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Biomedical subjects

C C Chao

Publications and source records attributed to C C Chao.

At least 109 records · Page 6Linked to original sources

CD14 receptor-mediated uptake of nonopsonized Mycobacterium tuberculosis by human microglia.

This study was carried out to determine the role of CD14 receptors in the uptake of nonopsonized Mycobacterium tuberculosis by human microglia. Treatment of microglial cell cultures with antibodies to CD14 or with soluble CD14 significantly blocked infection by M. tuberculosis H37Rv, suggesting that CD14 receptors could facilitate entry of nonopsonized tubercle bacilli into macrophages within the brain.

Antigens, CD↗

Tumor necrosis factor-alpha production by human fetal microglial cells: regulation by other cytokines.

Tumor necrosis factor (TNF)-alpha has been postulated to play an important physiologic as well as pathologic role within the developing brain. In the present study, we found that human fetal microglial cells released abundant amounts of TNF-alpha upon stimulation with lipopolysaccharide (LPS). Treatment of microglial cell cultures with antibodies specific to interleukin (IL)-6, IL-10, and transforming growth factor (TGF)-beta augmented LPS-stimulated release of TNF-alpha. Each of these cytokines dose-dependently suppressed TNF-alpha release. Also, TNF-alpha mRNA expression was inhibited by each of these cytokines. By way of contrast, treatment of microglial cell cultures with IL-alpha or IL-1 beta alone or in the presence of LPS, resulted in increased release of TNF-alpha, and IL-1 stimulated the expression of TNF-alpha mRNA. These findings suggest that these cytokines are likely to modify the beneficial and harmful effects of TNF-alpha within the developing brain.

Base Sequence↗

Cloning and expression of an endothelin receptor subtype B from human prostate that mediates contraction.

Recent evidence suggests a role for endothelin (ET) in contraction of human prostate [J. Urol. 149:495-499 (1993)]. Although both ETA and ETB receptors have been shown to mediate contraction of smooth muscle, the molecular identity of the contractile ETB receptor is controversial. The aim of this study was to examine the receptor subtype that mediates ET-induced contraction in prostate from patients with benign prostatic hyperplasia. Saturation binding with 125I-ET-1 and 125I-ET-3 in prostate stromal cells (PSC) indicated the presence of receptors with subnanomolar affinity for these radioligands, with equivalent receptor densities. Inhibition of specific 125I-ET-1 or 125I-ET-3 binding in PSC revealed a rank order of potency of ET-1 - ET-3 = sarafotoxin S6c >> BQ-123. These data are consistent with a predominance of ETB receptors in PSC. The functional effects of ET stimulation of PSC were examined in a collagen gel contraction assay. ET-1 and ET-3 caused contraction of underlying collagen gel matrices with EC50 values of 0.4 +/- 0.04 and 0.7 +/- 0.2 nM, respectively. To determine the molecular nature of the contractile ETB receptor in PSC, reverse transcription-polymerase chain reactions were conducted with oligonucleotide primers to the 5' and 3' ends of the coding sequence of the full length human ETB receptor. DNA sequence analysis of the 1.3-kilobase DNA product showed 99% homology to other human ETB receptor cDNAs. The encoded protein has a deduced amino acid sequence identical to that of other human ETB receptors, with the exception of two conservative substitutions. Expression of the PSC ETB cDNA in COS-7 cells resulted in a binding profile similar to that observed in parent cells. Polymerase chain reaction analysis revealed the presence of prepro-ET-1 mRNA in PSC. Collectively, these data indicate that PSC from patients with benign prostatic hyperplasia express ETB receptors that mediate ET-induced contraction.

Amino Acid Sequence↗

Glia, cytokines, and neurotoxicity.

Activated glial cells (microglia and astrocytes) are a hallmark of several neurodegenerative disorders. A growing body of evidence supports the hypothesis that activation of glial cells by cytokines contributes to neurotoxicity. Although the precise mechanisms underlying glia-mediated neurotoxicity are unclear, it has been proposed that the generation of toxic free radicals or other neurotoxins is involved. This review focuses on the role of immune mediators released by activated glial cells in causing or preventing neuronal injury. Also, techniques used to assess neurotoxicity are discussed. It is hoped that research in this field will yield insights that will result in new therapies for neurodegenerative disorders.

Cytokines↗

Role of interleukin-6 and transforming growth factor-beta in anorexia nervosa.

