Quantitative Flow Cytometry Assay Research Articles

Secretion of serine peptidase by a clinical strain ofCandida albicans: influence of growth conditions and cleavage of human serum proteins and extracellular matrix components

Candida albicans expresses a vast number of hydrolytic enzymes, playing roles in several phases of yeast-host interactions. Here, we identified two novel extracellular peptidase classes in C. albicans. Using gelatin-sodium dodecyl sulfate polyacrylamide gel electrophoresis two gelatinolytic activities were detected at physiological pH: a 60-kDa metallopeptidase, completely blocked by 1,10-phenanthroline, and a 50-kDa serine peptidase inhibited by phenylmethylsulfonyl fluoride. In an effort to establish a probable functional implication for these novel peptidase classes, we demonstrated that the 50-kDa secretory serine peptidase was active over a broad pH range (5.0-7.2) and was capable to hydrolyze some soluble human serum proteins and extracellular matrix components. Conversely, when this isolate was grown in yeast carbon base supplemented with bovine serum albumin, a secretory aspartyl peptidase activity was measured, instead of metallo- and serine peptidases, suggesting that distinct medium composition induces different expression of released peptidases in C. albicans. Additionally, we showed by quantitative proteolytic measurement, flow cytometry and immunoblotting assays that the brain heart infusion medium might repress the Sap1-3 production. Collectively, our results showed for the first time the capability of an extracellular proteolytic enzyme other than aspartic-type peptidases to cleave a broad spectrum of relevant host proteinaceous substrates by the human pathogen C. albicans.

Direct transport across the plasma membrane of mammalian cells ofLeishmaniaHASPB as revealed by a CHO export mutant

Leishmania HASPB is a lipoprotein that is exported to the extracellular space from both Leishmania parasites and mammalian cells via an unconventional secretory pathway. Exported HASPB remains anchored in the outer leaflet of the plasma membrane mediated by myristate and palmitate residues covalently attached to the N-terminal SH4 domain of HASPB. HASPB targeting to the plasma membrane depends on SH4 acylation that occurs at intracellular membranes. How acylated HASPB is targeted to the plasma membrane and, in particular, the subcellular site of HASPB membrane translocation is unknown. In order to address this issue, we screened for clonal CHO mutants that are incapable of exporting HASPB. A detailed characterization of such a CHO mutant cell line revealed that the expression level of the HASPB reporter molecule is unchanged compared to CHO wild-type cells; that it is both myristoylated and palmitoylated; and that it is mainly localized to the plasma membrane as judged by confocal microscopy and subcellular fractionation. However, based on a quantitative flow cytometry assay and a biochemical biotinylation assay of surface proteins, HASPB transport to the outer leaflet of the plasma membrane is largely reduced in this mutant. From these data, we conclude that the subcellular site of HASPB membrane translocation is the plasma membrane as the reporter molecule accumulates in this location when export is blocked. Thus, these results allow us to define a two-step process of HASPB cell surface biogenesis in which SH4 acylation of HASPB firstly mediates intracellular targeting to the plasma membrane. In a second step, the plasma membrane-resident machinery, which is apparently disrupted in the CHO mutant cell line, mediates membrane translocation of HASPB. Intriguingly, the angiogenic growth factor FGF-2, another protein secreted by unconventional means, is shown to be secreted normally from the HASPB export mutant cell line. These observations demonstrate that the export machinery component defective in the export mutant cell line functions specifically in the HASPB export pathway.

Manipulation of CD98 expression affects both trophoblast cell fusion and amino acid transport activity during syncytialization of human placental BeWo cells.

The physiological importance of CD98 surface antigen in regulating placental trophoblast cell fusion and amino acid transport activity has been studied in parallel in a cell model of syncytialization (the cytotrophoblast cell line BeWo following increased intracellular cAMP by forskolin treatment) using antisense oligonucleotides. CD98 protein abundance (determined by Western blot) was decreased by 50 % following antisense oligonucleotide transfection. Transfection with antisense oligonucleotide altered the responses of BeWo to forskolin. Cell fusion (determined by a quantitative flow cytometry assay) was inhibited by 57 %, and both human chorionic gonadotropin secretion and L-leucine influx through system L were suppressed. These findings show that CD98 is involved in the process of cell fusion necessary for syncytiotrophoblast formation and that during this physiologically important event, amino acid transport activity is also regulated through expression of this membrane protein.

