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S Huang

Publications and source records attributed to S Huang.

At least 469 records · Page 26Linked to original sources
PubMed1995

Early interleukin 12 production by macrophages in response to mycobacterial infection depends on interferon gamma and tumor necrosis factor alpha.

Interleukin 12 (IL-12) produced by macrophages immediately after infection is considered essential for activation of a protective immune response against intracellular pathogens. In the murine Mycobacterium bovis Bacillus Calmette-Guérin (BCG) model we assessed whether early IL-12 production by macrophages depends on other cytokines. In vitro, murine bone marrow-derived macrophages produced IL-12 after infection with viable M. bovis BCG or stimulation with LPS, however, priming with recombinant interferon gamma (rIFN-gamma) was necessary. In addition, IL-12 production by these macrophages was blocked by specific anti-tumor necrosis factor alpha (TNF-alpha) antiserum. Macrophages from gene deletion mutant mice lacking either the IFN-gamma receptor or the TNF receptor 1 (p55) failed to produce IL-12 in vitro after stimulation with rIFN-gamma and mycobacterial infection. In vivo, IL-12 production was induced in spleens of immunocompetent mice early during M. bovis BCG infection but not in those of mutant mice lacking the receptors for IFN-gamma or TNF. Our results show that IL-12 production by macrophages in response to mycobacterial infection depends on IFN-gamma and TNF. Hence, IL-12 is not the first cytokine produced in mycobacterial infections.

Animals↗
PubMed1995

The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors.

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence-activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38-, HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.

Antigens, CD↗
PubMed1995

Crystal structure of spinach chloroplast fructose-1,6-bisphosphatase at 2.8 A resolution.

The three-dimensional structure of the spinach chloroplast fructose-1,6-bisphosphatase (Fru-1,6-Pase) has been solved by the molecular replacement method at 2.8 A resolution and refined to a crystallographic R factor of 0.203. The enzyme is composed of four monomers and displays pseudo D2 symmetry. Comparison with the allosteric Fru-1,6-Pase from pig kidney shows orientationally displaced dimers within the quaternary structure of the chloroplast enzyme. When the C1C2 dimers of the two enzymes are superimposed, the C3C4 dimer of the chloroplast enzyme is rotated 20 degrees and 5 degrees relative to the C3C4 dimer of the R and T forms of the pig kidney enzyme, respectively. This new quaternary structure, designated as S, may be described as a super-T form and is outside of the pathway of the allosteric transition which occurs in the pig kidney enzyme, which shows a 15 degrees rotation between T and R forms. Chloroplast Fru-1,6-Pase, unlike the pig kidney enzyme, is insensitive to allosteric transformation by AMP. Structural changes in the AMP binding site involving mainly helices H1, H2, and H3 and the loop between H1 and H2 at the dimer interface interfere with binding of the phosphate of AMP. Finally, the location of cysteines residues provides a basis for a preliminary discussion of the activation of the enzyme by reduction of cysteines via the ferredoxin-thioredoxin f system; this process is complementary to activation by pH changes, Mg2+ or Ca2+, Fru-1,6-P2, and possibly Fru-2,6-P2.

Adenosine Monophosphate↗
PubMed1995

Structural aspects of the allosteric inhibition of fructose-1,6-bisphosphatase by AMP: the binding of both the substrate analogue 2,5-anhydro-D-glucitol 1,6-bisphosphate and catalytic metal ions monitored by X-ray crystallography.

The crystal structures of the T form pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) complexed with AMP, the substrate analogue 2,5-anhydro-D-glucitol 1,6-bisphosphate (AhG-1,6-P2), and Mn2+ at concentrations of 5, 15, 100, and 300 microM have been determined and refined at resolutions of 2.1-2.3 A to R factors which range from 0.180 to 0.195, respectively. Two metal ions per active site have been identified, one at a binding site of high affinity (metal site 1'), the second in a low affinity site (metal site 2'). The 1-phosphate group of the substrate analogue coordinates to the metal ion at site 1', but not at site 2'. In these four complexes, the distances between the two metal ions are all within 0.2 A of 4.3 A. In the previously determined R form structure of Fru-1,6-Pase complexed with AhG-1,6-P2 and Mn2+, there are also two metal ions in the active site at metal sites 1 and 2. The metal ion at site 1 is only 0.6 A displaced from the metal ion at site 1' in the T form and is also coordinated to the 1-phosphate group of AhG-1,6-P2. However, the second metal ion is located in two distinct sites which are 1.4 A apart in the T and R form structures. In the R form the Mn2+ at site 2 is coordinated to the 1-phosphate group of the substrate analogue. This metal ion is apparently required to orient the phosphate group for nucleophilic attack at the phosphorus center.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1995

Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells.

