Journal: Journal of Virology

Article Title: Longitudinal Examination of the Intestinal Lamina Propria Cellular Compartment of Simian Immunodeficiency Virus-Infected Rhesus Macaques Provides Broader and Deeper Insights into the Link between Aberrant MicroRNA Expression and Persistent Immune Activation

doi: 10.1128/JVI.00189-16

Figure Lengend Snippet: THC treatment prevented miR-150 downregulation (A) and IRAK1 upregulation (B) in CD8+ T cells 24 h after in vitro activation with PMA-ionomycin compared to those treated with vehicle (VEH). (C) The quantification of Western blotting revealed significant THC-mediated reduction of IRAK1 protein levels in CD8+ T cells compared to those treated with vehicle. miR-150 expression was normalized to snoU6. The error bars represent standard errors of the means of fold changes within each group. T, THC; V, vehicle (DMSO).

Article Snippet: For in vivo T-cell and in vitro CD8 + T-cell immunophenotyping, ∼100-μl aliquots were stained with appropriately diluted, directly conjugated monoclonal antibodies to CD3 (Pacific blue, SP34-2), CD4 (SK3, peridinin chlorophyll protein-Cy5.5), and CD8 (phycoerythrin-Texas red, 3B5), CD69 (FN50 and V450), and Ki67 (556026, fluorescein isothiocyanate) (BD Biosciences, San Jose, CA).

Techniques: In Vitro, Activation Assay, Western Blot, Expressing

Journal: Journal of Virology

Article Title: Longitudinal Examination of the Intestinal Lamina Propria Cellular Compartment of Simian Immunodeficiency Virus-Infected Rhesus Macaques Provides Broader and Deeper Insights into the Link between Aberrant MicroRNA Expression and Persistent Immune Activation

doi: 10.1128/JVI.00189-16

Figure Lengend Snippet: Percentage of CD4+ (A) and CD8+ (B) T cells among lamina propria leukocytes (LPLs) isolated at different time points after SIV infection. The cells were first gated on singlets followed by lymphocytes and CD3, and then they were gated on CD3+ CD4+ and CD3+ CD8+ T-cell subsets. Data analysis using Kruskal-Wallis test identified significant differences among the different groups for both CD4 (P = 0.0003) and CD8 (P = 0.0020) T cells. Post hoc analysis using Dunn's multiple-group comparison identified both CD4+ and CD8+ T cells at 180 dpi to be significantly different from the preinfection group. *, P < 0.05. The error bars represent standard errors of means for CD4+ or CD8+ T-cell percentages within each group.

Article Snippet: For in vivo T-cell and in vitro CD8 + T-cell immunophenotyping, ∼100-μl aliquots were stained with appropriately diluted, directly conjugated monoclonal antibodies to CD3 (Pacific blue, SP34-2), CD4 (SK3, peridinin chlorophyll protein-Cy5.5), and CD8 (phycoerythrin-Texas red, 3B5), CD69 (FN50 and V450), and Ki67 (556026, fluorescein isothiocyanate) (BD Biosciences, San Jose, CA).

Techniques: Isolation, Infection

Journal: Journal of Virology

Article Title: Longitudinal Examination of the Intestinal Lamina Propria Cellular Compartment of Simian Immunodeficiency Virus-Infected Rhesus Macaques Provides Broader and Deeper Insights into the Link between Aberrant MicroRNA Expression and Persistent Immune Activation

