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ABSTRACT: Molecular regulation of HIV transcription is a multifaceted process dictated in part by the abundance of cellular transcription factors that induce or repress HIV promoter activity. β-Catenin partners with members of the T cell factor (TCF)/LEF transcription factors to regulate gene expression. The interaction between β-catenin and TCF-4 is linked to inhibition of HIV replication in multiple cell types, including lymphocytes and astrocytes. Here, we evaluated the molecular mechanism by which β-catenin/TCF-4 repress HIV replication. We identified for the first time multiple TCF-4 binding sites at -336, -143, +66, and +186 relative to the transcription initiation site on the HIV long terminal repeat (LTR). Two of the sites (-143 and +66) were present in approximately 1/3 of 500 HIV-1 isolates examined. Although all four sites could bind to TCF-4, the strongest association occurred at -143. Deletion and/or mutation of -143, in conjunction with β-catenin or TCF-4 knockdown in cells stably expressing an LTR reporter construct, enhanced basal HIV promoter activity by 5-fold but had no effect on Tat-mediated transactivation of the HIV LTR. We also found that TCF-4, β-catenin, and the nuclear matrix binding protein SMAR1 tether at the -143-nucleotide (nt) site on the HIV LTR to inhibit HIV promoter activity. Collectively, these data indicate that TCF-4 and β-catenin at -143 associate with SMAR1, which likely pulls the HIV DNA segment into the nuclear matrix and away from transcriptional machinery, leading to repression of basal HIV LTR transcription. These studies point to novel avenues for regulation of HIV replication by manipulation of β-catenin signaling within cells.
Journal of Virology 06/2012; 86(17):9495-503. · 5.40 Impact Factor
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ABSTRACT: The Wnt/β-catenin pathway is involved in diverse cell functions governing development and disease. β-Catenin, a central mediator of this pathway, binds to members of the TCF/LEF family of transcription factors to modulate hundreds of genes. Active Wnt/β-catenin/TCF-4 signaling plays a significant role in repression of HIV-1 replication in multiple cell targets, including astrocytes. To determine the mechanism by which active β-catenin/TCF-4 leads to inhibition of HIV replication, we knocked down β-catenin or TCF/LEF members in primary astrocytes and astrocytomas transiently transfected with an HIV long terminal repeat (LTR)-luciferase reporter that contained an integrated copy of the HIV LTR-luciferase construct. Knockdown of either β-catenin or TCF-4 induced LTR activity by 2- to 3-fold under both the episomal and integrated conditions. This knockdown also increased presence of serine 2-phosphorylated RNA polymerase II (Pol II) on the HIV LTR as well as enhanced its processivity. Knockdown of β-catenin/TCF-4 also impacted tethering of other transcription factors on the HIV promoter. Specifically, knockdown of TCF-4 enhanced binding of C/EBPβ, C/EBPδ, and NF-κB to the HIV LTR, while β-catenin knockdown increased binding of C/EBPβ and C/EBPδ but had no effect on NF-κB. Approximately 150 genes in astrocytes were impacted by β-catenin knockdown, including genes involved in inflammation/immunity, uptake/transport, vesicular transport/exocytosis, apoptosis/cellular stress, and cytoskeleton/trafficking. These findings indicate that modulation of the β-catenin/TCF-4 axis impacts the basal level of HIV transcription in astrocytes, which may drive low level/persistent HIV in astrocytes that can contribute to ongoing neuroinflammation, and this axis also has profound effects on astrocyte biology.