Previous studies from our laboratory demonstrated an inverse relationship between the expression level of inducible nitric oxide synthase (iNOS) and the metastatic potential of murine K-1735 melanoma cells. The purpose of this study was to provide direct evidence that the expression of iNOS suppresses metastatic potential of melanoma cells. Highly metastatic K-1735 clone 4 cells (C4.P), which express low levels of iNOS, were transfected with a functional iNOS (C4.L8), inactive-mutated iNOS (C4.S2), or neomycin-resistance (C4.Neo) genes in medium containing 3 mM NG-methyl-L-arginine (NMA). Positive transfectants were identified by Southern and Northern blot analyses and homogeneous staining with a specific anti-iNOS monoclonal antibody. Semiconfluent cultures of C4.P (parental), C4.Neo.3 (control transfection), C4.S2.3 (inactive iNOS), and C4.L8.5 (functional iNOS) were harvested, and viable cells were injected intravenously into syngeneic C3H/HeN mice and allogeneic BALB/c nude mice. C4.P, C4.Neo.3, and C4.S2.3 cells were highly metastatic whereas C4.L8.5 cells were not metastatic. Experiments with [125I]dUrd-labeled tumor cells demonstrated that the initial arrest in the lung microvasculature did not differ among the lines, but that C4.L8.5 cells died by 48-72 h after injection. Enhanced survival of all K-1735 C4 cells (including C4.L8.5) was found in mice given twice daily injections of 20 mg NMA. The C4.L8.5 cells produced slow growing subcutaneous tumors in nude mice, whereas the other three lines produced fast growing tumors. In vitro studies confirmed that in the absence of NMA the expression of iNOS in C4.L8.5 cells induced apoptosis. Collectively, these data demonstrate that the expression of recombinant iNOS in melanoma cells is associated with apoptosis, suppression of tumorigenicity, and abrogation of metastasis.

Amino Acid Oxidoreductases↗
PubMed1995

Involvement of IFN-gamma in Bacillus Calmette-Guérin-induced but not in tumor-induced sensitization to TNF-induced lethality.

In healthy mice, murine (m) TNF is fairly lethal, whereas human (h) TNF (a selective murine TNF-R55 agonist) is rather harmless. However, we and others observed that mice suffering from a bacterial infection, such as Bacillus Calmette-Guérin (BCG), or bearing i.m. some types of tumor, develop a hypersensitivity to the IL-6-inducing and lethal properties of hTNF. This is a cardinal problem as it severely limits the potential use of hTNF-R55-specific agonists for systemic treatment of human cancer. Using mice carrying a targeted disruption in the gene encoding the IFN-gamma receptor (IFN-gamma Ro/o), we here report that endogenous IFN-gamma plays a crucial role in the development of TNF hypersensitivity during BCG infection. Indeed, both the lethality and the IL-6 induced by hTNF were drastically reduced in IFN-gamma Ro/o mice as compared with control mice. These results demonstrate that the enhancement of TNF effects is at least an equally important mechanism by which IFN-gamma contributes to BCG-induced hypersensitivity as the previously described augmentation of TNF production. Experiments in athymic nude mice, either depleted of NK cells or not, revealed that the latter cell population is an important source of the sensitizing IFN-gamma during BCG infection. In contrast, IFN-gamma Ro/o mice were as susceptible as control mice to the sensitizing effects of tumors. mTNF, which interacts with both mTNF-R55 and mTNF-R75 and causes lethality on its own, is as toxic in IFN-gamma Ro/o mice as in wt control mice; this means that TNF-induced IFN-gamma does not play a role in mTNF-induced lethality.

Animals↗
PubMed1995

Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response.

Mice with homologous disruption of the gene coding for the ligand-binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN-gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells.

