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PubMed2004

The methanol method for the quantification of ascorbic acid and dehydroascorbic acid in biological samples.

We present a fast to perform spectrophotometric method for the quantification of ascorbic acid and its oxidized form dehydroascorbic acid in biological samples. The assay detects a chromophore formed during the reaction of dehydroascorbic acid with methanol in phosphate/citrate buffer. This reaction can also be employed for the determination of ascorbate (vitamin C) in the presence of ascorbate oxidase. The major advantage of the developed protocol for the determination of both forms of vitamin C is a simple spectrophotometrical single end point determination. It is demonstrated that the methanol method is an improvement compared with a commercially available test kit for the determination of vitamin C. Using the methanol method, a dose-dependent increase in intracellular ascorbic acid was determined upon incubation of L-929 cells and RAW 264.7 macrophages with increasing concentrations of extracellular ascorbate. In blood serum, vitamin C was determined at concentrations between 46 and 97 microM. Supplementation with different amounts of ascorbate showed satisfying recovery. In L-929 cells, even unphysiologically high amounts of reactive nitrogen species were unable to completely oxidize intracellular vitamin C.

Animals↗
PubMed2001

Interleukin-8 up-regulation by neutrophil elastase is mediated by MyD88/IRAK/TRAF-6 in human bronchial epithelium.

Cystic fibrosis is characterized in the lungs by neutrophil-dominated inflammation mediated significantly by neutrophil elastase (NE). Previous work has shown that NE induces interleukin-8 (IL-8) gene expression and protein secretion in bronchial epithelial cells. We sought to determine the intracellular mechanisms by which NE up-regulates IL-8 in bronchial epithelial cells. The data show that stimulation of 16HBE14o(-) cells with NE induced IL-8 protein production and gene expression. Both responses were abrogated by actinomycin D, indicating that regulation is at the transcriptional level. Electrophoretic mobility shift assays demonstrated that nuclear factor kappaB (NFkappaB) was activated in 16HBE14o(-) cells stimulated with NE. Western blot analysis demonstrated that activation of NFkappaB by NE was preceded by phosphorylation and degradation of IkappaB proteins, principally IkappaBbeta. In addition, we observed that interleukin-1 receptor-associated kinase (IRAK) was degraded in 16HBE14o(-) cells stimulated with NE. Quantification of IL-8 reporter gene activity by luminometry demonstrated that dominant negative MyD88 (MyD88Delta) or TRAF-6 (TRAF-6Delta) inhibited IL-8 reporter gene expression in response to NE. Furthermore, MyD88Delta inhibited NE-induced IRAK degradation. These results show that NE induces IL-8 gene up-regulation in bronchial epithelial cells through an IRAK signaling pathway involving both MyD88 and TRAF-6, resulting in degradation of IkappaBbeta and nuclear translocation of NFkappaB. These findings may have implications for therapeutic treatments in the cystic fibrosis condition.

Adaptor Proteins, Signal Transducing↗
PubMed1976

A new culture model facilitating rapid quantitative testing of mitotic spindle inhibition in mammalian cells.

A new culture model, which facilitated both mass screening of potential anticancer drugs acting on microtubules and quantitative experiments with known "antitubulins," was found to have the following advantages: use of mammalian cells (either transformed or not), simplicity of the techniques (phase-contrast microscopy or simple microscopy after Giemsa staining), and ease with which it lent itself to quantification. The model was based on the uniform multimicronucleation response induced by antitubulins in MO cells. The specificity (towards antitubulins) of this response was ascertained by the use of many substances, including most of the known antitubulins and a number of nonrelated cytostatic or cytotoxic compounds. The uniformity of the response was established with the use of time-lapse observation of large numbers of cells and quantitative approaches. The results obtained in this model with the standard antitubulins (colchicine, vinblastine, vincristine) showed similar effects. The major difference between colchicine and the Vinca alkaloids was that colchicine was less reversible, which might be an indication of stronger intracellular binding. The Vinca alkaloids acted synergistically with colchicine when threshold subactive doses were combined, although it is known that they bind at a different site on tubulins. A number of substances that have been claimed or were suspected to interfere with microtubules were tested. The results showed that the following substances were indeed active with MO cells: colchicine, vinblastine, vincristine, podophyllotoxin, rotenone, griseofulvin, mercaptoethanol, benomyl, methyl benzimidazol-2-yl carbamate, and R 17934. Compounds that were inactive on these mammalian cells in culture included isopropyl carbanilate and melatonin, both of which have been shown to be active in other systems.