Journal of Virology 12/2011; 86(4):1911-21. · 5.40 Impact Factor
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ABSTRACT: Upon activation, a subset of mature human CD8(+) T cells re-expresses CD4 dimly. This CD4(dim)CD8(bright) T cell population is genuine and enriched in antiviral CD8(+) T cell responses. The signaling pathway that leads to CD4 re-expression on mature CD8(+) T cells is not clear. Given that Wnt/beta-catenin signaling plays a critical role in the transition of CD4(-)CD8(-) to CD4(+)CD8(+) thymocytes, we determined whether beta-catenin mediates CD4 expression on mature CD8(+) T cells. We demonstrate that active beta-catenin expression is 20-fold higher on CD4(dim)CD8(bright) than CD4(-)CD8(+) T cells. Activation of beta-catenin signaling, through LiCl or transfection with a constitutively active construct of beta-catenin, induced CD4 on CD8(+) T cells by approximately 10-fold. Conversely, inhibition of beta-catenin signaling through transfection with a dominant-negative construct for T cell factor-4, a downstream effector of beta-catenin signaling, diminished CD4 expression on CD8(+) T cells by 50% in response to T cell activation. Beta-catenin-mediated induction of CD4 on CD8(+) T cells is transcriptionally regulated, as it induced CD4 mRNA, and T cell factor/lymphoid enhancer factor sites were identified within the human CD4 promoter. Further, beta-catenin expression induced the antiapoptotic factor BcL-xL, suggesting that beta-catenin may mediate protection against activation-induced cell death. Collectively, these data demonstrate that beta-catenin is critical in inducing CD4 expression on mature CD8(+) T cells, suggesting that it is a common pathway for CD4 upregulation among thymocytes and mature CD8(+) T cells.
The Journal of Immunology 08/2010; 185(4):2013-9. · 5.79 Impact Factor
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ABSTRACT: Purified samples of CD45+ hematopoietic cells are a prerequisite for chimerism analysis in transplantation therapies, and are useful in various research and clinical settings such as functional and molecular analysis or disease diagnosis. Recently, we have established a flow-based adhesion molecule-dependent process for the purification of these cells from human bone marrow. However, for practical purposes, it is desirable to apply this approach to process small volumes of human blood. CD45+ cell purities were >94% when PBMNCs and plasma depleted blood were perfused through P-selectin coated microtubes. However, P-selectin surface failed to capture CD45+ cells when fresh blood prior to washing was perfused. The process requires a pre-step of plasma removal which otherwise inhibits interactions of cell surface PSGL-1 with immobilized P-selectin due to the presence of soluble PSGL-1 in plasma. We conclude that P-selectin can be used in a compact flow device to isolate and purify CD45+ cells directly from human peripheral blood. The process is simple, rapid, cost effective and represents a physiologic approach to the capture and purification of CD45+ MNCs from peripheral blood.
Blood Cells Molecules and Diseases 01/2009; 42(2):136-9. · 2.35 Impact Factor
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ABSTRACT: Recently, a flow-based selectin-dependent method for capture and enrichment of specific type blood cells (CD34+ hematopoietic stem and progenitor cells) from bone marrow and peripheral blood has been described. Using a similar approach, here we show isolation of CD45+ blood cells directly from human bone marrow to a high purity (90%-97%). The process mimics a ubiquitous mechanism of cell trafficking in the body for the recruitment of neutrophils during inflammation. The method is straightforward, rapid, and may represent a practical alternative to CD45+ cell enrichment procedures required for chimerism analysis following allogenic stem-cell transplantation.
American Journal of Hematology 05/2008; 83(8):627-9. · 4.67 Impact Factor
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ABSTRACT: Clinical infusion of haematopoietic stem and progenitor cells (HSPCs) is vital for restoration of haematopoietic function in many cancer patients. Previously, we have demonstrated an ability to mimic physiological cell trafficking in order to capture CD34-positive (CD34+) HSPCs using monolayers of the cell adhesion protein P-selectin in flow chambers. The current study aimed to determine if HSPCs could be captured directly from circulating blood in vivo. Vascular shunt prototypes, coated internally with P-selectin, were inserted into the femoral artery of rats. Blood flow through the cell capture device resulted in a wall shear stress of 4-6 dynes/cm(2). After 1-h blood perfusion, immunofluorescence microscopy and flow cytometric analysis revealed successful capture of mononuclear cells positive for the HSPC surface marker CD34. Purity of captured CD34+ cells showed sevenfold enrichment over levels found in whole blood, with an average purity of 28%. Robust cell capture and HSPC enrichment were also demonstrated in devices that were implanted in a closed-loop arterio-venous shunt conformation for 2 h. Adherent cells were viable in culture and able to differentiate into burst-forming units. This study demonstrated an ability to mimic the physiological arrest of HSPCs from blood in an implantable device and may represent a practical alternative for adult stem cell capture and enrichment.