Animals↗
PubMed1995

Identification of novel promoter and repressor elements in the 5'-flanking regions of the rat insulin-like growth factor-I gene.

Although the major endocrine source of insulin-like growth factor-I (IGF-I) is the liver, little is known about IGF-I transcriptional mechanisms in this tissue. To evaluate the role of cis-regulatory elements, rat hepatocytes in primary culture were transfected with DNA constructs containing IGF-I 5'flanking sequences, fused to a luciferase reporter. We demonstrate the presence of a novel promoter approximately 0.5 kb upstream from exon 1 transcription initiation sites, together with a repressor element in this region, and a downstream repressor element which can modulate the activity of both endogenous and heterologous promoters.

Animals↗
PubMed1995

Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation.

Expression of mRNA for the neutrophil (PMN) chemokine, KC, in rat models of lung injury suggests a role for this chemokine in pulmonary inflammation. We addressed this hypothesis at the protein level by functionally characterizing recombinant rat KC (rKC) in vitro and in vivo. In vitro, rKC induced PMN chemotaxis and increased the expression of CD11b/CD18 on PMNs. Recombinant KC also induced a respiratory burst (quantitated by flow cytometry) in rat PMNs, similar to that caused by its human structural homologue, gro/melanoma growth-stimulating activity, on human PMNs, but less than that caused by IL-8 on human PMNs. Intratracheal instillation of rKC induced dose-dependent PMN influx into airspaces (average PMNs in bronchoalveolar lavage: vehicle = 1.5%, n = 4; rKC (1 microgram) = 11.5%, n = 2; rKC (10 micrograms) = 77.3%, n = 2). A neutralizing anti-KC Ab reduced the chemotactic activity of rat bronchoalveolar lavage fluid collected after the intratracheal administration of LPS (48.3 +/- 8% of control, n = 4). Anti-KC neutralizing Ab markedly inhibited PMN accumulation (71 +/- 6%) within the lungs in response to an intratracheal challenge of LPS. We conclude that rat KC is a major but not exclusive mediator of PMN activation and recruitment during LPS-induced pulmonary inflammation.

Amino Acid Sequence↗
PubMed1995

Role of protein kinase C alpha in the induction of carcinoembryonic antigen by transforming growth factor beta 1.

Previous studies showed that transforming growth factor beta 1 (TGF beta 1) regulates the expression of carcinoembryonic antigen (CEA) and CEA-cross-reactive glycoproteins (CEA-GLYs) in human colon carcinoma cells through a signal-transducing pathway associated with protein kinase C (PKC) (Chakrabarty, J. Cell. Physiol., 1992, 152:494-499). In this study we determined the role of the PKC alpha isoform in the regulation of CEA and CEA-GLYs expression by TGF beta 1. Expression of PKC alpha antisense RNA, through transfection experiments with an antisense PKC alpha expression vector, resulted in down-modulation of PKC alpha RNA and protein expression. TGF beta 1 was unable to stimulate the expression and secretion of CEA in cells in which the expression of PKC alpha protein was substantially reduced. The ability of TGF beta 1 to stimulate the expression of the 95- and 55-kDa CEA-GLYs, however, was not affected. We therefore conclude that TGF beta 1 regulates the secretion and expression of CEA through a signal-transducing pathway associated with PKC alpha. TGF beta 1 may also regulate the expression of CEA-GLYs through signal-transducing pathways associated with other PKC isoforms.

Carcinoembryonic Antigen↗
PubMed1995

Expression of antisense epidermal growth factor receptor RNA downmodulates the malignant behavior of human colon cancer cells.

Human colon cancer (Moser) cells produce and secrete epidermal growth factor (EGF) and respond to EGF via an autocrine/paracrine mode through the cell surface EGF receptor (EGFR). In this report we show that EGF promotes the malignant behavior of the Moser cells in vitro in terms of growth in soft agarose and invasion of Matrigel-coated porous membranes. Expressing antisense EGFR RNA in the Moser cells (through transfection with an inducible antisense EGFR expression vector) downmodulated the expression of cell surface EGFR and EGFR mRNA with a concurrent inhibition of growth in soft agarose and invasion of Matrigel-coated membranes. In addition, the ability of exogenously applied EGF in promoting the malignant behavior of these cells was circumvented. We conclude that antisense EGFR RNA was a potent agent in circumventing the in vitro malignant properties of the Moser cells.

