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H Dang

Publications and source records attributed to H Dang.

At least 37 records · Page 2Linked to original sources
PubMed1999

Binding of nuclear proteins to the negative regulatory element of the IL-2 gene in lymphocytes from rheumatic patients.

T lymphocytes from several autoimmune diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) exhibit deficient mitogenic response in terms of proliferation and IL-2 production. The expression of the IL-2 gene is regulated by various transcription factors. One of these factors suppresses IL-2 expression and binds to the negative responsive element in the IL-2 gene 5' flanking region (NRE-A). The authors hypothesized that the decreased production of IL-2 by T cells from RA and SLE patients is at least partially caused by high expression of the NRE-A binding protein. To test this hypothesis T cells from healthy donors and patients with RA and SLE were stimulated. Using the electrophoretic mobility shift assay we detected NRE-A DNA-binding proteins in the nuclei of the stimulated cells. No difference was found between NRE-A DNA binding in nuclear extracts of T cells taken from healthy donors and those taken from patients. The specificity of the DNA-protein interactions was ascertained through the use of unlabeled DNA competitors. No correlation was found between DNA-binding and the patients' disease duration or medication. In conclusion, decreased IL-2 biosynthesis by T lymphocytes from RA and SLE patients can not be explained by abnormal expression of the NRE-A DNA-binding protein.

Adult↗
PubMed1999

Monocyte rescue of human T cells from apoptosis is CD40/CD154 dependent.

The induction of T-cell apoptosis is regulated in part by monocytes (CD14+ cells). Human peripheral blood monocytes inhibited the spontaneous cell death of activated T cells in vitro. The inhibition of T-cell apoptosis did not require autologous monocytes. Inhibition required direct contact with monocytes and was not due to a soluble factor. Furthermore, treatment of monocytes with actinomycin D, cycloheximide and paraformaldehyde abrogated the anti-apoptotic activity of these cells. Blocking antibody to CD40 and CD154 (CD40 ligand) decreased the ability of monocytes to aid in T-cell survival, whereas, blocking LFA-1/I-CAM-1, Fas ligand and the CD4/major histocompatibility complex class II interaction did not affect the influence of monocytes on T-cell survival. This shows that monocytes rescue of activated T cells from apoptosis is dependent upon CD40/CD154 interaction.

Antibodies, Blocking↗
PubMed1998

Fas (CD95)-transduced signal preferentially stimulates lupus peripheral T lymphocytes.

Fas (CD95) is a cell surface receptor whose biological function in circulating peripheral T cells is not well understood. To address the question of abnormal T cell sensitivity to Fas stimulation in systemic lupus erythematosus (SLE), we studied Fas-transduced stimulation and apoptosis in peripheral blood T cells from patients with SLE and normal control. Immobilized anti-Fas monoclonal antibodies (mAb) (imCH-11; IgM type) significantly stimulated SLE T cell proliferation compared to T cells from normal donors and patients with rheumatoid arthritis (p < 0.003 and p < 0.005, respectively). The soluble form of CH-11 and other immobilized anti-Fas mAb (UB-2, ZB-4; IgG type) failed to stimulate lupus T cells while immobilized human Fas ligand did. Furthermore, imCH-11 induced IL-2 and IL-6 mRNA expression. However, imCH-11 activation failed to induce expression of the T cell activation surface molecules CD25 and CD69. Addition of exogenous ceramide, a second messenger for Fas-mediated apoptosis signaling, also induced T cell proliferation in SLE and normal controls. Moreover, fumonisin B1, a specific ceramide synthase inhibitor, and caspase inhibitors markedly suppressed imCH-11 induced T cell proliferation, suggesting that the ceramide pathway may be involved in Fas-transduced stimulation signals in SLE T cells. These results show that SLE T cells have an alteration in the Fas signal transduction pathway leading to cell proliferation. This defect may be important in Fas-mediated peripheral immune homeostasis.

Adult↗
PubMed1998

Bcl-2 family expression in salivary glands from patients with primary Sjögren's syndrome: involvement of Bax in salivary gland destruction.

