Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CYTOMEGALOVIRUS COINFECTION IS ASSOCIATED WITH INCREASED VASCULAR-HOMING CD57+ CD4 T CELLS IN HIV INFECTION

doi: 10.4049/jimmunol.1900734

Figure Lengend Snippet: IL-15 and TNF enhance the chemoattraction of CD57+ CD4 Tmem toward CX3CL1-expressing endothelial cells. (A) Percentage of CD57− and CD57+ CD4 Tmem from CMV-seronegative (n = 8) and CMV-seropositive (n = 12) donors that express CX3CR1. Significance determined by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. (B) MFI of CX3CR1 staining on CD57− or CD57+ CD4 Tmem from HIV-uninfected control donors (n = 12) after 48h of stimulation with medium control or IL-15 (20ng/ml). Significance determined by Wilcoxon signed rank test. Differences in fold change (FC) determined by Mann-Whitney test. *P ≤ 0.05. (C) Schematic diagram of the transwell assay system. Confluent monolayers of human aortic endothelial cells (HAoECs) were cultured in the presence or absence of TNF (10ng/ml) for 7 days in wells of a 24-well plate. Purified T cells from CMV-seropositive PLWH (n = 11) were exposed to IL-15 (20ng/ml) or medium control for 2 days prior to placement in the upper chamber of a 5μm transwell situated onto the HAoEC monolayer. In some cases, T cells were treated with AZD8797 (500nM) for 1 hour prior to co-culture. After 3 hours, T cells were separately harvested from the upper and lower chambers and analyzed by flow cytometry. (D) Representative dotplots showing CX3CR1 and CD57 expression on CD4 T cells in transwell assay from upper and lower chambers in indicated conditions. (E) Percentage of CD4 T cells recovered from the upper and lower chambers expressing CD57 after 3 hours. Significance determined by Wilcoxon signed rank test. (F) Absolute number of CD57+ CD4 T cells in the lower chamber expressed as a percent of total (upper chamber + lower chamber) CD57+ CD4 T cells (“Percent migrated”) in indicated conditions. Significance determined by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. (G) The proportion of CD57+ CD4 Tmem that expresses CX3CR1 predicts the percent migrated of CD57+ CD4 T cells in the assay after IL-15 and TNF co-treatment (minus no treatment baseline). Significance determined by simple linear regression. (H) (Left) Enrichment in the percentage of CD57+ CD4 Tmem (lower chamber minus upper chamber) versus the percentage change in percent migrated following AZD8797 treatment of CD57+ CD4 Tmem in the IL-15 and TNF co-treatment condition. Significance determined by Spearman analysis. (Right) The percentage change in percent migrated following AZD8797 treatment of CD57+ CD4 Tmem in the IL-15 and TNF co-treatment condition for donors whose CD57+ CD4 Tmem enrichment was at the median value or below and those who enrichment was greater than the median value. Significance determined by Mann-Whitney test.

Article Snippet: Primary HAoECs (PromoCell) were treated for 7 days with recombinant human TNF (R&D Systems).

Techniques: Expressing, Staining, Control, MANN-WHITNEY, Transwell Assay, Cell Culture, Purification, Co-Culture Assay, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Long non‐coding RNA GAPLINC promotes angiogenesis by regulating miR‐211 under hypoxia in human umbilical vein endothelial cells

doi: 10.1111/jcmm.14678

Figure Lengend Snippet: Effects of the expression level of GAPLINC on VEGFR and DLL4 expression in HUVECs. The treatment condition as indicated. Cells were lysed and proteins were transferred to PVDF membrane and stained with antibodies. A, Representative Western blot of VEGFR and DLL4 was shown. B, The summary of expression of VEGFR and DLL4 in different condition were shown. The results are presented as mean ± SD for three independent experiments. Each experiment performed in triplicate. *: P < .05, **: P < .01, ***: P < .001, ****: P < .0001

Article Snippet: The primary antibodies raised against GAPDH (1:1000, Goodhere), Bcl2 (1:1000, Proteintech), VEGFR (1:2000, Boster) and DLL4 (1:300, Proteintech) were incubated with the membrane overnight.

Techniques: Expressing, Membrane, Staining, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: HIV impairs TNF-alpha mediated macrophage apoptotic response to Mycobacterium tuberculosis.

doi: 10.4049/jimmunol.179.10.6973

Figure Lengend Snippet: FIGURE 2. TNF- release and the role of TNF- in MTb-mediated macrophage apoptosis. A, MTb-mediated macrophage apoptosis is dependent on TNF-. Differentiated U937 macrophages were incubated with MTb isolates in the presence and absence of specific neutralizing Ab to TNF- (anti-TNF; mAb 210, 5 g/ml) or isotype control (isotype), and after 24 h, apoptosis was determined by ELISA. Data are expressed as percent change in apoptosis compared with unstimulated macrophages (n 3). B, Reduced MTb-mediated TNF- release by HIV human macrophages. Differentiated human U937 and HIV U1 macrophages were incubated with individual MTb isolates at a multiplicity of 10:1 for 24 h, in the presence and absence of a general cellular phosphatase inhibitor, okadaic acid (OA; 50 ng/ml). Cell-free culture supernatants were assayed for TNF- by ELISA. C, Reduced TNF- mRNA transcripts in HIV macrophages. Differentiated U937 and HIV U1 macrophages were incubated for 6 h in the presence or absence of irradiated virulent MTb (iMTbRv). Specific TNF- mRNA was detected by RT-PCR, with -actin as an internal control for mRNA loading. Representative gel of three experiments providing identical results. D, Recovery of apoptosis in HIV macrophages by exogenous TNF- or endogenous macrophage TNF- release. Differentiated HIV U1 macrophages were incubated with irradiated virulent H37Rv MTb (iMTbRv) in the presence or absence of exogenous recombinant human TNF- (100 ng/ml), okadaic acid (OA; 50 ng/ml), or neutralizing anti-TNF Ab (mAb 210) for 24 h at 37°C. Apoptosis was determined by Cell Death ELISA. Data is shown as percentage change in OD from unstimulated condition. All quantitative data values represent mean SEM. , p 0.05 compared with iMTbRv alone; , p 0.05 compared with iMTbRv OA.

Article Snippet: Staurosporine (protein kinase C inhibitor; 5 M) was used as a positive control for apoptosis, and for select experiments, TNF- neutralizing Abs or isotype controls (mAb 210, R&D Systems) were added to culture supernatants to neutralize TNF- bioactivity before addition of MTb.

Techniques: Incubation, Control, Enzyme-linked Immunosorbent Assay, Irradiation, Reverse Transcription Polymerase Chain Reaction, Recombinant