Antineoplastic Agents↗
PubMed1987

Immunofluorescent quantification of ribonucleotide reductase M1 subunit and correlation with DNA content by flow cytometry.

The cytoplasmic enzyme ribonucleotide reductase is essential for DNA synthesis, and its activity is strongly correlated with cellular proliferation. This paper describes a flow cytometric technique for the simultaneous measurement of DNA content and the M1 subunit of ribonucleotide reductase. Data are presented for cycling cultured human leukemic lymphoblasts in which M1 is constitutively expressed, and peripheral blood lymphocytes in which it is only detectable with certainty after mitogen stimulation. The choice of fixation procedure strongly influenced the amount of M1 subunit detected. Paraformaldehyde (PF) at concentrations of 2% (w/v in PBS) or greater provided optimal results. Fixation at 37 degrees C was significantly more effective in preserving M1 than fixation at room temperature or 4 degrees C. These variables are shown to have affected cytoplasmic retention during postfixation processing. Their relevance to the flow cytometric measurement of other intracellular components by this procedure are discussed.

Cell Cycle↗
PubMed2006

RT-PCR analysis of the expression of POMC and its processing enzyme PC1 in amphibian melanotropes.

The frog intermediate lobe comprises two functionally distinct cell subtypes, referred to as secretory and storage melanotropes, which differ in their ultrastructure, secretory, and synthetic rates, and display dissimilar responses to hypothalamic regulatory factors. All these differences make melanotrope subtypes an excellent model to analyze the expression and regulation of genes involved in the control and maintenance of the secretory state of endocrine cells. However, quantification of the expression levels of genes involved in the secretory process requires the characterization of a gene whose expression remains constant irrespective of the secretory state of the cells. In this study, we have cloned the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from frog pituitary and have evaluated its suitability as internal standard in gene expression studies in melanotropes. A semiquantitative RT-PCR system developed to this end revealed that secretory melanotropes and storage melanotropes possess similar expression levels of GAPDH, whereas, as expected, secretory melanotropes showed higher levels of POMC transcripts than storage cells. Furthermore, we found that the expression of the convertase PC1, an intracellular protease involved in POMC processing, parallels that of POMC, thus suggesting that the higher secretory rate of the POMC-derived peptide alpha-MSH exhibited by secretory melanotropes is supported by their higher PC1 expression levels. In addition, we have shown that both POMC and PC1 mRNAs are up-regulated by the hypothalamic factor TRH in melanotrope cell cultures. In contrast, the inhibitory factor NPY reduced the expression level of the convertase but did not modify that of POMC. Taken together, these results demonstrate that PC1 expression is regulated in melanotropes by both stimulatory (TRH) and inhibitory (NPY) hypothalamic signals, in a manner which essentially parallels that observed for the precursor POMC.

Amino Acid Sequence↗
PubMed2006

Detection of Chlamydia in the peripheral blood cells of normal donors using in vitro culture, immunofluorescence microscopy and flow cytometry techniques.

BACKGROUND: Chlamydia trachomatis (Ct) and Chlamydia pneumoniae (Cp) are medically significant infectious agents associated with various chronic human pathologies. Nevertheless, specific roles in disease progression or initiation are incompletely defined. Both pathogens infect established cell lines in vitro and polymerase chain reaction (PCR) has detected Chlamydia DNA in various clinical specimens as well as in normal donor peripheral blood monocytes (PBMC). However, Chlamydia infection of other blood cell types, quantification of Chlamydia infected cells in peripheral blood and transmission of this infection in vitro have not been examined. METHODS: Cp specific titers were assessed for sera from 459 normal human donor blood (NBD) samples. Isolated white blood cells (WBC) were assayed by in vitro culture to evaluate infection transmission of blood cell borne chlamydiae. Smears of fresh blood samples (FB) were dual immunostained for microscopic identification of Chlamydia-infected cell types and aliquots also assessed using Flow Cytometry (FC). RESULTS: ELISA demonstrated that 219 (47.7%) of the NBD samples exhibit elevated anti-Cp antibody titers. Imunofluorescence microscopy of smears demonstrated 113 (24.6%) of samples contained intracellular Chlamydia and monoclonals to specific CD markers showed that in vivo infection of neutrophil and eosinophil/basophil cells as well as monocytes occurs. In vitro culture established WBCs of 114 (24.8%) of the NBD samples harbored infectious chlamydiae, clinically a potentially source of transmission, FC demonstrated both Chlamydia infected and uninfected cells can be readily identified and quantified. CONCLUSION: NBD can harbor infected neutrophils, eosinophil/basophils and monocytes. The chlamydiae are infectious in vitro, and both total, and cell type specific Chlamydia carriage is quantifiable by FC.