Cell Division↗
PubMed1995

Suppression of tumor growth and metastasis of murine renal adenocarcinoma by syngeneic fibroblasts genetically engineered to secrete the JE/MCP-1 cytokine.

The murine monocyte chemoattractant protein 1, JE/MCP-1, like its human counterpart monocyte chemotactic and activating factor (MCAF), attracts monocytes-macrophages to tumor tissues. In previous studies we reported that expression of the JE/MCP-1 gene in murine colon carcinoma cells reduced their tumorigenicity and suppressed their metastatic potential. We now demonstrate that the growth and metastasis of the renal adenocarcinoma cell line RENCA are reduced when it was admixed with syngeneic fibroblasts engineered to secrete the JE/MCP-1 cytokine before injection. Culture supernatants of JE/MCP-1-expressing cells plus lipopolysaccharide (LPS) synergistically activated tumoricidal properties in syngeneic macrophages against RENCA cells. This activity was blocked by anti-JE/MCP-1 antibody, indicating that JE/MCP-1 was involved in priming the macrophages to respond to LPS. Moreover, alveolar macrophages isolated shortly after iv injections of JE/MCP-1 transfected cells were cytotoxic to RENCA cells in vitro. Collectively, these data suggest that in addition to its chemotactic properties, JE/MCP-1 can synergize with bacterial endotoxins to activate macrophages, thus providing a rationale for the use of the JE/MCP-1 protein as a modality for treatment of metastasis.

3T3 Cells↗
PubMed1995

Immune defence in mice lacking type I and/or type II interferon receptors.

Mice lacking the receptor for type I interferon (IFN-alpha beta, A129 mice), for type II interferon (IFN-gamma, G129 mice) or for both receptors (AG129 mice) have been generated by embryonic stem cell mediated gene targeting and inter-crossing A129 x G129, respectively. The role of the two IFN systems in controlling a range of infections has been studied using these mice. Type I IFN is shown to be responsible for the immune defence against most viral infections tested (Lymphocytic Choriomeningitis Virus, Semliki Forest Virus, Theiler's Virus, Vesicular Stomatitis Virus), type II IFN seems to be of little importance. In Vaccinia Virus and Theiler's Virus infection, however, both IFN systems were found to play a nonredundant role. IFN-gamma was critical for the defence against intracellular bacteria (Mycobacterium, Listeria) and parasites (Leishmania), whereas IFN-alpha beta was not. IFN-alpha beta is produced by virus-infected cells within hours and plays an important role in preventing virus spread early. Production of IFN-gamma on the other hand needs activation of the immune system and plays a major role later, i.e. mostly during the immune response. Data obtained with the mice described here show that both IFN systems seem to have evolved to complement each other in the host defence against a wide variety of infectious agents.

Animals↗
PubMed1995

Expansion of gamma delta+ T cells in BALB/c mice infected with Leishmania major is dependent upon Th2-type CD4+ T cells.

T cells belong to either the alpha beta+ or gamma delta+ lineage as defined by their antigen receptor. Although both T-cell subsets have been shown to be involved in the immune response to the parasite Leishmania major, very little is known about possible interactions between these two populations. In this study, using a mouse model of infection with L. major, we showed that expansion of a subset of gamma delta+ T cells in vivo is dependent upon the presence of alpha beta+ CD4+ T cells. Moreover, this effect appears to be mediated via the secretion of lymphokines by CD4+ cells with a T-helper 2 (Th2) functional phenotype. Results showing that activation of Th2-type cells in mice treated with anti-immunoglobulin D antibodies or infected with Nippostrongylus brasiliensis also results in gamma delta+ T-cell expansion suggest that this effect of the Th2-type CD4+ cells is a general phenomenon not restricted to infection with L. major.

Animals↗
PubMed1995

Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery.