Involvement of nuclear transcription factor-κB in low-dose doxorubicin-induced drug resistance of cervical carcinoma cells

Administration of suboptimal doses of anticancer drugs not only fails to control tumor but often results in increased drug resistance of tumor cells. However, little is known about the effects of transient exposure to a minimally cytotoxic dose of chemotherapy on the development of drug resistance of the tumors. Previous studies have shown that upregulation of drug-exporter proteins (ATP-binding-cassette proteins) may be one of the key mechanisms involved in inducible drug resistance. In this study, we demonstrated that upregulation of NF-κB is another possible mechanism. SiHa cells were exposed to low-dose doxorubicin (100 nM; ic 30) for 3 hr, and then were continuously cultured in drug-free culture media (designated as SiHa/DR cells). SiHa/DR cells at up to 9 passages showed increased resistance to doxorubicin and cross-resistance to cisplatin. Results of quantitative real-time PCR and flow cytometry assay indicated that the increased drug-resistance in SiHa/DR cells was not due to upregulation of drug-exporter proteins or to the decrease of intracellular concentration of anticancer drugs. Both the basal and drug-induced NF-κB activity were shown to be increased in SiHa/DR cells by EMSA and NF-κB-driven luciferase reporter gene assay. Suppression of NF-κB activation by transfection of a dominant negative IκBα prevented the development of drug resistance, indicating that the upregulated NF-κB activity was positively correlated with the low-dose doxorubicin-induced drug resistance. These results suggest that even a transient exposure to a small dose of anticancer drugs may induce drug resistance in some cancer cells via upregulation of NF-κB activity.

Hypoxia alters expression and function of syncytin and its receptor during trophoblast cell fusion of human placental BeWo cells: implications for impaired trophoblast syncytialisation in pre-eclampsia

The fundamental process of placental trophoblast cell fusion (syncytiotrophoblast formation or syncytialisation) which is a characteristic of this tissue is poorly understood. Pre-eclampsia is associated with placental hypoxia and suppressed syncytiotrophoblast formation. We therefore have studied the effect of low-oxygen tensions on the rate of cell fusion, relative abundance of mRNAs encoding syncytin and its receptor, amino acid transport system B 0, which are thought to be involved in trophoblast cell fusion (as well as the activity of amino acid transport through this system) in a cell model of syncytialisation (BeWo cells following forskolin treatment). Forskolin-induced cell fusion (determined by a quantitative flow cytometry assay) was reversibly suppressed in 2% oxygen compared to 20% oxygen. This was associated with suppressed secretion of human chorionic gonadotropin. Forskolin stimulated relatively less syncytin mRNA (determined by reverse transcription–polymerase chain reaction) in 2% than in 20% oxygen and there was no stimulation after 48 h in 2% oxygen. There was a spontaneous, time-dependent increase of amino acid transporter B 0 mRNA in vehicle, which was suppressed by 2% oxygen and by forskolin treatment in 20% oxygen. Forskolin-induced changes in amino acid transport system B 0 function were not seen in cells cultured for 48 h in 2% oxygen. These observations suggest that under conditions of low ambient oxygen, dysregulation of expression of syncytin and of its receptor may suppress the normal process of cell fusion necessary for syncytiotrophoblast formation and contributes to syncytiotrophoblast abnormalities characteristic of pre-eclampsia.

CD4 T cell surface CCR5 density as a host factor in HIV-1 disease progression.

We have recently shown that the number of CCR5 molecules at the surface of peripheral blood CD4 T cells (CCR5 density) correlates with the viral RNA plasma level in HIV-1-infected individuals. As viral load is a strong predictor of outcome in HIV infection, the present study examines the correlation between CCR5 density and HIV-1 disease progression. Using a quantitative flow cytometry assay, we measured CCR5 density in HIV-1-infected adults and control healthy volunteers. The CCR5 genotype (presence of a Delta 32 allele) was also determined. CCR5 density was stable over time on non-activated, HLA-DR(-)CD4 T cells of infected individuals. In a study cohort of 25 patients, asymptomatic and non-treated, we observed a correlation between CCR5 density on HLA-DR(-)CD4 T cells and the CD4 T cell slope (P = 0.026), which was independent of the presence or absence of the Delta 32CCR5 deletion. In particular, slow progressors expressed lower CCR5 densities than non-slow progressors (P = 0.004) and non-infected control subjects (P = 0.002). These results are compatible with the hypothesis that CCR5 density, which is a key factor of HIV-1 infectability, determines in-vivo HIV production, and thereby the rate of CD4 cell decline. Consequently, CCR5 density quantitation could be a new valuable prognostic tool in HIV-1 infection. Moreover, these data emphasize the therapeutic potential of treatments that reduce functional CCR5 density.

Neutrophil activation in preeclampsia and isolated intrauterine growth restriction

Objective: Neutrophils have been implicated in the pathogenesis of preeclampsia. Because preeclampsia and intrauterine growth restriction result from similar placental lesions, the aim of this study was to investigate neutrophil activation in isolated intrauterine growth restriction relative to preeclampsia and uncomplicated pregnancy. Polymorphonuclear neutrophil activation was analyzed by measuring cell surface and soluble cell adhesion molecule expressions. Study Design:L -Selectin (CD62L ) and CD11b surface expressions on polymorphonuclear neutrophils were analyzed in 13 women with preeclampsia, 11 women with isolated intrauterine growth restriction, and 17 age- and gestation-matched control women by means of a standardized quantitative flow cytometry assay. Serum levels of soluble L -selectin were measured by enzyme-linked immunosorbent assay. Results: Neutrophils from women with isolated intrauterine growth restriction and women with preeclampsia displayed higher levels of CD11b and lower levels of CD62L than did neutrophils from healthy pregnant women. Soluble L -selectin serum levels were significantly increased in the preeclampsia and intrauterine growth restriction groups relative to normal values. No significant difference in the levels of CD11b, CD62L , and soluble L -selectin were observed between women with isolated intrauterine growth restriction and those with preeclampsia. Leukocyte activation was not correlated with disease severity. Conclusion: The observed alteration in polymorphonuclear neutrophil adhesion molecule expressions and increased serum soluble L -selectin levels are consistent with activation of peripheral blood neutrophils occurring in isolated intrauterine growth restriction in a manner similar to that seen in preeclampsia. This evidence of neutrophil activation may help to advance our understanding of the disease process in isolated intrauterine growth restriction. (Am J Obstet Gynecol 2000;183:1558-63.)

A quantitative flow cytometry assay for the preclinical testing and pharmacological monitoring of rabbit antilymphocyte globulins (rATG)

Rabbit antilymphocyte or antithymocyte globulins (rATG) are currently used as immunosuppressive agents in clinical organ and bone marrow transplantation, but because of differences in antigen source and purification processes between manufacturers, determination of the dose to be administered remains empirical. Dosage may be adjusted to peripheral blood T cell counts or limited by side effects such as neutropenia or thrombocytopenia. We report here a measurement by indirect immunofluorescence of the amount of antibodies from rATG that bind to different blood cell types. A concentration-related immunofluorescence signal was obtained with reasonable replicability, allowing comparison of different rATGs by reference to the same rATG batch as internal standard. Ratios of antilymphocyte to undesirable antibodies (directed against antigens expressed on neutrophils, platelets or red cells) could be calculated for each rATG preparation. Finally, the method is applicable to the measurement of free antibodies in sera from patients treated with rATG and to the analysis of the effects of in vivo absorption of rATG during its administration. Marked differences in antibody content per mg of rIgG account for the differences in rATG dosage that were empirically established for clinical applications.

Free and Total Platelet Glycoprotein IIb/IIIa Measurement in Whole Blood by Quantitative Flow Cytometry during and after Infusion of c7E3 Fab in Patients Undergoing PTCA

A quantitative flow cytometry assay was used to evaluate the ex vivo kinetics of c7E3 Fab platelet effect in 16 patients undergoing PTCA treated with abciximab and compared with aggregometry assay. Immunolabeling of platelets was directly assessed on whole blood, using in parallel two monoclonal antibodies (Mabs) raised against GPIIIa, Mab1, the binding of which is inhibited by c7E3 Fab, and Mab2, the binding of which is not affected by c7E3 Fab. We found a severe and sustained inhibition of both GPIIb/IIIa receptors and platelet functions. The inter-individual variation in response to abciximab was low. A significant transient increase at H24 and H48 in the binding of Mab2 was found as an unexpected result, and confirmed in vitro. Results demonstrate that flow cytometry is a reliable method in agreement with aggregation. In addition, our results show that it is a standardized tool and a time-saving technique.

Correlation between kinetics of soluble CD4 interactions with HIV-1-Env-expressing cells and inhibition of syncytia formation: implications for mechanisms of cell fusion and therapy for AIDS.

To study the kinetics of the interactions between soluble (s) CD4 and HIV-1-Env-expressing cells in relation to subsequent events leading to cell fusion and inhibition of syncytia formation. Vaccinia-HIV-1 (Env)-infected CD4- T-cells were used to study the kinetics of sCD4-gp120/41 interactions and syncytia formation (with CD4+ T-cells) under identical conditions. sCD4 association and dissociation rates for HIV-1-Env-expressing cells, and quantification of sCD4-induced gp120 shedding was determined by a quantitative flow cytometry assay. Syncytia inhibition was measured in the continuous presence of sCD4, or after washing of HIV-1-Env-expressing cells following pre-incubation with sCD4. The kinetics of syncytia inhibition correlated with sCD4 binding when sCD4 was maintained during the culture. When Env-expressing cells, which had been pre-incubated with sCD4, were washed to remove unbound sCD4, no syncytia formation inhibition was observed, even following sCD4-induced shedding of greater than 50% of surface gp120 molecules. The lack of syncytia inhibition seen after removal of unbound sCD4, even after pre-incubation of cells under saturation and gp120 shedding conditions, indicated that sufficient numbers of fusogenic molecules remained on the sCD4-treated cells. In addition, fast dissociation of pre-bound sCD4 occurred in culture. These results are important for understanding HIV-1-Env-mediated cell fusion and AIDS therapy.