Adult↗
PubMed2004

Cross-sectional and longitudinal analysis of myelin-reactive T cells in patients with multiple sclerosis.

Activated myelin-specific T cells are thought to mediate inflammatory tissue damage in multiple sclerosis (MS). Applying a large panel of myelin antigens, we demonstrate the direct ex vivo detection of viable IFN-gamma/TNF-alpha producing CD4+/CD69+ T cells 6 hours after antigenic challenge, by intracellular flow cytometry in 3/33 MS patients and 2/26 healthy controls with calculated frequencies of (mean +/- SEM): 0.031% +/- 0.002% versus 0.037% +/- 0.029%. By comparison, the recently developed IL-7 modified proliferation assay revealed i) a higher number of individuals showing myelin reactivity (17/37 MS patients and 12/24 healthy individuals) and ii) a significant difference in the response to myelin basic protein (MBP) between the two groups in a longitudinal analysis, indicating a higher activity of myelin-specific T cells in MS patients. Our data provide new perspectives in detecting pathogenetically relevant T cells, but clearly demonstrate the different conclusions which must be drawn from various approaches concerning the quantification of autoreactive T cells.

Adult↗
PubMed1986

In situ activity and phosphorylation of tyrosine hydroxylase in the median eminence.

Intracellular activation and phosphorylation of tyrosine hydroxylase (TH; E.C. 1.14.16.2) in the median eminence of the rat brain were investigated. The in situ activity of TH was assayed by the accumulation of L-dihydroxyphenylalanine (DOPA) in the median eminence of hypothalamic fragments incubated in the presence of NSD 1015. When hypothalamic fragments were incubated with veratridine (0-10(-3) M), maximal stimulation of TH activity was observed at 10(-4) M. The mean concentration of DOPA in the median eminence of hypothalamic fragments incubated with 10(-4) M veratridine was 3 times that seen in its absence. Phosphorylation of TH in the median eminence was evaluated by autoradiographic quantification of [32P]TH in 32P-labelled median eminence tissue. The amount of [32P]TH in 32P-labelled median eminence incubated with 10(-4) M veratridine was 2 times that seen in the absence of veratridine. These data are consistent with the view that in the median eminence phosphorylation and activation of TH are linked events and that phosphorylation may be a means of regulating the biosynthesis of dopamine in this region of the brain.

Animals↗
PubMed2003

Escherichia coli ghost production by expression of lysis gene E and Staphylococcal nuclease.

The production of bacterial ghosts from Escherichia coli is accomplished by the controlled expression of phage phiX174 lysis gene E and, in contrast to other gram-negative bacterial species, is accompanied by the rare detection of nonlysed, reproductive cells within the ghost preparation. To overcome this problem, the expression of a secondary killing gene was suggested to give rise to the complete genetic inactivation of the bacterial samples. The expression of staphylococcal nuclease A in E. coli resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments shorter than 100 bp. Two expression systems for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. With regard to the absence of reproductive cells from the ghost fractions, the reduction of viability could be determined as being at least 7 to 8 orders of magnitude. The lysis process was characterized by electrophoretic analysis and absolute quantification of the genetic material within the cells and the culture supernatant via real-time PCR. The ongoing degradation of the bacterial nucleic acids resulted in a continuous quantitative clearance of the genetic material associated with the lysing cells until the concentrations fell below the detection limits of either assay. No functional, released genetic units (genes) were detected within the supernatant during the lysis process, including nuclease expression.

Bacteriolysis↗
PubMed1991

Synchronous triggering of trout sperm is followed by an invariable set sequence of movement parameters whatever the incubation medium.

