Journal: PLoS ONE

Article Title: Activation of Nucleotide Oligomerization Domain 2 (NOD2) by Human Cytomegalovirus Initiates Innate Immune Responses and Restricts Virus Replication

doi: 10.1371/journal.pone.0092704

Figure Lengend Snippet: A . U373 cells were transiently transfected with pcDNA4/HisMax, pcDNA4/EGFP, pcDNA4/HisMax-hNOD2 or pcDNA4/HisMax-hRIPK2 plasmid. 24 h after transfection cells were infected with pp28-luciferase HCMV. Luciferase activity was measured in cell lysates at 72 hpi. B . Cell lysates from 4 A were used to determine protein expression of HCMV-immediate early (IE1/IE2), early (UL44), and late (pp65) genes. Levels of NOD2 and RIPK2 proteins were measured to confirm NOD2 overexpression; β-actin served as loading control. Western blot data are representative of three independent experiments. Asterisks (*) denote endogenous NOD2 and RIPK2 proteins. C – D. U373 cells were transiently transfected with pcDNA4/HisMax, hNOD2 or hRIPK2 plasmid. 24 h after transfection cells were infected with HCMV Towne and total RNA was isolated at 72 hpi. Levels of IFN-β and IL8 mRNAs were measured by qRT-PCR in non-infected (mock) and HCMV-infected (HCMV) cells. E. HFFs stably expressing empty vector (HFF-control) or HA-tagged NOD2 (HFF-NOD2) were either untreated or treated with 2 μg/ml doxycycline and expression of NOD2 was determined at 48 h using anti-HA antibody. F. HFFs stably expressing empty vector (control) or NOD2 (HFF-NOD2) were induced with doxycycline for 24 h followed by infection with HCMV Towne. Cells were incubated in doxycycline containing media and cell-free supernatants were collected at 96 hpi. Virus progeny released into the supernatants were quantified after infection of fresh HFFs (second cycle) with equal amount of supernatants from control or NOD2-overexpressing HFFs using luciferase assay at 3 dpi (Y-axis on left, in blue) and by real-time PCR (Y-axis on right, in red) in the supernatants of newly-infected HFFs (Fig. 4F). Viral protein expression was determined in newly-infected HFFs at 3 dpi (Fig. 4 G). H, J. Levels of IFN-β and IL8 mRNAs were determined in HFFs-control or HFF-NOD2 cells using qRT-PCR at 96 hpi. I. Levels of IFN-β secreted into the media from HFF-control and HFF-NOD2 cells (first cycle of infection) were measured at 24 hpi using IFN-β ELISA kit. The data shown are the average of three experiments ± SD (*p<0.05, **p<0.01, ***p<0.001, one-way ANOVA test).

Article Snippet: Human interferon β (IFN-β) specific ELISA kit (PBL InterferonSource) was used to measure levels of secreted IFN-β from HFF-control and HFF-NOD2 overexpressing cells according to manufacturer's instructions.

Techniques: Transfection, Plasmid Preparation, Infection, Luciferase, Activity Assay, Expressing, Over Expression, Control, Western Blot, Isolation, Quantitative RT-PCR, Stable Transfection, Incubation, Virus, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Journal of Human Immunity

Article Title: Increased prevalence of autoantibodies neutralizing IFNλ2/3 in young individuals with cystic fibrosis