Entry of human adenovirus into host cells involves interaction of virus particles with two distinct receptors. The initial binding event is mediated by the fiber protein, while subsequent interaction of the penton base protein with alpha v integrins promotes virus internalization and/or penetration. Although these interactions in epithelial and endothelial cells have been well characterized, relatively little is known as to whether these events occur during virus infection of human peripheral blood mononuclear cells. We demonstrate that freshly isolated peripheral blood monocytes and T lymphocytes express very small amounts of alpha v integrins and also are resistant to adenovirus infection. Exposure of monocytes to hematopoietic growth factors granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor induced expression of cell surface alpha v integrins, promoted the binding of penton base protein, and also rendered these cells susceptible to adenovirus-mediated gene delivery. Stimulation of T cells with a mitogen, phytohemagglutinin, or a cell-activating agent, phorbol myristate acetate, induced expression of alpha v integrins and also enhanced adenovirus-mediated gene delivery. These studies further indicate that alpha v integrins play a crucial role in adenovirus infection and also provide a useful strategy for enhancing adenovirus-mediated gene delivery into human peripheral blood mononuclear cells.

Adenoviruses, Human↗
PubMed1995

Antiviral defense in mice lacking both alpha/beta and gamma interferon receptors.

Alpha/beta interferon (IFN) and gamma IFN exert widely overlapping biological effects. Still, mice with individually inactivated alpha/beta or gamma receptors exhibit variably severely reduced resistance to infection and altered immune responses. To investigate to what extent the two IFN systems are functionally redundant, we generated mice with a combined receptor defect (AG129 mice). Like mice with individual mutations, AG129 mice had no apparent anomalies, confirming that in the mouse the IFN system is not essential for normal development. These mice showed an additive phenotype with respect to antiviral defense and exhibited an increased susceptibility to lymphocytic choriomeningitis virus (LCMV) and notably vaccinia virus infection. Because of unlimited replication and subsequent rapid exhaustion of cytotoxic T lymphocyte (CTL) precursors, these mice were unable to mount a CTL response to LCMV. CD8(+)-mediated immunopathology was absent in LCMV-infected mice, and virus persisted. Vaccinia virus replicated much faster in AG129 mice, and a 10(4)-fold lower dose of vaccinia virus was sufficient to prime these mice. With the normal priming dose of 10(6) PFU, cytopathic effects and overwhelming infection possibly causing partial exhaustion of CTL interfered with the anti-vaccinia virus response. Even though global antiviral immunoglobulin G (IgG) titers were within normal ranges, the IgG subclass distribution was heavily biased toward IgG1.

Animals↗
PubMed1995

Airway neutrophilia and chemokine mRNA expression in sulfur dioxide-induced bronchitis.

Airway inflammation in acute and chronic bronchitis includes a prominent neutrophil influx. Using a rat model of sulfur dioxide (SO2)-induced bronchitis, we investigated the role of the polymorphonuclear leukocyte (PMN) chemokines macrophage inflammatory protein-2 (MIP-2) and KC. Adult female rats were exposed to 230 ppm SO2 for 5 h/day for periods of 1 day to 5 wk. Immunohistochemical identification of rat PMNs in trachea cryostat sections allowed quantitation of a marked neutrophil influx into airways of bronchitic rats (PMNs/trachea ring = 55 +/- 26.2 [1 day SO2] versus 3.6 +/- 2.7 [air]; n = 5, P < or = 0.05). Northern analysis of trachea homogenates demonstrated induction of KC and MIP-2 mRNA expression after 1 day of SO2 and persistence of increased expression after longer exposure periods examined. Pretreatment of rats with dexamethasone (0.5 mg/kg) prior to a 1-day acute SO2 exposure prevented induction of chemokine mRNA and abrogated neutrophil influx completely (PMNs/trachea ring = 6.6 +/- 8.8 versus air controls; n = 5, P = 0.96). To determine if chemokine inhibition by dexamethasone could be further studied in vitro, the rat alveolar macrophage cell line NR8383 was treated with dexamethasone (10(-7) M) before stimulation with lipopolysaccharide (10 micrograms/ml). Pretreatment with dexamethasone substantially decreased induction of both MIP-2 and KC mRNA in response to lipopolysaccharide, indicating the potential utility of in vitro systems to identify additional anti-inflammatory agents. These studies support the hypothesis that the chemokines MIP-2 and KC mediate airway neutrophil influx in both acute and chronic SO2-induced bronchitis in the rat.

Acute Disease↗