British Journal of Haematology 04/2008; 140(6):673-81. · 4.94 Impact Factor
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ABSTRACT: Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematologic disorders and in basic stem cell research. Primitive CD34+ HSPCs have demonstrated stronger rolling adhesion on selectins than mature CD34- mononuclear cells (MNCs). We have exploited this differential rolling behavior to capture and purify HSPCs from bone marrow by perfusing MNCs through selectin-coated microtubes.
Bone marrow MNCs were perfused through the cell-capture microtubes coated with adhesion molecules. We washed the device lumen and visualized and estimated captured cells by video microscopy. Adherent cells were eluted by high shear, calcium-free buffer, and air embolism. We used immunofluorescence staining followed by flow cytometry to analyze CD34+ HSPCs.
CD34+ HSPC purity of cells captured in adhesion molecule-coated devices was significantly higher than the fraction of CD34+ cells found in bone marrow MNCs [mean (SE) 2.5% (0.8%)]. P-selectin-coated surfaces yielded 16% to 20% CD34+ cell purity, whereas antibody-coated surfaces yielded 12% to 18%. Although CD34+ cell purity was comparable between selectin and antibody surfaces, the total number of CD34+ HSPCs captured was significantly higher in P-selectin devices (approximately 5.7 x 10(4) to 7.1 x 10(4)) than antibody devices (approximately 1.74 x 10(4) to 2.61 x 10(4)).
P-selectin can be used in a compact flow device to capture HSPCs. Selectin-mediated capture of CD34+ HSPCs resulted in enrichment approximately 8-fold higher than the CD34+ cell population from bone marrow MNCs. This study supports the hypothesis that flow-based, adhesion molecule-mediated capture may be a viable alternative approach to the capture and purification of HSPCs.
Clinical Chemistry 02/2008; 54(1):77-85. · 7.91 Impact Factor
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ABSTRACT: Cryptococcus gattii (Cg) is an emerging pathogen of both healthy and immunocompromised patients worldwide. Understanding the molecular genetic basis of virulence and physiology of this pathogen will be critical for defining its pathogenic mechanisms. The purine biosynthetic gene, URA5 encoding orate phosphorybosyltransferase (OPRTase), has been successfully used as a selectable marker for gene disruption by transformation and homologous recombination in Cg. Here, we report the characterization of ura5 auxotrophy and URA5 reversion phenomenon at the molecular, genetic, and structural levels, and use of ura5-->URA5 reversion as a tool for reconstitution of gene of interest and auxotrophic marker to their native loci. We identified a single mutation of GG(128)T-->GAT with substitution of glycine to aspartic acid at amino acid position 43 resulting in ura5 auxotrophy. The ura5-->URA5 reversion on CSM lacking uracil (CSM-U) was found to be a rare phenomenon with a reversion frequency of 0.000002%, and sequence analysis of URA5 from all the reverted strains revealed mutation of GA(128)T-->GGT back to its ancestral state. The URA5 allele in the reverted strains was fully functional, as demonstrated by the excellent growth of these strains on medium lacking uracil, as well as by the ability of this allele to efficiently transform ura5 mutant to restore prototrophy. The deduced Cg URA5 protein modeled on the known crystal structures of OPRTase from Salmonella typhimurium (1LH0_A, 1STO) and from Escherichia coli (1ORO_A) indicated that the glycine 43 of Cg URA5 was situated on a conserved loop, and it's substitution to more globose aspartic acid may have resulted in URA5 inactivation in auxotrophic strain. The advantages of this approach for the generation of a reconstituted strain are (1) that it restores the functionality of the native URA5, (2) that it eliminates an additional biolistic delivery of exogenous URA5, and (3) that it allows easy selection of reconstituted strains with homologous integration. This strategy was successfully used for the generation of Cg can2+CAN2/URA5 homologous reconstituted strains, which grew in ambient air to the wild-type level while can2 mutant exhibited severe growth defect under similar conditions.