The movement of live trout spermatozoa is very brief (25 sec at 20 degrees C) and conditions have been developed to get synchronous initiation of sperm motility which allowed quantification of the major parameters of sperm movement during the motility phase. Recorded flagellar beat frequencies decreased steadily from values of 55 Hz at the beginning to 20 Hz at the end of the motility phase. Sperm forward velocities followed a similar pattern from 250 to 20 microns.sec-1 in the same conditions and the diameters of sperm trajectories were reduced from 370 to 40 microns. Thus none of the characteristics of sperm movement was constant during the motile phase which ended abruptly by a straightening of the flagella. The decrease in flagellar beat frequencies and sperm velocities are much greater than what could be extrapolated from the decrease of intracellular ATP (Christen R. et al: Eur. J. Biochem, 166: 667-671, 1987) or from measurements of ATP-dependence of reactivated sperm velocities (Okuno M. and Morisawa N.: In Biological Functions of Microtubules and Related Structures. New York: Academic Press, pp. 151-162, 1982). Therefore, the cessation of flagellar beating at 25 sec is not directly the result of the low concentration of intracellular ATP. The decrease in the diameters of sperm trajectories which occurred during the first part of the motility phase was correlated with [Ca]i measurements (Cosson M.P. et al, Cell Motil. Cytoskeleton, 14:424-434, 1989). The effect of Ca2+ at the axonemal level does not indicates that Ca2+ influx is previous to flagellar beating but rather suggests a classical Ca2+ regulation of the flagellar assymetry. The short duration of the motility phase and the characteristics of sperm movement were very similar in various conditions (high external K+, low pH media) where increased external Ca2+ or divalent ions were shown to overcome K+ and H+ inhibition of sperm motility, both conditions which have been shown to depolarize the plasma membrane potential (Gatti J.L. et al: J. Cell Physiol., 143:546-554, 1990). The present study of the parameters of sperm movement suggests that once motility is initiated, a defined set of axonemal events will take place whatever the external conditions.

Animals↗
PubMed1994

The substrate specificities of four different lysophospholipases as determined by a novel fluorescence assay.

A novel fluorescence assay for quantifying lysophospholipase activity is described which utilizes a commercially available acrylodated intestinal fatty-acid-binding protein (ADIFAB) and non-radiolabelled substrate. Quantification of enzyme activity is based on the decrease in ADIFAB fluorescence at 432 nm in the presence of nanomolar concentrations of non-esterified ('free') fatty acids. Lysophospholipase activity measured by the ADIFAB assay and a conventional radiometric assay yield comparable results and have comparable levels of sensitivity (approximately 10 pmol/min per ml). The ADIFAB assay has the advantageous features of continuous monitoring of enzyme activity and the availability of a broad range of potential substrates, because non-radiolabelled lysophospholipids can be employed in the assay. The hydrolytic activities of four lysophospholipases were determined, including a bacterial secreted phospholipase A2/lysophospholipase, the human-eosinophil-secreted lysophospholipase, a human intracellular lysophospholipase (peak 3) isolated from HL-60 cells and a high-molecular-mass cytosolic phospholipase A2/lysophospholipase from a mouse mammary carcinoma. Each of these enzymes was found to have a distinctive hydrolytic profile as determined by an array of lysophospholipids differing in their polar headgroups and sn-1 fatty-acyl substituents.

Carrier Proteins↗
PubMed2005

A mathematical model to determine molecular kinetic rate constants under non-steady state conditions using fluorescence recovery after photobleaching (FRAP).

Fluorescence recovery after photobleaching (FRAP) analyses of binding and unbinding of molecules that interact with insoluble scaffolds, such as the cytoskeleton and nuclear matrix, in living cells commonly assume that this process is at equilibrium over the time scale of fluorescence recovery. This assumption breaks down for relatively fast intracellular processes like focal adhesion assembly at the leading edge of a migrating cell, or changes of transcriptional activation in the nucleus, that can occur in a matter of a few minutes. In this paper, we formulate a mathematical model that permits FRAP to be used to determine kinetic rate constants of molecules that interact with insoluble cellular structures under non-steady state conditions. We show that unlike steady state FRAP, fluorescence recovery time scales under these unsteady conditions are determined not only by unbinding rates, but also by the overall assembly and disassembly dynamics of the structural scaffold which supports these binding interactions. Experimental data from FRAP analysis and quantification of scaffold assembly dynamics may be combined and used with our mathematical model to estimate kinetic rate constants, as well as the apparent rate constant of scaffold assembly and disassembly.