Anorexia nervosa is a serious eating disorder characterized by extreme weight loss and abnormalities of the neuroendocrine and immune systems. To determine the potential role of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-beta) in anorexia nervosa, serum concentrations of these cytokines were measured in patients with anorexia nervosa during starvation and after weight gain. Serum IL-6 and TGF-beta concentrations were both significantly elevated during starvation and returned to levels comparable to those of normal-weight controls by the end of therapy. In contrast, serum TNF-alpha levels were undetectable in all patients and controls. Cytokines may play previously unsuspected roles in anorexia nervosa and its complications.

Adolescent↗

Iron mobilization from crocidolite asbestos by human lung carcinoma cells.

Neutron-activated crocidolite, containing 55Fe and 59Fe, was used to determine whether iron was mobilized from crocidolite phagocytized by cultured human lung carcinoma cells (A549 cells). Cells were treated with neutron-activated crocidolite in medium at pH 6.8 or 7.4 for 24 h. The mobilization of iron into two subcellular fractions, 10,000g supernatant (total iron) or < 10,000 MW [low-molecular-weight (LMW)] was monitored using scintillation counting. Iron was mobilized from crocidolite at a rate similar to that observed in vitro when citrate was incubated with crocidolite for 24 h at pH 7.4, but the amount mobilized was greater when cells were cultured at pH 6.8 than at 7.4. Iron mobilization was not due to the medium nor did it appear to be due to differences in the amount of crocidolite phagocytized. At the highest concentration of crocidolite used for treatment at pH 7.4 (4.5 micrograms/cm2), a total of 3600 pmol iron/10(6) cells was mobilized of which 54 pmol/10(6) cells was in a LMW fraction. After estimation of the volume of the cells, this was calculated to be equivalent to an intracellular concentration of 1.4 mM iron of which 22 microM was in the LMW fraction. Cell survival decreased linearly as the iron mobilized into the LMW fraction increased, independent of the pH of the culture medium being used. These results suggest that iron mobilization from crocidolite into a LMW fraction may represent "iron overload" in cells which have phagocytized the fibers and may be responsible for crocidolite-dependent cytotoxicity and possibly other crocidolite-dependent biological effects.

Asbestos, Crocidolite↗

Use of a monoclonal antibody to detect DNA damage caused by the anticancer drug cis-diamminedichloroplatinum (II) in vivo and in vitro.

A monoclonal antibody, MAb62-5, was prepared and used to detect DNA damage due to the anticancer drug cis-diamminedichloroplatinum (II) (or cisplatin). ELISA competition indicated that the binding of MAb62-5 to cisplatin-DNA was competitively inhibited (50% control) by 210 nM of cisplatin bound to DNA, cisplatin/nucleotide (D/N) = 0.2. Using a DNA mobility shift assay, MAb62-5 binding activity was inhibited by 50% by approximately 50-fold molar excess of cisplatin-DNA adducts (D/N = 0.08), whereas there was less than 5% inhibition by UV-DNA adducts or mock-treated DNA. In addition, MAb62-5 showed a similar affinity to the cisplatin-DNA adducts as compared to an endogenous cisplatin-damaged DNA recognition protein. Using ELISA with this antibody, we have demonstrated a 2-fold enhancement in excision repair of cisplatin-DNA adducts in resistant HeLa cells. This is supported by the measurement of repair-associated DNA strand breaks using alkaline elution and host cell reactivation of transfected plasmid DNA carrying cisplatin damage. These findings also provide explanation for the complexicity of immunoassay in cells.

Antibodies, Monoclonal↗

Enhanced excision repair of DNA damage due to cis-diamminedichloroplatinum(II) in resistant cervix carcinoma HeLa cells.

We have previously reported a cisplatin-resistant HeLa cell line which exhibits overproduction of nuclear proteins preferential for cisplatin-modified DNA (Chao et al., Cancer Res. 51:601-605, 1991; Biochem. J. 277: 875-878, 1991). In this study, excision repair of cisplatin-DNA adducts in a resistant and a revertant cell lines was investigated using in situ detection of cisplatin-DNA adducts by an immunoassay and the measurement of repair-associated DNA strand breaks by a sensitive alkaline elution method. The resistant cells exhibited a 2-fold decrease in the accumulation of cisplatin-DNA adducts; whereas, the revertant cells showed a similar level of cisplatin-DNA adducts as the parental cells in the parallel experiment. Immediately following cisplatin treatment, the resistant and the revertant cells accumulated respectively approximately 50% and 90% cisplatin-DNA adducts of the parental cells. However, the kinetic patterns of repair rate following peak accumulation of cisplatin-DNA adducts (which took approximately 4 h) was the same in the three cell lines. This finding was supported by the measurement of repair-associated DNA strand breaks using alkaline elution which showed 1.6- and 1.5-fold increase in the resistant and the revertant cells respectively. In addition, following transfection with plasmid DNA carrying cisplatin damage, the resistant and the revertant cells displayed a 2.4- and 1.4-fold enhancement in host cell reactivation, respectively. Furthermore, the acquired resistance in HeLa cells was partially reversed by nontoxic aphidicolin, a DNA polymerase-alpha and DNA repair inhibitor. The results strongly suggest the improved excision repair of cisplatin-DNA adducts as a mechanism of phenotypic resistance of cells to cisplatin.

