Journal: PLoS Pathogens

Article Title: β-catenin regulates HIV latency and modulates HIV reactivation

doi: 10.1371/journal.ppat.1010354

Figure Lengend Snippet: (A) 3–4 human donors were used to establish a primary T CM model of HIV latency using HIV strain NL4-3 (left) or REJO (right). Latently infected cells were stimulated with αCD3/αCD8 activation beads alone (black) or in the presence of 2 μM Bio (blue). The percentage of CD4 negative HIV p24 positive cells is shown. (B) T CM cells latently infected with HIV REJO were stimulated with the indicated drugs in combination with 2 μM Bio (blue). Reactivation as measured by the percentage of CD4 negative HIV p24 positive cells was normalized to reactivation with the drug in the absence of Bio. Mean and SEM of 3 donors is shown. Significance was determined using paired t -tests for all panels, * p<0.05, ** p<0.01.

Article Snippet: CD8+ depleted PBMCs were isolated using the RosetteSep Human CD8 depletion cocktail (StemCell) and suspended in RPMI supplemented with 30 U/ml IL-2 and antiretrovirals raltegravir (1 μM) and nelfinavir (0.5 μM).

Techniques: Infection, Activation Assay

Journal: PLoS Pathogens

Article Title: β-catenin regulates HIV latency and modulates HIV reactivation

doi: 10.1371/journal.ppat.1010354

Figure Lengend Snippet: (A) CD8-depleted PBMCs from n = 5 HIV positive donors on suppressive cART therapy were treated for 48 hours with 50 nM β-catenin inhibitor ADV, αCD3/αCD28 T-cell activating beads alone or combined with 50 nM ADV. Extracellular (released virions, right) HIV RNA copies were quantified. Absolute RNA copy numbers in vehicle control and ADV treated cultures are shown, with lines connecting samples from the same donor. Symbols corresponding to donors in are used for panels a-g. (B) Fold change of RNA copies in ADV treated cultures over vehicle control are shown from released virus. (C) Fold change of RNA copies in ADV co-treated cultures over αCD3/αCD28 single treatment. (D) As in (A), cells were treated with αCD3/αCD28 beads alone or combined with 2 μM 6Bio. HIV RNA quantities are shown with lines connecting cultures from the same donor. (E) Fold change of HIV RNA copies in 6Bio co-treated cells over αCD3/αCD28 single treatment. (f-g) Downstream target of β-catenin, Bcl-xL, was quantified by flow cytometry in cells treated with ADV, αCD3/αCD28, or αCD3/αCD28 with ADV/6Bio, to confirm the modulation of β-catenin by these drugs. Fold change in mean fluorescence intensity of Bcl-xL is shown compared to vehicle control or αCD3/αCD28 treated cells, for single or dual treated cells, respectively. (H) Viability and T cell activation markers in cells following drug treatments were quantified by flow cytometry. The proportion of CD3+ CD4+ T cells expressing Ki67, CD69, CD38/HLA-DR, or LIVE/DEAD stain are plotted for cells treated with the indicated treatments. Significance was determined using paired t -tests for all panels, * p<0.05, ** p<0.01.

Article Snippet: CD8+ depleted PBMCs were isolated using the RosetteSep Human CD8 depletion cocktail (StemCell) and suspended in RPMI supplemented with 30 U/ml IL-2 and antiretrovirals raltegravir (1 μM) and nelfinavir (0.5 μM).