To investigate the molecular mechanism of glandular parenchyma destruction in Sjögren's syndrome (SS), Bcl-2, Bax, Bcl-X, and Bak expression were studied. SS (n = 18) and control salivary glands (n = 6) were examined by immunohistochemistry. Apoptosis was assessed by in situ DNA nick end labeling. Infiltrating mononuclear cells in the SS salivary gland showed elevated Bcl-2. These mononuclear cells expressed increased Bax but did not undergo apoptosis. Both SS and control salivary gland ductal epithelial cells expressed Bcl-2, Bax, and Bcl-X. SS, but not normal, salivary gland acinar cells expressed Bax and underwent apoptosis. These results suggest that elevated Bax expression in SS salivary gland acinar cells may play an important role in the apoptotic pathway. In contrast, Bcl-2 expression in SS infiltrating mononuclear cells and ductal cells may contribute to their survival.

Apoptosis↗
PubMed1998

Alpha1A-adrenergic receptors mediate vasoconstriction of the isolated spiral modiolar artery in vitro.

Several lines of evidence suggest that cochlear blood flow is under the control of the sympathetic nervous system and that this control is mediated via alpha-adrenergic receptors. The goal of the present study was to determine whether alpha-adrenergic receptors mediate vasoconstriction of the spiral modiolar artery and, if so, to determine which subtype dominates this response. Vascular diameter was measured with video microscopy in the isolated superfused spiral modiolar artery in vitro. The diameter of the spiral modiolar artery under control conditions was 61 +/- 2 microm (n = 60). Spontaneous vasomotion was observed in most specimens. Addition of norepinephrine to the superfusate caused a phasic vasoconstriction and an increase in the amplitude of vasomotion. These effects were limited to the vicinity of arteriolar branch points of the spiral modiolar artery. Norepinephrine-induced vasoconstriction occurred with EC50 of (1.9 +/- 0.4) x 10(-5) M (n = 44) and the vascular diameter was maximally reduced by a factor of 0.87 +/- 0.01 (n = 29). Neither the phasic nature nor the EC50 of the norepinephrine-induced vasoconstrictions was altered in the presence of the beta2-adrenergic receptor antagonist 10(-5) M ICI118551 or the nitric oxide synthase inhibitor 10(-4) M NOARG. In contrast, the alpha2-adrenergic receptor antagonist 10(-7) M yohimbine and the alpha1-adrenergic receptor antagonist 10(-9) and 10(-8) M prazosin caused a significant shift in the dose-response curve. The affinity constants (K(DB)) for yohimbine and prazosin were (5+/-2) x 10(-8) M (n=4) and (2.0+/-0.7) x 10(-10) M (n=18), respectively. The alpha1A-adrenergic receptor antagonist 10(-8) M 5-methyl urapidil and the alpha1D-adrenergic receptors antagonist 5 x 10(-6) M BMY7378 caused a significant shift in the dose-response curve. The K(DB) values for 5-methyl urapidil and for BMY7378 were (2.7 +/- 0.7) x 10(-10) M (n = 8) and (4.4 +/- 2.7) x 10(-7) M (n = 8), respectively. Further, total RNA was isolated from microdissected spiral modiolar arteries and the presence of transcripts for alpha1-adrenergic receptor subtypes was determined by reverse transcription polymerase chain reaction (RT-PCR). Primers specific for gerbil alpha1-adrenergic receptor subtypes were developed using RNA from rat and gerbil brain. Analysis of RNA extracted from the spiral modiolar artery revealed RT-PCR products of the appropriate size for the alpha1A-adrenergic receptor, however, no evidence for the alpha1B- and alpha1D-adrenergic receptor was found. Further, analysis of RNA extracted from blood, which was a contaminant of the microdissected spiral modiolar arteries, revealed no RT-PCR products. Sequence analysis of the RT-PCR product of the alpha1A-adrenergic receptor from the spiral modiolar artery confirmed its identity. Identity between the 175 nt gerbil sequence fragment and the known rat, mouse and human alpha1A-adrenergic receptor sequences was 90.9, 92.0 and 85.2%, respectively. These observations demonstrate that the spiral modiolar artery contains alpha1A-adrenergic receptors which mediate vasoconstriction at branch points.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1998

Contributions of N-linked glycosylation to the expression of a functional alpha7-nicotinic receptor in Xenopus oocytes.

