2 and IL-7 promoted maximal growth of the long-term cultured DETC line 7-17. When tested in combinations, synergistic growth-promoting effects were seen with IL-2 and IL-4 or IL-7, and with IL-7 and IL-4 or TNF alpha. Dose-response experiments demonstrated that TNF alpha, which is produced by keratinocytes, enhances IL-7-induced DETC proliferation, but inhibits IL-2-induced proliferation. The mouse keratinocyte-derived cell line Pam 212 was used to test these cytokines for their capacities to regulate keratinocyte growth. Only gamma IFN, which is produced by DETC, inhibited proliferation in a dose-dependent fashion. These results illustrate three reciprocal pathways by which epidermal cytokines regulate the growth of epidermal cells: 1) a paracrine mechanism by which keratinocyte-derived cytokines (e.g., IL-7 and TNF alpha) promote the growth of DETC, 2) an autocrine mechanism by which DETC-derived cytokines (e.g., IL-2 and IL-4) support their own growth, and 3) a reciprocal pathway in which a cytokine produced by resident epidermal leukocytes (e.g., gamma IFN) modulates the growth of keratinocytes.

Animals↗
PubMed1993

Isolation and culture of human hepatocytes from resected liver tissue as a bioreactor for a hybrid artificial liver.

To use cultured human hepatocytes as a hybrid artificial liver, effective methods for isolating and culturing the hepatocytes from resected surgical specimens were investigated. Two different procedures for isolating hepatocytes, perfusion and agitation with a collagenase solution (Method 1) and perfusion with a mixed solution of collagenase and dispase (Method 2), were examined. The yield of isolated hepatocytes obtained by Method 2 (13.31 x 10(6) cells/g of liver) was significantly higher than that by Method 1 (0.94 x 10(6)). The warm ischemia time (0-90 min) of the liver fragments obtained did not disturb the viability and yield of the isolated hepatocytes. The gluconeogenesis and urea synthesis of the cultured human hepatocytes were well preserved for 10 days. These results show that for prolonged human hepatocyte culture (10 days), isolation from resected human liver tissues by a combination of the proteolytic enzymes collagenase and dispase was effective and warm ischemia was tolerated for up to 90 min, which indicates the possibility of using cultured human hepatocytes as a hybrid artificial liver.

Artificial Organs↗
PubMed1992

Folate receptor allows cells to grow in low concentrations of 5-methyltetrahydrofolate.

The folate receptor mediates the uptake of 5-methyltetrahydrofolate in certain cultured cells by a process called potocytosis. When these cells are grown in physiological concentrations of folate, the receptor increases the efficiency of vitamin uptake by 30-fold. We now show that PAM 212 cells, a mouse keratinocyte cell line, are unable to grow in 1 nM 5-methyltetrahydrofolate unless they express a functional folate receptor. These results suggest that under certain conditions, tissue cells in the body may depend on the folate receptor to obtain enough 5-methyltetrahydrofolate for growth.

Animals↗
PubMed1992

Langerhans cells are the major source of mRNA for IL-1 beta and MIP-1 alpha among unstimulated mouse epidermal cells.

Immunocompetent cells of the epidermis can interact by the elaboration and recognition of cytokines. Although much new information has been reported concerning the cytokines secreted by keratinocytes, little is known about cytokines derived from Langerhans cells (LC). To address this deficiency, we examined cytokine mRNA profiles in different epidermal preparations from BALB/c mice, taking advantage of the sensitive technique of polymerase chain reaction (PCR), after reverse transcription of mRNA. In assays of epidermal sheets separated from dermis by ammonium thiocyanate, mRNA for IL-1 alpha, IL-1 beta, IL-6, IL-7, tumor necrosis factor alpha (TNF alpha), TNF beta, granulocyte macrophage/colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) were unequivocally present. By contrast, faint bands were detected for IL-4, IL-5, and interferon gamma (IFN gamma), and no PCR signal was detected for IL-2. Importantly, assays of epidermal cells (EC) dissociated with trypsin revealed similar mRNA profiles. To determine the effects of cell isolation, fluorescence-activated cell sorter (FACS)-purified Ia- EC were first analyzed; all of the previously cited cytokine mRNA were present except for IL-1 beta and MIP-1 alpha. EC depleted of LC by a second technique, lysis using anti-Ia monoclonal antibody and complement, revealed similar profiles, with substantially reduced PCR signals for IL-1 beta and MIP-1 alpha. Finally, FACS-purified LC (Ia+ EC) clearly expressed IL-1 beta and MIP-1 alpha mRNA, a finding that was verified by Southern blotting using internal oligo probes. We conclude that these cell-isolation procedures did not produce substantial alterations in basal mRNA profiles and that LC are the principal source of mRNA for IL-1 beta and MIP-1 alpha among unstimulated EC in mice.

Animals↗
PubMed1992

Cytokine expression by epidermal cell subpopulations.

Epidermal cells (EC) are a rich source of cytokines that can regulate the function of cells in skin and in other tissues. To organize the array of data pertaining to cytokine expression by EC subpopulations, we have tabulated such data according to cell source, state of cell activation, and type of assay employed. This information forms a background for our own studies, in which reverse transcriptase-polymerase chain reaction (RT-PCR) was used to show that Langerhans cells (LC) are the principal source of mRNA for interleukin 1 beta and macrophage inflammatory protein-1 alpha (MIP-1 alpha) among unstimulated mouse EC.

Animals↗