Fluorescence Recovery After Photobleaching↗
PubMed2005

Use of tissue ultrafiltration for treatment of compartment syndrome: a pilot study using porcine hindlimbs.

OBJECTIVES: To demonstrate the efficacy of compartment syndrome ultrafiltration for the treatment of acute compartment syndrome in an animal model. Our hypothesis is the removal of interstitial fluid will result in a reduction of intramuscular pressure compared with untreated controls in a model of bilateral induced compartment syndrome. DESIGN: Controlled experimental model. SETTING: Animal research facility. PATIENTS/PARTICIPANTS: Three pairs of porcine hindlimbs. INTERVENTION: Acute compartment syndrome was created in the pig hindlimb by infusion of saline to maintain the intramuscular pressure 30 mm Hg greater than the animal's mean arterial pressure for 8 hours. After a 2-hour reperfusion interval, ultrafiltration (removal of fluid through 1 mm diameter porous catheters, connected to -500 mm Hg suction) was commenced in 1 limb only and continued for 9.5 hours. MAIN OUTCOME MEASURES: Intramuscular pressure, ultrafiltrate volume, ultrafiltrate and serum levels of creatine kinase and lactate dehydrogenase, histologic measurement of extracellular and intracellular edema, as well as the degree of cellular necrosis. RESULTS: Intramuscular pressure tended to be lower on the treated side at the end of the treatment period [treated leg: 9.3 +/- 4.0 mm Hg (+/- SE), control leg: 19.3 +/- 1.4 mm Hg, P = 0.03]. Analysis of ultrafiltrate fluid showed that levels of creatine kinase and lactate dehydrogenase were elevated compared with serum levels. Creatine kinase levels in serum were measured at 4150 +/- 780 U/L, whereas ultrafiltrate levels of creatine kinase were 28,700 +/- 17,700 U/L (+/- SE) (P = 0.1). Lactate dehydrogenase was measured at 1950 +/- 180 U/L in serum, but markedly elevated in ultrafiltrate [160,000 +/- 88,900 U/L (+/- SE), P = 0.05]. Quantification of cellular and interstitial dimensions showed no difference in control and experimental limbs. Quantification of the degree of muscle necrosis revealed 6.1 +/- 2.7% necrosis in the treated limb compared to 11.3 +/- 1.6% necrosis in the control group (P = 0.02, df = 2, 1-tailed paired t test). CONCLUSION: This pilot study demonstrates the feasibility of tissue ultrafiltration for reduction of intramuscular pressure in this porcine model. Further studies are underway. Compartment syndrome ultrafiltration may be useful prophylactically in patients at risk for acute compartment syndrome. Sampling of interstitial fluid and frequent measurement of intramuscular pressure may allow earlier diagnosis and treatment of acute compartment syndrome, whereas the reduction of tissue pressure by compartment syndrome ultrafiltration may prevent acute compartment syndrome from occurring. Additionally, compartment syndrome ultrafiltration will not hinder the ability of clinicians to use the clinical examination and pressure monitoring as the gold standard.

Animals↗
PubMed2006

Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Given the role of folate in many disorders, intracellular distribution of folate vitamers is of potential clinical importance. In particular, accumulation of non-methyltetrahydrofolates due to altered partitioning of folate metabolism at the level of methylenetetrahydrofolate is of interest. METHODS: We describe a positive-electrospray liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows determination of erythrocyte folate vitamer distribution by accurately measuring both 5-methyltetrahydrofolate (5-methylTHF) and non-methyl folate vitamers. Whole blood lysates are deconjugated in ascorbic acid solutions, deproteinized, purified using folate-binding protein affinity columns, concentrated by solid-phase extraction (SPE) and evaporation, and separated on a C18 column within 6 min. RESULTS: The limit of quantification for both 5-methylTHF and non-methylTHF was 0.4 nmol/L (signal-to-noise >10). Intra- and inter-assay CVs for 5-methylTHF were 1.2% and 2.8%, respectively. Intra- and inter-assay CVs for non-methylTHF as a group were 1.6% and 1.5%, respectively. Recovery results were 97-107%. We measured 8-72% non-methyl folate vitamers in volunteers (n=5) with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype. Concentrations ranged from 117 to 327 nmol/L and 23 to 363 nmol/L for 5-methylTHF and non-methylTHF vitamers, respectively. We measured 0-2% non-methylTHF vitamers in MTHFR 677 CC genotype volunteers. In addition, we found that storage of whole-blood samples in ascorbic acid at low pH resulted in 53-90% loss of the non-methylTHF fraction. CONCLUSION: This LC-MS/MS method accurately determines erythrocyte 5-methylTHF and non-methyl folate vitamers.

