Journal: PLoS ONE

Article Title: Immunogenicity, Protective Efficacy, and Non-Replicative Status of the HSV-2 Vaccine Candidate HSV529 in Mice and Guinea Pigs

doi: 10.1371/journal.pone.0121518

Figure Lengend Snippet: BALB/c mice (n = 10/group) were immunized with HSV529 (10 4 CCID 50 , 10 5 CCID 50 , or 10 6 CCID 50 ) or PBS by the i.m. route on days 0 and 21. Sera were collected on days 21 (D21; n = 10) and 41 (D41; n = 5). (A) HSV-2-specific IgG1 and IgG2a antibody titers in the sera were determined by ELISA using a lysate prepared from HSV-2-infected Vero cells and secondary antibodies specific for mouse IgG1 and IgG2a. (B) HSV-2 neutralizing antibodies in the sera were measured by preincubating dilutions of heat-inactivated sera with 100 CCID 50 of live HSV-2 (strain G) virus for 1 hour prior to infection of Vero cell cultures. Infected cells were detected with anti-HSV glycoprotein D antibodies. The serum dilution that neutralized 50% of the virus (SN 50 ) was determined by plotting the neutralization activity versus the serum dilutions. Splenic lymphocytes secreting IFNγ (C) or IL-5 (D) in response to ex vivo stimulation with heat-inactivated HSV-2 (strain G) were counted using an ELISPOT assay. Error bars represent standard error of the mean.

Article Snippet: The plates were washed and the locations where cells secreting INFγ or IL-5 during incubation were stained with a biotinylated anti-mouse IFNγ or IL-5 antibody (BD Pharmigen, Le Pont de Claix, France), a streptavidin-horseradish peroxidase conjugate (Southern Biotechnology), and a chromogenic substrate (amino ethyl carbazole; Sigma-Aldrich, St. Louis, MO).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Neutralization, Activity Assay, Ex Vivo, Enzyme-linked Immunospot

Journal: Journal of Neuroinflammation

Article Title: Murine astrocytes produce IL-24 and are susceptible to the immunosuppressive effects of this cytokine

doi: 10.1186/s12974-019-1444-1

Figure Lengend Snippet: IL-24 augments the expression of suppressive cytokine signaling components by murine astrocytes and limits inflammatory cytokine release by these cells. a Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 30 min and the presence of phosphorylated STAT3 and STAT1 was determined by immunoblot analysis. Expression of β-actin is shown as a loading control and these immunoblots are representative of two separate experiments. b Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 2 or 4 h, and SOCS3 mRNA expression was determined by semi-quantitative RT-PCR. Expression of the housekeeping gene product GAPDH is shown, and relative SOCS3 expression was determined by densitometric analysis and normalized to untreated cells. Data is expressed as the mean ± the SEM of three independent experiments, and an asterisk indicates a statistically significant difference from unchallenged cells at each time point ( p < 0.05). c Astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 8 h prior to immunoblot analysis for SOCS3 protein expression. Expression of the housekeeping gene β-actin is shown and relative SOCS3 protein expression was determined by densitometric analysis normalized to untreated cells. Asterisks indicate statistically significant differences from unchallenged cells (p < 0.05). d Astrocytes were untreated or treated with IL-24 (0.5, 3, 10, 30, or 100 ng/mL) for 4 h prior to challenge with bacterial LPS (5 ng/mL) or vehicle control for 12 h, and IL-6 secretion was determined by specific capture ELISA. Asterisks indicate a statistically significant difference ( p < 0.05) from similarly challenged cells in the absence of IL-24 ( n = 3). e Primary astrocytes were untreated or treated with recombinant IL-24 (10, 30, or 100 ng/mL) for 4 h prior to being uninfected or infected with Nm for 48 h before cell viability analysis via MTS assay. Data is presented as the mean absorbance ± SEM for three experiments. 0.1% Triton X-100 was used as a positive control, and an asterisk indicates a statistically significant difference from unchallenged cells in the absence of IL-24 ( p < 0.05)

Article Snippet: Commercially available Duoset® ELISA kits were used to measure IL-10 and TNF-α secretion (R&D Systems), while murine IL-6 secretion was measured using a rat anti-mouse IL-6 capture antibody (Clone MP5-20F3) and a biotinylated rat anti-mouse IL-6 detection antibody (Clone MP5-C2311) (BD Biosciences).

Techniques: Expressing, Recombinant, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Infection, MTS Assay, Positive Control

Journal:

Article Title: Toll-Like Receptor 9 Enhances Nephritogenic Immunity and Glomerular Leukocyte Recruitment, Exacerbating Experimental Crescentic Glomerulonephritis

doi: 10.2353/ajpath.2010.100153

Figure Lengend Snippet: Systemic immune responses to the nephritogenic antigen measured in WT and TLR9−/− mice, 21 and 7 days after the administration of CpG-ODN and sheep anti-mouse GBM globulin and control WT mice administered GpC and sheep anti-mouse GBM globulin (represented by the dotted line). On day 21, antigen-stimulated production of IFN-γ (A), IL-17A (C), and IL-6 (D) was increased in WT mice administered CpG-ODN and sheep anti-mouse GBM globulin compared with TLR9−/− mice. There was no change in IL-2 (B), IL-4 (E), or IL-10 (F) production between the groups and control WT mice administered GpC and sheep anti-mouse GBM globulin. Mean values obtained for controls and WT mice administered GpC and sheep anti-mouse GBM globulin are represented by the dotted line (mean and SEM values for WT mice treated with GPC and sheep anti-mouse GBM globulin were as follows: IFN-γ, 236.8 ± 78.9 pg/4 × 106cells; IL-2, 252.9 ± 23.2 pg/4 × 106cells; IL-17A, 24.2 ± 6.2 pg/4 × 106cells; IL-6, 22.8 ± 5.6 pg/4 × 106cells; IL-4, 59.3 ± 12.4 pg/4 × 106cells; and IL-10, 66.0 ± 13.6 pg/4 × 106cells). IFN-γ (G) and IL-17A (H) cytokine production were increased in WT compared with TLR9−/− mice 7 days after the administration of CpG-ODN and sheep anti-mouse GBM globulin. **P < 0.01; ***P < 0.001.

Article Snippet: For the IL-2 ELISA, rat anti-mouse IL-2 (JES6-1A12; DNAX) and biotinylated anti-mouse IL-2 (JES6-5H4; DNAX) were used.

Techniques: