Biomedical subjects

Marina Zaitseva

Publications and source records attributed to Marina Zaitseva.

10 recordsLinked to original sources

PubMed2006

In vitro culture significantly alters gene expression profiles and reduces differences between myometrial and fibroid smooth muscle cells.

Cultured myometrial (M) and fibroid (F) smooth muscle cells (SMCs) have been widely used as a model for the study of F growth. The aim of this study was to compare gene expression profiles using microarrays between six paired M and F tissues from hysterectomy specimens, as well as cells isolated from the same tissues and cultured for up to three passages. A total of 2055 genes were differentially expressed by ANOVA between all experimental groups. Among them, 128 genes were found to be statistically different between M and F tissues. More than 1100 genes were significantly changed between tissues and cultured cells, with 648 genes common between both M and F cells at P0 and P3. Expression profiles of six genes including estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were also validated using real-time PCR. These data demonstrate that large changes occur in SMC gene expression in culture, reducing differences between M and F cells. They also show that ERalpha and PR levels are reduced in cells compared with whole tissue. These results indicate that although M and F cell cultures provide an important tool to study these tumours, in vitro studies must be carefully planned and evaluated to provide meaningful results.

Cells, Cultured↗

PubMed2005

Increased CXCR4-dependent HIV-1 fusion in activated T cells: role of CD4/CXCR4 association.

Activation of peripheral CD4+ T cells resulted in augmented fusion with X4 human immunodeficiency virus type 1 (HIV-1) envelope-expressing cells without parallel increases in the surface expression of CD4 or CXC chemokine receptor 4 (CXCR4). Our study used biochemical methods and biological assays to correlate the increased fusion potential of activated T cells with changes in CXCR4 isoforms and CD4-CXCR4 association. Western blot analyses of CXCR4, precipitated from resting T cells, identified several CXCR4 species with molecular weights of 47, 50, 62, and 98 kDa. After 24 h stimulation with phytohemagglutinin/interleukin-2, a marked reduction was seen in the 47-kDa, with a concomitant increase in the amounts of 50 and 62-64 kDa CXCR4. T cell activation also induced an increase in the coprecipitation of CXCR4 with CD4. The 62-kDa CXCR4 predominantly coprecipitated with CD4 and was shown to be ubiquitinated. Stripping of CD4 from the cell surface with pronase treatment prior to cell lysis only partially reduced coprecipitation of CD4 with the 62-kDa CXCR4, revealing a pool of intracellular CD4-CXCR4 complexes. Coprecipitation of CXCR4 with CD4 was reduced in activated cells treated with Brefeldin A and Monensin, suggesting that late endosomes play a role in intracellular association of CXCR4 with CD4. Confocal microscopy confirmed the colocalization of CD4 and CXCR4 within CD63+ endocytic compartments. These findings demonstrated a correlation between the enhanced susceptibility of activated T cells to HIV-1 fusion and accumulation of ubiquitinated 62-64 kDa CXCR4 species, which preferentially associated with CD4. The CD4-CXCR4 complexes may shuttle between late endosomes and the cell surface.

Antigens, CD↗

PubMed2005

Up-regulation of IL-7, stromal-derived factor-1 alpha, thymus-expressed chemokine, and secondary lymphoid tissue chemokine gene expression in the stromal cells in response to thymocyte depletion: implication for thymus reconstitution.

Three in vivo adult mouse models were established to study which signals are required to restore the postnatal thymus. Single administration of dexamethasone, estradiol, or exposure to sublethal dose of gamma irradiation served as prototype thymus-ablating therapies. In all models, transient thymic atrophy was manifested due to the loss of the predominant portion of CD4- CD8- double negative and CD4+ CD8+ double positive thymocytes and was followed by a complete regeneration of the thymuses. Acute atrophy/regeneration was observed in the dexamethasone and irradiation models; in the estradiol-treated animals, slow kinetics of atrophy and regeneration was observed. Importantly, in both acute and chronic models, high levels of IL-7 mRNA were detected in the thymuses isolated from mice during maximum atrophy. In addition, chemokine gene array analysis of involuted thymuses revealed high levels of mRNA expression of stromal-derived factor-1alpha (SDF-1alpha), thymus-expressed chemokine (TECK), and secondary lymphoid tissue chemokine (SLC) but not of other chemokines. The levels of IL-7, SDF-1alpha, TECK, and SLC mRNA inversely correlated with the kinetics of regeneration. RT-PCR analysis of stromal cells purified from involuted thymuses confirmed increased IL-7, SDF-1alpha, and SLC gene expression in MHC class II+ CD45- epithelial cells and increased IL-7 and TECK gene expression in class II+ CD45+ CD11c+ dendritic cells. Thus, our data showed for the first time that expression of IL-7, SDF-1alpha, TECK, and SLC mRNA is induced in the thymic stroma during T cell depletion and may play an important role in the reconstitution of the adult thymus.

Animals↗

PubMed2004

Estrogen receptor-alpha agonists promote angiogenesis in human myometrial microvascular endothelial cells.

OBJECTIVE: The relative role of the two estrogen receptors, ERalpha and ERbeta, in mediating angiogenic responses in adult human endothelium is unknown. The aim of this study was to determine whether novel ERalpha-selective agonists, propyl pyrazole triol (PPT) and the tetrahydrochrysene (R,R-THC), up-regulate the expression of vascular endothelial growth factor receptor-2 (VEGFR-2), and promote VEGF-stimulated endothelial cell proliferation in primary cultures of adult female microvascular endothelial cells co-expressing endogenous ERalpha and ERbeta. METHODS: Confluent primary cultures of microvascular endothelial cells isolated from human myometrium were incubated with 17beta-estradiol (1 and 10 nM), PPT (10 nM to 3 microM), or R,R-THC (10 nM to 3 microM) for 18 hours and VEGFR-2 expression measured by biotin-VEGF165 binding and flow cytometry. Endothelial cell proliferation was assessed in microvascular endothelial cells after incubation with 17beta-estradiol (10 nM), PPT (100 nM), and R,R-THC (100 nM) for 6 days using a tetrazolium-based bioassay. RESULTS: Both PPT and R,R-THC increased VEGFR-2 expression on myometrial microvascular endothelial cells in a dose-dependent manner, reaching a maximum at 1 microM. Approximately 40% of myometrial microvascular endothelial cell isolates only express ERbeta and do not express ERalpha, and in these neither PPT, R,R-THC, nor 17beta-estradiol increased VEGF binding. PPT- or R,R-THC-stimulated increase in VEGF binding was significantly different between ERalpha+ and ERalpha- microvascular endothelial cell samples (P < .001 and P < .05, respectively). PPT, R,R-THC, and 17beta-estradiol significantly augmented VEGF-stimulated microvascular endothelial cell proliferation in ERalpha+ (P < .05), but not in ERalpha- samples. CONCLUSIONS: This angiogenic effect of 17beta-estradiol on adult female microvascular endothelial cells is mediated by ERalpha, rather than ERbeta.

Cell Division↗

PubMed2003

HIV coreceptors: role of structure, posttranslational modifications, and internalization in viral-cell fusion and as targets for entry inhibitors.

The human immunodeficiency virus (HIV) envelope glycoprotein forms trimers on the virion surface, with each monomer consisting of two subunits, gp120 and gp41. The gp120 envelope component binds to CD4 on target cells and undergoes conformational changes that allow gp120 to interact with certain G-protein-coupled receptors (GPCRs) on the same target membranes. The GPCRs that function as HIV coreceptors were found to be chemokine receptors. The primary coreceptors are CCR5 and CXCR4, but several other chemokine receptors were identified as "minor coreceptors", indicating their ability support entry of some HIV strains in tissue cultures. Formation of the tri-molecular complexes stabilizes virus binding and triggers a series of conformational changes in gp41 that facilitate membrane fusion and viral cell entry. Concerted efforts are underway to decipher the specific interactions between gp120/CD4, gp120/coreceptors, and their contributions to the subsequent membrane fusion process. It is hoped that some of the transient conformational intermediates in gp120 and gp41 would serve as targets for entry inhibitors. In addition, the CD4 and coreceptors are primary targets for several classes of inhibitors currently under testing. Our review summarizes the current knowledge on the interactions of HIV gp120 with its receptor and coreceptors, and the important properties of the chemokine receptors and their regulation in primary target cells. We also summarize the classes of coreceptor inhibitors under development.

Anti-HIV Agents↗

PubMed2003

Peptides trap the human immunodeficiency virus type 1 envelope glycoprotein fusion intermediate at two sites.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells requires folding of two heptad-repeat regions (N-HR and C-HR) of gp41 into a trimer of N-HR and C-HR hairpins, which brings viral and target cell membranes together to facilitate membrane fusion. Peptides corresponding to the N-HR and C-HR of gp41 are potent inhibitors of HIV infection. Here we report new findings on the mechanism of inhibition of a N-HR peptide and compare these data with inhibition by a C-HR peptide. Using intact envelope glycoprotein (Env) under fusogenic conditions, we show that the N-HR peptide preferentially binds receptor-activated Env and that CD4 binding is sufficient for triggering conformational changes that allow the peptide to bind Env, results similar to those seen with the C-HR peptide. However, activation by both CD4 and chemokine receptors further enhances Env binding by both peptides. We also show that a nonconservative mutation in the N-HR of gp41 abolishes C-HR peptide but not N-HR peptide binding to gp41. These results indicate that there are two distinct sites in receptor-activated Env that are potential targets for drug or vaccine development.

Amino Acid Sequence↗

PubMed2002

Stromal-derived factor 1 expression in the human thymus.

Stromal-derived factor-1 (SDF-1), the only known ligand for the chemokine receptor CXCR4, is broadly expressed in cells of both the immune and central nervous systems, and it can induce the migration of resting leukocytes and hemopoietic progenitors. SDF-1 mRNA was previously detected in human thymus-derived stromal cells, but its role in thymopoiesis was unknown. Here we show that SDF-1 is expressed in medullar epithelial cells forming Hassall's corpuscles (HC). In search of the cell type that may be attracted by SDF-1(+) cells in the medulla, we determined that dendritic cells (DC) could be found in situ in close proximity to SDF-1(+) epithelial cells in HC. In HIV-1-infected SCID-hu thymuses, DC contained apoptotic cells and were located within enlarged HC. It was further demonstrated that uptake of apoptotic thymocytes by immature DC induced an increase in CXCR4 expression and SDF-1-mediated chemotaxis. Our data suggest a role for SDF-1 in the elimination of apoptotic thymocytes.

Animals↗

PubMed2002

Estrogen receptor-alpha and -beta expression in microvascular endothelial cells and smooth muscle cells of myometrium and leiomyoma.

The two estrogen receptors, ERalpha and ERbeta, are likely to have roles in the pathophysiology of fibroid development. They have been detected in myometrial and leiomyoma (fibroid) tissue, but the cell types expressing ERalpha and ERbeta have not been determined. ERs have also been detected in human endothelial cells. The aims of the present study were to determine whether pure populations of myometrial microvascular endothelial cells (MEC) express ERalpha and ERbeta, to compare MEC ERalpha/ERbeta expression with that of pure populations of myometrial smooth muscle cells (SMC) and to determine if ERalpha/ERbeta are differentially expressed in MEC and SMC of myometrium and fibroids from nine paired samples. Using RT-PCR (for ERalpha and ERbeta) and Western blotting (for ERalpha only), we demonstrated that all cultures of early passage myometrial and fibroid SMC (>99% pure) expressed ERalpha but not ERbeta, while myometrial and fibroid MEC (>99% CD31+) constitutively expressed ERbeta. However, both myometrial and fibroid MEC showed variable expression of ERalpha, with approximately 60% of MEC samples expressing ERalpha. While the majority (6/9) of MEC from myometrial and fibroid pairs demonstrated the same pattern of ERalpha expression, 3/9 pairs showed discordant ERalpha expression. These results show that ERalpha and ERbeta are differentially expressed in SMC and MEC of human myometrium and fibroids. Since ERalpha and ERbeta mediate opposing transcriptional activities, any effect of estrogen on the growth and development of fibroids is likely to be complex and may involve both SMC and MEC.

Adult↗

PubMed2002

Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates.

Human immunodeficiency virus type 1 (HIV-1) entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1 entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Suboptimal temperature (31.5 degrees C) was used to prolong fusion intermediates as monitored by confocal microscopy. After transfer to 37 degrees C, these fusion intermediates progressed to syncytium formation with enhanced kinetics compared with effector-target (E/T) cell mixtures that were incubated only at 37 degrees C. gp41 peptides DP-178, DP-107, and IQN17 blocked fusion more efficiently (5- to 10-fold-lower 50% inhibitory dose values) when added to E/T cells at the suboptimal temperature prior to transfer to 37 degrees C. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37 degrees C only if preincubated with E/T cells at the suboptimal temperature. Similar fusion inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41 and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site.

Animals↗

PubMed2002

17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

Estrogen has a cardiovascular protective role in women due in part to its effect on the vasculature. The roles of the two estrogen receptors (ERs), ERalpha and ERbeta, in the vascular actions of estrogen are unclear, as are effects of estrogen on microvascular endothelial cells (MEC) derived from sex steroid-responsive tissues. The present study demonstrates that 17beta-estradiol, but not progesterone, increases vascular endothelial growth factor (VEGF) receptor (VEGFR) expression on human myometrial MEC measured using biotin-recombinant human (rh) VEGF(165) and flow cytometry. This response occurred in a time- and dose-dependent manner, with significantly increased rhVEGF(165) binding at 3 h and maximal responses between 0.1 and 10 nmol/liter 17beta-estradiol, which was blocked by the antiestrogen ICI 182,780. Approximately 60% of samples demonstrated this response to 17beta-estradiol. All samples of myometrial MEC expressed both ERbeta mRNA and protein demonstrated by semiquantitative RT-PCR and Western blotting. However, ERalpha mRNA and protein were expressed in only 13 of 21 MEC samples. There was a significant association between ERalpha expression in myometrial MEC and their ability to respond to 17beta-estradiol by increasing rhVEGF(165) binding. 17beta-estradiol increased VEGFR-2 expression in ERalpha-expressing MEC isolates, which also demonstrated increased rhVEGF(165) binding, but failed to have these effects on ERalpha negative samples. Similarly, 17beta-estradiol augmented VEGF-induced MEC proliferation in ERalpha-expressing MEC samples, which was blocked by ICI 182,780. These observations suggest that 17beta-estradiol increases VEGFR-2 expression on human myometrial MEC promoting endothelial cell proliferation, an effect that varies between subjects and appears to be mediated primarily by ERalpha.

Adult↗