doi: 10.70962/jhi.20250268

Figure Lengend Snippet: At least 10% of young individuals with CF harbor neutralizing anti-IFNλ2/3 IgG autoAbs. (A) IFNλ1/2/3 neutralization by plasmas from individuals with AD ( n = 39), various adults (CT; n = 68), or pwCF (CF; n = 84 samples from n = 51 individuals). Dotted lines indicate thresholds set using standard outlier calculations. Neutralizing plasmas are orange; numbers represent unique individuals; positive individuals are labeled. (B) Anti-IFN-III and anti-IFN-I IgGs in CF plasmas positive (neut; n = 9) or negative (non-neut; n = 6) for IFNλ2/3 neutralization. (C) Spearman’s correlation between IFNλ2/3 neutralization (A) and IgG (B). (D) Anti-IFNλ2/3 IgG subclasses in CF plasmas ( n = 5). (E) Area under the curve from PIV5-GFP replication kinetics in A549 cells pretreated with IFNλ2 (or not) in the presence of pwCF plasmas positive (pos) or negative (neg) for IFNλ2/3 neutralization. Values are triplicate means from two experiments. Error bars indicate standard deviations. For the IFNλ2+pos and IFNλ2+neg groups, data are from two patients (squares/triangles) from two experiments. (F) Fluorescence images of infected cells from E. (G) Random forest regression of clinical variables in pwCF positive ( n = 4) or negative ( n = 16) for anti-IFNλ2/3 autoAbs. Dots represent importance scores per iteration (maximum 5,000); error bars reflect importance spread. Abbreviations: MFI FC, median fluorescence intensity fold-change; IVIG, intravenous immunoglobulin; IBD, inflammatory bowel disease; CFRD, CF-related diabetes; ABPA, allergic bronchopulmonary aspergillosis; SA, Staphylococcus aureus ; PA, Pseudomonas aeruginosa . Statistical analyses were two-way ANOVA with the Šidák correction (B), Spearman correlation (C), and Mann–Whitney test (E) (ns, nonsignificant; *P < 0.05; ****P < 0.0001).

Article Snippet: Protein targets were: IFNλ1 (NBP2-34996; Novusbio), IFNλ2 (8417-IL; R&D Systems), IFNλ3 (5259-IL; R&D Systems), IFNα2 (NBP2-34971, Novusbio), and IFNβ (11420-1; PBL Assay Science).

Techniques: Neutralization, Labeling, Fluorescence, Infection, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Plasmacytoid dendritic cells have divergent effects on HIV infection of initial target cells and induce a pro-retention phenotype

doi: 10.1371/journal.ppat.1009522

Figure Lengend Snippet: (A) Normalised p24 expression in resting TEM cells infected with HIV and treated with neutralising antibody to ICAM-1 for 2 hours prior to addition of pDCs (n = 4). (B-C) The effect of virus depleted supernatants (from TEM-pDC cocultures or pDCs) in modulating p24 and CD69 expression in HIV-infected TEM cells (n = 6). (D) Normalised p24 expression and (E) percentage of CD69 expression in resting TEM cells (n = 5) upon modulation of IFN-signaling and/or TNF-signaling via: the addition of exogenous IFNα8 and/or TNFα; blocking the IFNAR1, IFNα and β and/or TNFR1 and TNFα via neutralizing antibodies (a-IFN and a-TNF). (F) Normalised p24 expression in resting TEM cells infected with HIV and treated with maraviroc for 2 hours prior to addition of pDCs (n = 4). (G) Detection of integrated HIV DNA by PCR in infected memory CD4 T cell subsets in the absence (HIV) and presence of pDCs (HIV+pDCs) (n = 4). (H-I) Detection of TEM DNA + cells via DNAScope in the absence (HIV) and presence of pDCs (HIV+pDCs) (n = 4). (J-K) Reversal of viral latency in J-Lat cells measured as increased HIV GFP expression upon co-culture with pDCs or after treatment with supernatants derived from TEM-pDCs. For all data, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by repeated measures ANOVA with Tukey post-hoc test (A-F) or paired two-sided t-test (G).

Article Snippet: These reagents included: an inhibitor of the intercellular adhesion molecule and component of the immunologic synapse (anti-ICAM-1, 10μg/mL, R&D systems, clone BBIG-I1); the CCR5 inhibitor maraviroc (10μM, AIDS Reagent Program); the reverse transcription inhibitor Zidovudine (50μM, AIDS Reagent Program); neutralising antibodies to receptors of IFN (IFNAR, 20μg/mL, PBL Assay Science, clone MMHAR-2) and TNF (TNFR1, 10μg/mL, R&D systems, clone 16803) in addition to neutralising antibodies for secreted IFNα8 (20000U/mL, PBL Assay Science), IFNβ (5000U/mL, PBL Assay Science) and TNFα (1μg/mL, R&D systems) to block signalling pathways of INF and TNF; recombinant (r) human TNFα (10ng/mL, R&D systems) and rIFNα8 (PBL Assay Science) was used at 350ng/mL for MDDCs and MDMs and at a graded concentration of 300, 600 and 900ng/mL for TCM, TTM and TEM based on the amount of IFNα detected by ELISA in infected MDDCs ( ) and T cell subsets ( ) upon their co-culture with pDCs.

Techniques: Expressing, Infection, Virus, Blocking Assay, Co-Culture Assay, Derivative Assay