The alpha7 subunit of the neuronal nicotinic acetylcholine receptor, when expressed in Xenopus oocytes, forms homooligomeric ligand-gated ion channels that are blocked by a snake toxin, alpha-bungarotoxin. The amino-terminal extracellular domain of the alpha7 sequence has three consensus sites for asparagine-linked glycosylation (N46DS, N90MS, and N133AS). In this study, we show that alpha7 expressed either in vivo or in vitro is a glycoprotein of 57 kDa. In addition, we demonstrate by site-directed mutagenesis that all three consensus sites are used for glycosylation. To elucidate the role(s) of asparagine-linked glycosylation in the formation and function of the alpha7 receptor, wild-type and glycosylation-deficient alpha7 subunits were expressed in COS cells and oocytes. We examined biochemical and physiological properties of expressed receptors and found that alpha7 glycosylation mutations do not affect homooligomerization and surface protein expression of the alpha7 receptor but do affect surface expression of alpha-bungarotoxin binding sites and the function of the receptor. Our data indicate that asparagine-linked glycosylation is required for the expression of a functional alpha7 receptor in oocytes.

Amino Acid Sequence↗
PubMed1998

[Alteration of fatty acid components of erythrocyte membrane and insulin resistance in diabetic patients].

OBJECTIVE: To study the effect of alteration of erythrocyte membrane fatty acid components and fluidity on insulin resistance (ISR) in NIDDM patients. METHODS: The fatty acid(FA) components and fluidity of erythrocyte membrane in 52 NIDDM patients with and without hyper-insulinemia were examined by a high-performance liquid chromatographic method and flourescence polarization technique. RESULTS: The content and composition erythrocyte membrane arachidonate (AA) were significantly lower in NIDDM patients than in controls, and also markedly lower in patients with hyperinsulinemia than those without hyperinsulinemia. The diabetics had increased erythrocyte membrane microviscosities as compared with controls, and membrane microviscosities were higher in diabetics with hyperinsulinemia than those without hyperinsulinemia. AA contents of erythrocyte membrane were negatively correlated with membrane membrane microviscosities and positively with ISI in NIDDM patients. CONCLUSION: In NIDDM patients, hyperinsulinemia and ISI bring about AA metabolic abnormalities and decreased AA contents in cell membrane. Alteration of cell membrane FA components results in the changes of membrane fluidity, and the latter can increase IRS.

Aged↗
PubMed1998

CD4 mononuclear cell infiltrates and Fas/Fas ligand positive mammary gland cells in breast tissue from a patient with Sjögren's syndrome.

We describe a 49-year-old patient with lip biopsy proven Sjögren's syndrome (SS) and keratoconjunctivitis sicca, who had dental caries, xerostomia, recurrent upper respiratory tract infections, arthritis in her hands, elbows and knees, and recurrent parotid inflammation. She developed bilateral breast nodules in 1988. Right breast nodules were excised in 1993 and 1995, but reappeared in 1996, requiring 2 more excisions. Breast tissue samples showed remarkable intralobular and perilobular mononuclear cell infiltrates that were predominantly CD4+ T cells and expressed bcl-2. A few cells stained CD20+ and CD8+. SS breast glandular epithelial cells stained more intensely for Fas compared to normal cells. CD4+ T cells and Fas mediated cell death may be involved in the mammary gland lesions in SS.

Breast↗
PubMed1997

Up-regulation of cytokine mRNA, adhesion molecule proteins, and MHC class II proteins in salivary glands of TGF-beta1 knockout mice: MHC class II is a factor in the pathogenesis of TGF-beta1 knockout mice.

Mice homozygous for a disrupted TGF-beta1 allele develop multiple lymphoproliferative disorders similar to those seen in the pseudolymphoma of Sjögren's syndrome. At 2 wk of age, these TGF-beta1 mutant mice begin to develop wasting syndrome and die at around 4 to 5 wk of age. We studied salivary glands from symptomatic mutant mice >14 days of age. Reverse transcriptase-PCR analysis showed up-regulation of proinflammatory cytokine genes such as IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, and IFN-gamma in these mutant mice. Enhanced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1), and MHC class II as well as CD4-positive T lymphocyte infiltration was detected by immunostaining. To elucidate the role of MHC class II, salivary glands from TGF-beta1/MHC class II double knockout mice were used to investigate the expression of adhesion molecules and MHC class II. In spite of the existence of basal intercellular adhesion molecule-1 expression on vessels, there was neither MHC class II expression, enhanced vascular cell adhesion molecule-1 expression, nor lymphocytic infiltration in the salivary glands. These results suggest that MHC class II plays a significant role in the pathogenesis of TGF-beta1 mutant mice. Although the mechanism that initiates multiple inflammatory diseases in these mice remains unclear, the context reported here would provide insight into the immunopathology of Sjögren's syndrome.