Techniques: Control, Virus, Flow Cytometry, Fluorescence, Activation Assay, Expressing, Staining

Journal: PLoS Pathogens

Article Title: β-catenin regulates HIV latency and modulates HIV reactivation

doi: 10.1371/journal.ppat.1010354

Figure Lengend Snippet: Schematic demonstrating multiple potential mechanisms by which β-catenin may modulate HIV transcription and latency, based on integrated findings from previous studies and data presented here. (1) β-catenin and TCF-4 form a complex with nuclear matrix-associated protein SMAR1 at the HIV LTR just upstream of the transcriptional start site and Sp-1, NFκB, and AP-1 binding sites. This complex pulls the HIV LTR towards the nuclear matrix, occluding access of RNA polymerase . (2) β-catenin positively regulates levels of TCF-4, which has been shown to block binding and transcriptional regulation of NFκB at the HIV LTR . (3) β-catenin further regulates c-Myc levels, which recruit HDAC enzymes, resulting in the viral promoter being more densely packed in chromatin . (4) β-catenin also mediates self-renewal and cell proliferation of memory T cells through CBP, which may contribute to perpetuating the reservoir of latently infected cells , (5) A source of β-catenin signaling are CD8+ T cells, which secrete Wnt proteins resulting in stimulation of the Wnt/β-catenin pathway in CD4+ T cells, which culminates in accumulation of β-catenin in the cytoplasm and translocation to the nucleus . This may explain the observed role of CD8+ T cells in maintaining HIV latency. Notably, other cells may serve as a source of Wnt proteins and β-catenin pathway modulating factors.

Article Snippet: CD8+ depleted PBMCs were isolated using the RosetteSep Human CD8 depletion cocktail (StemCell) and suspended in RPMI supplemented with 30 U/ml IL-2 and antiretrovirals raltegravir (1 μM) and nelfinavir (0.5 μM).

Techniques: Binding Assay, Blocking Assay, Infection, Translocation Assay

Journal: Toxicology mechanisms and methods

Article Title: Activation of Protein Kinase C and Protein Kinase D in Human Natural Killer Cells: Effects of Tributyltin, Dibutyltin, and Tetrabromobisphenol A

doi: 10.3109/15376516.2015.1070226

Figure Lengend Snippet: Effects of exposures to TBBPA on the phosphorylation/activation state of PKC and PKD in NK cells: Representative experiment for 10 min exposures to TBBPA. Experiment was repeated using cells from seven different donors.

Article Snippet: NK cells were purified by mixing 30–40 mL of buffy coat with 0.6 mL of RosetteSep human NK cell enrichment antibody cocktail (Stem Cell Technologies, Vancouver, BC, Canada).

Techniques: Activation Assay

Journal: Toxicology mechanisms and methods

Article Title: Activation of Protein Kinase C and Protein Kinase D in Human Natural Killer Cells: Effects of Tributyltin, Dibutyltin, and Tetrabromobisphenol A

doi: 10.3109/15376516.2015.1070226

Figure Lengend Snippet: Effects of incubations with K562 target cells on phosphorylation/activation state of PKC in NK cells: A) levels of phospho-PKC normalized to the control. B) Representative Western blot. Experiments were repeated using cells from five different donors. Asterisks indicate a significant difference as compared with control, p<0.05.

Article Snippet: NK cells were purified by mixing 30–40 mL of buffy coat with 0.6 mL of RosetteSep human NK cell enrichment antibody cocktail (Stem Cell Technologies, Vancouver, BC, Canada).

Techniques: Activation Assay, Western Blot

Journal: Toxicology mechanisms and methods

Article Title: Activation of Protein Kinase C and Protein Kinase D in Human Natural Killer Cells: Effects of Tributyltin, Dibutyltin, and Tetrabromobisphenol A

doi: 10.3109/15376516.2015.1070226

Figure Lengend Snippet: Effects of incubations with K562 target cells on phosphorylation/activation state of PKD in NK cells: A) levels of phospho-PKD normalized to the control. B) Representative Western blot. Experiments were repeated using cells from five different donors. Asterisks indicate a significant difference as compared with control, p<0.05.

Article Snippet: NK cells were purified by mixing 30–40 mL of buffy coat with 0.6 mL of RosetteSep human NK cell enrichment antibody cocktail (Stem Cell Technologies, Vancouver, BC, Canada).