doi: 10.1128/JVI.00189-16

Figure Lengend Snippet: (A) IRAK1 is a direct target of miR-150. Luciferase reporter vectors containing all four miR-150 binding sites (seed region) on the rhesus macaque 3′ UTR of IRAK1 mRNA or the corresponding construct with the binding sites deleted were cotransfected into HEK293 cells with 50 nM miRNA mimic. Firefly and Renilla luciferase activities were detected using the Dual-Glo luciferase assay system 72 h after transfection. The ratio of luciferase activities (Firefly/Renilla) was calculated and normalized to the wells transfected with only unmanipulated pmir-GLO vector. Triple asterisks indicate statistical significance (P < 0.001) compared to cells transfected with pmirGLO-delIRAK1 vector. IRAK1 protein expression is markedly elevated in the colon of chronic SIV-infected rhesus macaques (B and C) compared to that of the uninfected control macaque (D). Panels B, C, and D involve double labels with IRAK1 (green) and CD8 (red) for T cells. The gray channel represents differential interference contrast (DIC) to reveal tissue architecture. Colocalizations of green (IRAK1) and red (CD8) appear light yellow (arrows). In panels B and C, note the intense IRAK1 staining in the epithelium and lamina propria and markedly increased numbers of CD8+ IRAK1+ T cells (white arrowheads) in the colon of the SIV-infected macaques. In contrast, panel D shows that IRAK1 staining is extremely weak in the colonic epithelium and LPLs of the control macaque. All panels are ×40 magnification. Quantification of cells and regions of interest (ROI) labeled by IRAK1 was performed using Volocity 5.5 software after capturing images on a Leica confocal microscope. (E) Several ROI were hand drawn on the epithelial and LPL regions in the images from colon. Image analysis data were analyzed using nonparametric Wilcoxon's rank-sum test. (F) miR-150 can negatively regulate IRAK1 protein expression. Representative Western blot (F) and quantification (G) show the reduction in protein expression of IRAK1 (∼80 kDa) 96 h posttransfection of HEK293T cells with pre-miR-150-expressing plasmid (pEP-miR-150) (duplicate wells) compared to those transfected with pEP-Null control vector (duplicate wells). Kal is a marker (Bio-Rad kaleidoscope prestained marker).

Article Snippet: For in vivo T-cell and in vitro CD8 + T-cell immunophenotyping, ∼100-μl aliquots were stained with appropriately diluted, directly conjugated monoclonal antibodies to CD3 (Pacific blue, SP34-2), CD4 (SK3, peridinin chlorophyll protein-Cy5.5), and CD8 (phycoerythrin-Texas red, 3B5), CD69 (FN50 and V450), and Ki67 (556026, fluorescein isothiocyanate) (BD Biosciences, San Jose, CA).

Techniques: Luciferase, Binding Assay, Construct, Transfection, Plasmid Preparation, Expressing, Infection, Staining, Labeling, Software, Microscopy, Western Blot, Marker

Journal: Journal of Virology

Article Title: Longitudinal Examination of the Intestinal Lamina Propria Cellular Compartment of Simian Immunodeficiency Virus-Infected Rhesus Macaques Provides Broader and Deeper Insights into the Link between Aberrant MicroRNA Expression and Persistent Immune Activation

doi: 10.1128/JVI.00189-16

Figure Lengend Snippet: THC inhibits CD8+ T-cell activation in vitro. THC-treated CD8+ T cells show significantly reduced expression of the lymphocyte activation marker CD69 (A and B) and proliferation marker Ki67 (C). Note the significantly reduced percentage of CD3+ CD8+ CD69+ and CD3+ CD8+ Ki67+ cells in all five animals 24 h after in vitro activation with PMA-ionomycin. Mean percentages of CD8+ CD69+ (D) and CD8+ Ki67+ (E) are significantly lower in THC-treated than in vehicle (VEH)-treated cells. THC-mediated inhibition of in vitro CD8+ T-cell activation (A and B) and proliferation (C) in response to PMA-ionomycin stimulation is evidenced by the absence of cellular clumps (F) compared to cells treated with vehicle (DMSO) (G). The error bars represent standard errors of the means of fold changes within each group.

Article Snippet: For in vivo T-cell and in vitro CD8 + T-cell immunophenotyping, ∼100-μl aliquots were stained with appropriately diluted, directly conjugated monoclonal antibodies to CD3 (Pacific blue, SP34-2), CD4 (SK3, peridinin chlorophyll protein-Cy5.5), and CD8 (phycoerythrin-Texas red, 3B5), CD69 (FN50 and V450), and Ki67 (556026, fluorescein isothiocyanate) (BD Biosciences, San Jose, CA).

Techniques: Activation Assay, In Vitro, Expressing, Marker, Inhibition