Animals↗
PubMed1997

Fas and Fas ligand expression in the salivary glands of patients with primary Sjögren's syndrome.

OBJECTIVE: To assess the role of Fas-mediated apoptosis in the salivary glands of patients with primary Sjögren's syndrome (SS). METHODS: Expression of Fas, Fas ligand (FasL), and bcl-2 in salivary gland biopsy material was detected in situ by immunohistochemical staining and reverse transcriptase-polymerase chain reaction. DNA fragmentation in apoptotic cells was assessed by the enzymatic incorporation of labeled nucleotides (digoxigenin-dUTP). RESULTS: The acinar epithelial cells in SS were Fas+ and FasL+, and these cells died by apoptosis. The majority of infiltrating lymphocytes in SS were Fas+ and bcl-2+, while few lymphocytes expressed FasL. In situ detection of apoptosis showed minimal cell death of lymphocytes, particularly in dense periductal foci. Lymphocytic cell death was significantly lower (P < 0.0001) in these foci compared with that in the interstitium. CONCLUSION: Infiltrating lymphocytes in the focal lesions of the salivary glands of patients with SS are blocked in their ability to commit to apoptosis, even though they may express Fas. The presence of bcl-2 in these cells may explain their inability to undergo apoptosis. The acinar epithelial cells, in contrast, may undergo Fas-mediated apoptosis. These results suggest that the Fas death pathway may be an important mechanism leading to the glandular destruction found in SS.

Apoptosis↗
PubMed1997

Flow cytometric detection of Cryptosporidium oocysts in human stool samples.

Cryptosporidium parvum is an important pathogen that causes diarrhea in virtually all human populations. Improved diagnostic methods are needed to understand the risk factors, modes of transmission, and impact of cryptosporidiosis. In the present study, we fluorescently labeled and counted C. parvum oocysts by flow cytometry (FC) and developed a simple and efficient method of processing human stool samples for FC analysis. Formed stool (suspended in phosphate-buffered saline) from an asymptomatic, healthy individual was seeded with known concentrations of oocysts, and oocysts were labeled with a cell wall-specific monoclonal antibody and detected by FC. The method described herein resulted in a mean oocyst recovery rate of 45% +/- 16% (median, 42%), which consistently yielded a fourfold increase in sensitivity compared to direct fluorescent-antibody assay of seeded stool samples. However, in many instances, FC detected as few as 10(3) oocysts per ml. Thus, FC provides a reproducible and sensitive method for C. parvum oocyst detection.

Animals↗
PubMed1996

Autoimmunity associated with TGF-beta1-deficiency in mice is dependent on MHC class II antigen expression.

The progressive inflammatory process found in transforming growth factor beta1 (TGF-beta1)-deficient mice is associated with several manifestations of autoimmunity, including circulating antibodies to nuclear antigens, immune complex deposition, and increased expression of both class I and class II major histocompatibility complex (MHC) antigens. The contribution of MHC class II antigens to the genesis of this phenotype has been determined by crossing the TGF-beta1-null [TGF-beta1(-/-)] genotype into the MHC class II-deficient [MHC-II(-/-)] background. Mice homozygous for both the TGF-beta1 null allele and the class II null allele [TGF-beta1(-/-);MHC-II(-/-)] are without evidence of inflammatory infiltrates, circulating autoantibodies, or glomerular immune complex deposits. Instead, these animals exhibit extensive extramedullary hematopoiesis with progressive splenomegaly and adenopathy, surviving only slightly longer than TGF-beta1(-/-);MHC-II(+/+) mice. The role of CD4+ T cells, which are also absent in MHC class II-deficient mice, is directly demonstrated through the administration of anti-CD4 monoclonal antibodies in class II-positive, TGF-beta1(-/-) mice. The observed reduction in inflammation and improved survival emphasize the significance of CD4+ cells in the pathogenesis of the autoimmune process and suggest that the additional absence of class II antigens in TGF-beta1(-/-);MHC-II(-/-) mice may contribute to their extreme myeloid metaplasia. Thus, MHC class II antigens are essential for the expression of autoimmunity in TGF-beta1-deficient mice, and normally may cooperate with TGF-beta1 to regulate hematopoiesis.