Antibodies, Monoclonal↗

Ascorbic acid increases drug accumulation and reverses vincristine resistance of human non-small-cell lung-cancer cells.

A human lung-cancer PC-9 subline with acquired resistance to vincristine (VCR), a chemotherapeutic agent, was established with incremental increases of the drug. The resistant PC-9 subline (PC-9/VCR) shows a 12-fold increase in resistance to VCR and a unique cross-resistance pattern: high cross-resistance to the potent VCR analogue colchicine (6.9-fold) and vinblastine (2.5-fold); lower cross-resistance to actinomycin D (1.8-fold), cisplatin (1.2-fold) and adriamycin (1.3-fold) and a sensitivity to melphalan and VP-16 which is similar to that of the parental cell line. A reduced accumulation of VCR in the resistant cells was demonstrated. Interestingly, the VCR resistance of the PC-9/VCR cell line was partially reversed by ascorbic acid, and the drug uptake was enhanced. In contrast, ascorbic acid had no effect on drug tolerance and drug accumulation was not observed in either PC-9 parental cells or known multidrug-resistant (MDR) cells, suggesting that VCR resistance in PC-9/VCR cells results essentially from reduced drug accumulation. It is worth noting that, whereas reduced drug accumulation in the PC-9/VCR cells was susceptible to modulation by ascorbic acid, the increased efflux rate characteristic of the resistant cells was not. Further, there was a higher efflux rate in resistant cells than in parental cells. DNA Southern- and RNA Northern-blot hybridization analyses indicate that PC-9/VCR cells do not contain amplified mdr genes or overexpress P-glycoprotein. In addition, the calcium-channel blocker verapamil, which acts as a competitive inhibitor of drug binding and efflux, did not affect the resistant phenotype of PC-9/VCR cells. These findings suggest an ascorbic acid-sensitive drug uptake mechanism which is important in mediating VCR resistance per se in human lung-cancer cells; this differs from the P-glycoprotein-mediated MDR mechanism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Human microglial cell defense against Toxoplasma gondii. The role of cytokines.

Microglia may play an important role in host defense of the central nervous system against Toxoplasma gondii, and cytokines produced by these glial cells may participate in their antitoxoplasma activity. In our study, the antitoxoplasma activity of human fetal microglia was investigated. The RH strain of T. gondii multiplied readily in these glial cells. IFN-gamma/LPS-treated microglia limited (p < 0.01) T. gondii growth by reducing entry of this parasite rather than intracellular multiplication. More than 90% of the antitoxoplasma activity of activated microglia was blocked (p < 0.01) by neutralizing antibodies to TNF-alpha or IL-6 (but not to IL-1 or TGF-beta), suggesting that these proinflammatory cytokines play a role in the inhibitory process. Consistent with this hypothesis, treatment of microglia with TNF-alpha or IL-6 (in the presence or absence of IFN-gamma) inhibited (p < 0.01), in a dose-dependent manner, T. gondii growth. Inasmuch as NGMA did not affect cytokine-mediated antitoxoplasma activity of microglia, nitric oxide appears not to be involved in this host defense function of human fetal microglia. Results of our study suggest that the host defense activity of human microglia against T. gondii is dependent primarily on the activating properties of IFN-gamma, TNF-alpha, and IL-6.

Animals↗

Expression and regulation of adhesion molecules by gamma delta T cells from lymphoid tissues and intestinal epithelium.