Techniques: Activation Assay, Western Blot

Journal: Toxicology mechanisms and methods

Article Title: Activation of Protein Kinase C and Protein Kinase D in Human Natural Killer Cells: Effects of Tributyltin, Dibutyltin, and Tetrabromobisphenol A

doi: 10.3109/15376516.2015.1070226

Figure Lengend Snippet: Effects of exposures to TBT on the phosphorylation/activation state of PKC in NK cells: A) levels of phospho-PKC normalized to the control for 10 min exposures to TBT. B) Representative experiment for 10 min exposures to TBT. C) Representative experiment for 1 h exposures to TBT. D) Representative experiment for 6 h exposures to TBT. Experiments were repeated using cells from five different donors. Asterisks indicate a significant difference as compared with control, p<0.05.

Article Snippet: NK cells were purified by mixing 30–40 mL of buffy coat with 0.6 mL of RosetteSep human NK cell enrichment antibody cocktail (Stem Cell Technologies, Vancouver, BC, Canada).

Techniques: Activation Assay

Journal: Toxicology mechanisms and methods

Article Title: Activation of Protein Kinase C and Protein Kinase D in Human Natural Killer Cells: Effects of Tributyltin, Dibutyltin, and Tetrabromobisphenol A

doi: 10.3109/15376516.2015.1070226

Figure Lengend Snippet: Effects of exposures to TBT on the phosphorylation/activation state of PKD in NK cells: A) levels of phospho-PKD normalized to the control for 10 min exposures to TBT. B) Representative experiment for 10 min exposures to TBT. C) Representative experiment for 1 h exposures to TBT. D) Representative experiment for 6 h exposures to TBT. Experiments were repeated using cells from five different donors. Asterisks indicate a significant difference as compared with control, p<0.05.

Article Snippet: NK cells were purified by mixing 30–40 mL of buffy coat with 0.6 mL of RosetteSep human NK cell enrichment antibody cocktail (Stem Cell Technologies, Vancouver, BC, Canada).

Techniques: Activation Assay

Journal: Toxicology mechanisms and methods

Article Title: Activation of Protein Kinase C and Protein Kinase D in Human Natural Killer Cells: Effects of Tributyltin, Dibutyltin, and Tetrabromobisphenol A

doi: 10.3109/15376516.2015.1070226

Figure Lengend Snippet: Effects of exposures to DBT on the phosphorylation/activation state of PKC in NK cells: A) levels of phospho-PKC normalized to the control for 10 min exposures to DBT B) Representative experiment for 10 min exposures to DBT. C) levels of phospho-PKC normalized to the control for the 1 h exposures to DBT. D) Representative experiment for 1 h exposures to DBT. E) Representative experiment for 6 h exposures to DBT. Experiments were repeated using cells from six different donors for 10 min and 1h and form three different donors for 6 h. Asterisks indicate a significant difference as compared with control, p<0.05.

Article Snippet: NK cells were purified by mixing 30–40 mL of buffy coat with 0.6 mL of RosetteSep human NK cell enrichment antibody cocktail (Stem Cell Technologies, Vancouver, BC, Canada).

Techniques: Activation Assay

Journal: Toxicology mechanisms and methods

Article Title: Activation of Protein Kinase C and Protein Kinase D in Human Natural Killer Cells: Effects of Tributyltin, Dibutyltin, and Tetrabromobisphenol A

doi: 10.3109/15376516.2015.1070226

Figure Lengend Snippet: Effects of exposures to DBT on the phosphorylation/activation state of PKD in NK cells: A) levels of phospho-PKD normalized to the control for 10 min exposures to DBT. B) Representative experiment for 10 min exposures to DBT. C) Representative experiment for 1 h exposures to DBT. D) Representative experiment for 6 h exposures to DBT. Experiments were repeated using cells from five different donors (10 min), six different donors (1 h) and three different donors (6 h). Asterisks indicate a significant difference as compared with control, p<0.05.

Article Snippet: NK cells were purified by mixing 30–40 mL of buffy coat with 0.6 mL of RosetteSep human NK cell enrichment antibody cocktail (Stem Cell Technologies, Vancouver, BC, Canada).

Techniques: Activation Assay