Mycopathologia 12/2006; 162(6):401-9. · 1.65 Impact Factor
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ABSTRACT: Encapsulated yeast, Cryptococcus gattii (Cg) is a primary and emerging fungal pathogen in North America. It has a predilection for invading the central nervous system of both healthy and immunocompromised humans and animals. Recently, we initiated molecular pathogenesis studies in Cg strain NIH444 (ATCC 32609). In this report, we compared the biology and pathogenic potential of NIH444 to those of WM276, an Australian environmental isolate that is being used for the whole genome-sequencing project. Our data indicated that NIH444 is comparatively more virulent in a mouse model of cryptococcosis than is WM 276. We found robust mating of NIH444, and no mating of WM276, when tested against Cg MATa strain, NIH198. WM276 but not NIH444 was defective in filamentation and sporulation (haploid fruiting). Interestingly, NIH444 has a VGII/AFLP6 genotype similar to that of the genotype of the recent outbreak strains from Vancouver Island, British Columbia, Canada. Additionally, comparisons of nucleotide sequences of various genes also showed differences between NIH444 and WM276. Based on these observations, we conclude that NIH444 should remain the strain of choice for understanding Cg pathogenesis, especially on the North American continent.
Mycopathologia 11/2005; 160(3):207-15. · 1.65 Impact Factor
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ABSTRACT: We studied superoxide dismutases (SODs) in the encapsulated yeast Cryptococcus neoformans (Cn) variety gattii to analyse the role of mitochondrial MnSOD (SOD2) in fungal biology and virulence. SOD2 was cloned from a Cn cosmid library, sod2 mutant and sod2 + SOD2 reconstituted strains were constructed by homologous recombination, and two sod1sod2 double mutants were constructed by replacing SOD2 in the sod1 mutant with the sod2::HYG allele. The SOD2 protein (SOD2p) encoded 225 amino acids, with 36-66% identity with other fungal SOD2ps. SOD2 deletion rendered Cn highly growth-defective at 37 degrees C in 19-20% oxygen (normal air), and this defect was reversed by limiting oxygen to 1.3% as well in the presence of antioxidant, ascorbic acid. The sod2 mutant accumulated significantly more reactive oxygen species (ROS) at 37 degrees C as well at 30 degrees C in the presence of antimycin A, suggesting that SOD2p is the primary defence of Cn against the superoxide anion (O(2) (.-)) in the mitochondria. The sod2 was also highly susceptible to redox-cycling agents, high salt and nutrient limitations. The sod2 mutant was avirulent in intranasally infected mice and markedly attenuated in its virulence in intravenously infected mice. The virulence defect of sod2 mutant appeared related to its growth defects in high oxygen environment, but not resulting from increased sensitivity to oxidative killing by phagocytes. The sod1sod2 double mutants were avirulent in mice. Additionally, sod1sod2 double mutants showed a marked reduction in the activities of other known Cn virulence factors; and they were more susceptible to PMN killing than was the sod2 single mutant. Previously, we reported that the attenuation of sod1 mutant in mice was resulting from enhanced susceptibility to phagocyte killing, combined with a reduction in the activities of a number of virulence factors. Thus, SOD1p and SOD2p play distinct roles in the biology and virulence of Cn var. gattii via independent modes of action.
Molecular Microbiology 04/2005; 55(6):1782-800. · 5.01 Impact Factor
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ABSTRACT: The pathogenic yeast Cryptococcus neoformans (Cn) var. gattii causes meningoencephalitis in healthy individuals, unlike the better known Cn varieties grubii and neoformans, which are common in immunocompromised individuals. The virulence determinants and mechanisms of host predilection are poorly defined for var. gattii. The present study focused on the characterization of a Cu,Zn superoxide dismutase (SOD1) gene knock-out mutant constructed by developing a DNA transformation system. The sod1 mutant was highly sensitive to the redox cycling agent menadione, and showed fragmentation of the large vacuole in the cytoplasm, but no other defects were seen in growth, capsule synthesis, mating, sporulation, stationary phase survival or auxotrophies for sulphur-containing amino acids. The sod1 mutant was markedly attenuated in virulence in a mouse model, and it was significantly susceptible to in vitro killing by human neutrophils (PMNs). The deletion of SOD1 also resulted in defects in the expression of a number of virulence factors, i.e. laccase, urease and phospholipase. Complementation of the sod1 mutant with SOD1 resulted in recovery of virulence factor expression and menadione resistance, and in restoration of virulence. Overall, these results suggest that the antioxidant function of Cu,Zn SOD is critical for the pathogenesis of the fungus, but is dispensable in its saprobic life. This report constitutes the first instance in which superoxide dismutase has been directly implicated in the virulence of a fungal pathogen.
Molecular Microbiology 04/2003; 47(6):1681-94. · 5.01 Impact Factor