Chromatography, High Pressure Liquid↗
PubMed2006

Analysis of intact phosphoinositides in biological samples.

It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2)], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P(3)], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [(32)P]inorganic phosphate or [(3)H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS(3). Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.

Blood Platelets↗
PubMed2000

Endothelial preconditioning by transient oxidative stress reduces inflammatory responses of cultured endothelial cells to TNF-alpha.

Brief episodes of ischemia can render an organ resistant to subsequent severe ischemia. This 'ischemic preconditioning' is ascribed to various mechanisms, including oxidative stress. We investigated whether preconditioning exists on an endothelial level. Human umbilical vein endothelial cells (HUVECs) were transiently confronted with oxidative stress (1 mM H(2)O(2), 5 min). Adhesion molecules ICAM-1 and E-selectin and release of cytokines IL-6 and IL-8 to subsequent stimulation with TNF-alpha (2.5 ng/ml, 4 h) were measured (flow cytometry and immunoassay), as were nuclear translocation of the transcription factor NFkappaB (Western blotting, confocal microscopy) and redox status of HUVECs (quantification of glutathione by HPLC). TNF-alpha elevated IL-6 in the cell supernatant from 8.8 +/- 1 to 41 +/- 3 pg/ml and IL-8 from 0.5 +/- 0. 03 to 3 +/- 0.2 ng/ml. ICAM-1 was increased threefold and E-selectin rose eightfold. Oxidative stress (decrease of glutathione by 50%) reduced post-TNF-alpha levels of IL-6 to 14 +/- 3 and IL-8 to 1 +/- 0.2; the rise of ICAM-1 was completely blocked and E-selectin was only doubled. The anti-inflammatory effects of preconditioning via oxidative stress were paralleled by reduction of the translocation of NFkappaB on stimulation with TNF-alpha, and antagonized by the intracellular radical scavenger N-acetylcysteine. 'Anti-inflammatory preconditioning' of endothelial cells by oxidative stress may account for the inhibitory effects of preconditioning on leukocyte adhesion in vivo.

Apoptosis↗
PubMed2005

Chlamydia pneumoniae induces aponecrosis in human aortic smooth muscle cells.

BACKGROUND: The intracellular bacterium Chlamydia pneumoniae is suspected to play a role in formation and progression of atherosclerosis. Many studies investigated cell death initiation versus inhibition by Chlamydia pneumoniae in established cell lines but nothing is known in primary human aortic smooth muscle cells, a cell type among others known to be involved in the formation of the atherosclerotic plaque. Type of cell death was analyzed by various methods in primary aortic smooth muscle cells after infection with Chlamydia pneumoniae to investigate a possible pathogenic link in atherosclerosis. RESULTS: Chlamydiae were found to be localized up to 72 h post infection in aortic smooth muscle cells either as single bacteria or inside of large inclusions. Quantification of host cell death by lactate dehydrogenase release assay revealed strictly dose and time dependent lysis for all tested isolates of Chlamydia pneumoniae. Phosphatidylserine exposure was detected by flow cytometry in Chlamydia pneumoniae infected cells. Ultrastructure of Chlamydia pneumoniae infected human aortic smooth muscle cells showed extensive membrane- and organelle damage, chromatin condensation but no nuclear fragmentation. DNA fragmentation as well as cell membrane permeability was analyzed by TUNEL and NHS-biotin staining and occurred exclusively in cells carrying Chlamydia pneumoniae spots but not in smooth muscle cells with inclusions. These morphological features of cell death were not accompanied by an activation of caspase-3 as revealed by analysis of enzyme activity but involved mitochondrial membrane depolarization as shown by TMRE uptake and release of cytochrome c from mitochondria. CONCLUSION: This study provides evidence that Chlamydia pneumoniae induce a spot like infection in human aortic smooth muscle cells, which results in a chimeric cell death with both apoptotic and necrotic characteristics. This aponecrotic cell death may assist chronic inflammation in atherosclerotic blood vessels.

Aorta↗