Animals↗
PubMed1996

Lymphocyte apoptosis and apoptosis-associated gene expression in Sjögren's syndrome.

OBJECTIVE: To study the mechanism and regulation of apoptosis in peripheral blood T and B lymphocytes from patients with Sjögren's syndrome (SS). METHODS: The mode of in vitro lymphocyte death in the peripheral blood of patients with SS was determined by fluorescence microscopic analysis, terminal deoxynucleotidyl transferase assay, and DNA fragmentation analysis. Apoptotic cell death of T and B cells was determined at 48 hours of culture by fluorescence-activated cell sorter analysis of propidium iodidestained cells. Messenger RNA (mRNA) expression of bcl-2, bcl-x, bax, and c-myc in T and B cells was determined by enzyme-linked immunosorbent assay-polymerase chain reaction (ELISA-PCR). Expression of bcl-xL and bcl-xS was determined by Southern blot analysis of PCR products. Gene expression was calculated as the ratio of each gene message to the message of the GAPDH gene. Bcl-2 protein levels in SS T cells were determined by ELISA. RESULTS: SS T cells showed increased in vitro apoptosis compared with normal T cells (mean +/- SD 12.3 +/- 4.5% versus 7.3 +/- 2.0%; P < 0.01). Freshly isolated SS T cells showed increased expression of bcl-2 mRNA compared with normal controls (mean +/- SD 1.50 +/- 0.65 versus 0.88 +/- 0.23; P < 0.05). There was no significant difference in levels of bax or c-myc mRNA in T cells and B cells between SS patients and normal controls. When SS T lymphocytes were cultured in vitro for 72 hours, Bcl-2 protein levels decreased with time. CONCLUSION: SS T cells showed accelerated apoptosis in vitro. Freshly isolated SS T cells had increased expression of bcl-2. An increase in death-promoter signals and decrease in death-suppressor signals in vitro may have been responsible, in part, for the apoptosis in SS T lymphocytes.

Adult↗
PubMed1996

Abnormality of Oct-1 DNA binding in T cells from Sjögren's syndrome patients.

Primary Sjögren's syndrome (SS) is an autoimmune rheumatic disease characterized by T cell hypoactivity. To understand the diminished T cell response to activation signals, we measured nucleoprotein DNA-binding activities regulating gene expression during T cell activation using the electrophoretic mobility shift assay. Peripheral blood lymphocytes from 9/19 SS patients were found to be defective in their ability to bind an october sequence (Oct-1). This Oct-1-binding phenotype remained stable in culture for up to 3 days prior to activation. This abnormality was not seen in resting T cells nor T cells from patients with systemic lupus erythematosus, rheumatoid arthritis (RA), or SS accompanied by RA. The SS Oct-1 DNA-binding abnormality correlated significantly with an inability of cells to exit the Gzero/G1 cell cycle phase when stimulated in vitro. Importantly, nucleoprotein extracts showing decreased DNA-binding activity had normal amounts of Oct-1 proteins as determined by immunoprecipitation, implying a functional defect in the Oct-1 protein. Moreover, defective DNA binding was corrected by treatment with acid phosphatase.

Adult↗
PubMed1995

SLE-like autoantibodies and Sjögren's syndrome-like lymphoproliferation in TGF-beta knockout mice.

Mice bearing the TGF-beta 1 null mutation (-/-) develop lymphoid infiltrates in the heart, lungs, salivary glands, and other organs similar to those seen in the pseudolymphoma of Sjögren's Syndrome. We studied sera from -/- mice and found elevated Ab levels to dsDNA, ssDNA, and Sm ribonucleoprotein. No Abs to SSA/Ro or SSB/La and no IgM rheumatoid factor were found. Serum autoantibodies were predominately IgG and were specific as shown by ELISA inhibition studies. Antinuclear Ab patterns on Western blots varied from one mouse to the next, indicating a random process responsible for the diversity. Wild-type and heterozygote mice had no autoantibodies. Ig glomerular deposits were found in -/- mice, indicating that these autoantibodies may be pathogenic. Treatment of -/- mice with dexamethasone or TGF-beta 1 failed to suppress autoantibody production. These mice represent an overlap combining the autoimmune serology of SLE with the tissue infiltrates of SS. Our results support the concept that TGF-beta 1 is an important naturally occurring immunosuppressive cytokine whose absence can lead to a systemic autoimmune disease.

Animals↗