T cells bearing the gamma delta T cell receptor localize largely in epithelial tissues, but are also present at low frequency in organized secondary lymphoid organs. To assess the role of cell surface adhesion molecules in the traffic and tissue localization of gamma delta T cells, we compared the expression of these molecules on both alpha beta and gamma delta T cells in several lymphoid and non-lymphoid organs. In the gut epithelium, gamma delta cells express less LFA-1 (CD11a), Pgp-1 (CD44), and alpha 4 integrin than the corresponding alpha beta cells. In lymph nodes (LN) and Peyer's patches (PP), adhesion molecule expression by gamma delta cells is heterogeneous, with some of the cells having a phenotype similar to that of intraepithelial gamma delta cells and the rest expressing high levels of CD44 and L-selectin (CD62L) but lower beta 7 and alpha M290, a phenotype more like lymph node alpha beta cells. Therefore, the particular set of adhesion molecules expressed by a T cell is dependent, in part, on its anatomic location. Superimposed upon this, however, are differences in expression that are based on the type of T cell; LN and PP gamma delta T cells express less CD44 but much more beta 7, alpha M290 and ICAM-1 (CD54) than alpha beta T cells in the same organ. The differences in adhesion molecules between alpha beta and gamma delta cells are not due simply to differences in their activation status, because these molecules are regulated differently after activation through the T cell receptor (TcR)/CD3 complex. The differential expression of adhesion molecules on cells bearing a particular TcR V region suggests that distinct adhesion phenotypes may arise from prior contact with specific antigen and resultant cell activation in vivo. Lastly, the presence of high level expression of alpha 4 beta 7 and alpha M290 on L-selectinlo gamma delta cells in lymph nodes suggests that these gamma delta cells may be uniquely capable of migrating to the gut. The differences in adhesion molecule expression and regulation between gamma delta and alpha beta T cells could explain, in part, the distinct homing and tissue localization of these T cell subsets in vivo.

Animals↗

Role of cytokines in lipopolysaccharide-induced functional and structural abnormalities of astrocytes.

The mechanism underlying meningitis-associated brain injury is unclear. This study investigated the hypothesis that lipopolysaccharide (LPS) alters astrocyte function and structure via the release of proinflammatory cytokines. In enriched murine astrocyte cultures, LPS inhibited (P < 0.05) glutamine synthetase activity, 3H-gamma aminobutyric acid uptake, and DNA synthesis; LPS also induced ultrastructural changes. Antibodies to tumor necrosis factor-alpha, interleukin-1, and interleukin-6 blocked (P < 0.05) in part the LPS-induced inhibition of astrocyte function. Also, treatment of astrocyte cultures with cytokines significantly altered these astrocyte functions and ultrastructure. Taken together, the present findings support the hypothesis that LPS affects astrocyte function and structure via the release of proinflammatory cytokines, especially tumor necrosis factor-alpha.

Animals↗

Effect of long-term removal of iron from asbestos by desferrioxamine B on subsequent mobilization by other chelators and induction of DNA single-strand breaks.

The long-term removal of iron from crocidolite or amosite by desferrioxamine B (DF) at pH 7.5 or 5.0 was studied. Crocidolite or amosite (1 mg/ml) was suspended in 50 mM NaCl at pH 7.5 or 5.0 with the addition of 1 mM DF for up to 90 days. Although the rate of iron mobilization decreased with time, iron was continuously mobilized from both forms of asbestos at pH 5.0 or 7.5. The amount of iron mobilized from crocidolite was at least twice that mobilized from amosite at either pH. Iron was mobilized more rapidly from crocidolite at pH 5.0 than at 7.5 for the first 15 days, but at later times the amount being mobilized at pH 7.5 became equal to or slightly greater than that at 5.0. For amosite, the mobilization at pH 5.0 was always greater than that at pH 7.5. Next, the effect of iron removal from asbestos by DF on subsequent iron mobilization by a second chelator (EDTA or citrate) and on induction of DNA single-strand breaks (SSBs) was studied. Asbestos, treated for up to 15 days with DF at pH 7.5, was washed to remove ferrioxamine and excess DF, then incubated with EDTA or citrate (1 mM). The rates of iron mobilization from both forms of asbestos by a second chelator decreased as more and more iron was removed by DF. Induction of DNA SSBs also decreased, reflecting the unavailability of iron to catalyze the damage. The results suggest three things. First, if long-term mobilization of iron from asbestos occurs in vivo as has been observed in vitro, it may play a role in the long-term biological effects of asbestos. Second, more rapid mobilization of iron from asbestos fibers may occur when the fibers are phagocytized by cells and maintained in phagosomes where the pH is 4.0-5.0. Third, treatment of asbestos by iron chelators, such as DF, prior to exposure to cultured cells or whole animals, may reduce the biological effects of asbestos resulting from iron, but may not completely eliminate them.

Asbestos, Amosite↗

Role of endothelin receptor subtype B (ET-B) in myocardial ischemia.

Previous work indicated that endothelin (ET) may be involved in the pathogenesis of myocardial ischemia, although the relative importance of the ET receptor subtypes is presently not clear. The purpose of this study was to determine the role of myocardial ET-B receptors in mediating ischemic/reperfusion damage in isolated rat hearts. Saturation binding analyses were conducted with [125I]ET-1 and [125I]IRL-1620 to assess changes in ET-A and ET-B receptor binding. Total ET receptor density (Bmax) was greater in atrial versus ventricular tissue. ET-A Bmax was 8 to 10-fold greater than ET-B Bmax. In ischemic and ischemic/reperfused atrial tissue neither the equilibrium dissociation constant (Kd) nor Bmax for ET-B receptors was changed. The ET-B receptor Kd in ischemic or ischemic/reperfused ventricular tissue was also unchanged. In ischemic ventricular tissue there was a trend towards an increased ET-B Bmax, which was accentuated after ischemia/reperfusion. No changes were found in ET-A Bmax or Kd in ischemic ventricular or atrial tissue. The physiological importance of this receptor subtype in ischemic myocardium was determined using the selective ET-B agonist, sarafotoxin S6c. In non-ischemic tissue no effect on coronary flow or function were observed with sarafotoxin S6c. Furthermore, no changes were seen in ischemic time to contracture or any of the reperfusion indexes of myocardial damage. The sarafotoxin S6c utilized was active as it inhibited [125I]ET-3 binding to ET-B receptors (Ki = 0.1 nM). Thus, the pro-ischemic effect of ET-1 seems to be mediated by ET-A receptors. ET-B receptors do not appear to play a role in the pathogenesis of myocardial ischemia.

Animals↗

Morphine amplifies HIV-1 expression in chronically infected promonocytes cocultured with human brain cells.

Previous studies have shown that morphine promotes the replication of human immunodeficiency virus (HIV)-1 in peripheral blood mononuclear cell cocultures. In the present study, we tested the hypothesis that morphine would amplify HIV-1 expression in the chronically infected promonocytic clone U1 when cocultured with lipopolysaccharide-stimulated human fetal brain cells. Marked upregulation of HIV-1 expression was observed in these cocultures (quantified by measurement of HIV-1 p24 antigen levels in supernatants), and treatment of brain cells with morphine resulted in a bell-shaped dose-dependent enhancement of viral expression. The mechanism of morphine's amplifying effect appears to be opioid receptor-mediated and to involve enhanced production of tumor necrosis factor-alpha by microglial cells.

Acquired Immunodeficiency Syndrome↗

Cytomegalovirus replication in murine microglial cell cultures: suppression of permissive infection by interferon-gamma.

The pathogenesis of encephalitis due to cytomegalovirus (CMV), particularly the role of microglial cells in the spread or control of infection, remains incompletely defined. In this study, microglial cells were isolated from the brains of newborn mice and infected in vitro with murine CMV (MCMV). Microglial cells supported productive MCMV replication, and the MCMV-infected microglia manifested a cytopathic effect (CPE) characteristic of CMV infection. Exposure of microglia to interferon-gamma (IFN-gamma) 24 h before infection markedly suppressed virus production and resultant CPE in a dose-dependent fashion. Furthermore, the addition of IFN-gamma 2 h after infection demonstrated an antiviral effect equivalent to that achieved when IFN-gamma was administered 2 h before infection. These results demonstrate that murine microglial cells are fully permissive to MCMV replication and that IFN-gamma markedly suppresses virus expression in these cells.

Animals↗

Nitric oxide production and neurotoxicity mediated by activated microglia from human versus mouse brain.

Recent studies indicate that human macrophages lack a high-output inducible nitric oxide synthase (NOS) antimicrobial system. In the present study, microglial cells derived from fetal human versus neonatal mouse brain were compared in a coculture assay of human and murine neuronal cell injury. Neurotoxicity (reflected by lactate dehydrogenase release and impaired neuronal uptake of [3H] gamma-amino butyric acid) and nitric oxide (NO) production (assessed by measurement of nitrite) were observed only in cocultures containing interferon (IFN)-gamma-lipopolysaccharide (LPS)-stimulated murine microglia. Cultures of purified human fetal microglia, however, did produce low levels of NO upon stimulation with IFN-gamma-LPS. These findings support the proposal that human macrophages have an inefficient IFN-gamma-inducible NOS and suggest that in tissues, such as brain, this deficiency could be advantageous for neighboring cells.

Aged↗