Journal: Vaccines

Article Title: Assessment of Different Infectious Bovine Rhinotracheitis Marker Vaccines in Calves

doi: 10.3390/vaccines10081204

Figure Lengend Snippet: Antibody response of calves immunized with different BoHV-1 marker vaccines, challenge infected with virulent strain of BoHV-1, and treated with dexamethasone (DMS).

Article Snippet: Twenty-eight PCDs, the calves were subjected to dexamethasone (DMS, Dexadreson ® , MSD Animal Health S.r.l., Milan, Italy) treatment.

Techniques: Marker, Vaccines, Infection

Journal: International Journal of Molecular Sciences

Article Title: miR-132-3p Modulates DUSP9-Dependent p38/JNK Signaling Pathways to Enhance Inflammation in the Amnion Leading to Labor

doi: 10.3390/ijms23031864

Figure Lengend Snippet: The role of p38 and JNK in DUSP9 siRNA-induced expression of proinflammatory cytokines and COX2 as well as PGE2 in WISH cells. WISH cells were transfected with si- DUSP9 or NC for 24 h, followed by 24 h of treatment with DMSO, p38 inhibitor SB203580 (10 μM), or JNK inhibitor SP600125 (20 μM). RT-qPCR analysis of the expression of IL-1β , TNF-α , COX2 ( A , G ), IL-6 , and IL-8 ( B , H ). Magnetic Luminex Assays of the secretion of IL-1β, TNF-α ( C , I ), IL-6, and IL-8 ( D , J ). Western blot analysis of COX2 expression ( E , K ). ELISA analysis of PGE2 level ( F , L ). Data were shown as mean ± SEM, n = 3. * p < 0.05, ** p < 0.01 vs. control (0), # p < 0.05, ## p < 0.01 vs. si- DUSP9 .

Article Snippet: To verify the involvement of p38 and JNK signaling pathways in the induction of IL-1β, IL-6, IL-8, TNF-α, and COX2 as well as PGE2 secretion by DUSP9 siRNA, the cells were transfected with NC or si- DUSP9 for 24 h, followed by 24 h of treatment with dimethyl sulfoxide (DMSO), p38 inhibitor SB203580 (10 μM, Selleck, Houston, TX, USA), or JNK inhibitor SP600125 (20 μM, Selleck, Houston, TX, USA).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Luminex, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: PLoS ONE

Article Title: Target Of Rapamycin pathway in the white-rot fungus Phanerochaete chrysosporium

doi: 10.1371/journal.pone.0224776

Figure Lengend Snippet: A. Growth curve measured after germination of conidia from P . chrysosporium RP78 in liquid medium using nephelometric measurement with different concentrations of rapamycin. Those curves represent means ± SD of n = 3 replicates. B. Effect of rapamycin on hyphae growth in solid medium (agar plate). Rapamycin dissolved in DMSO was loaded on Whatman filter paper dots, DMSO was used as control, and both were used as in a disk diffusion test. Movie of the corresponding experiment is provided in .

Article Snippet: For each microplate well, 200 μl of sample were prepared: 10,000 spores were resuspended in 198 μL of malt 1% and 2 μL of rapamycin (LC laboratories, R-5000) were added for treatment or 2 μl of DMSO as mock for control.

Techniques: Control, Diffusion-based Assay

Journal: Viruses

Article Title: Dendritic Cells Pulsed with HAM/TSP Exosomes Sensitize CD4 T Cells to Enhance HTLV-1 Infection, Induce Helper T-Cell Polarization, and Decrease Cytotoxic T-Cell Response

doi: 10.3390/v16091443

Figure Lengend Snippet: Exosomes activate and elicit proinflammatory responses in dendritic cells. ( A ) Representative phenotype of myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC) population differences with exosome stimulation ( n = 4). GMFI levels of CD40, CD80, and CD86 in mDC and pDC populations untreated or stimulated with exosomes. Bars represent mean ± standard deviation (SD). ( B ) Expression of cytokines associated with Th1, Th2, Th17, and Treg polarization in total DCs stimulated with OSP2 cell-derived exosomes or LPS. Cytokines were grouped according to associated Th1 (IFN-γ, TNF-α, and IL-2), Th2 (IL-4, IL-12a, and IL-13), Th17 (IL-6 and IL-17a), and Treg (IL-10 and TGF-β) subsets. ( C ) Cytokine levels (pg/mL) in supernatants of mDCs ( left ) and pDCs ( right ) untreated or exosome-stimulated. Statistical differences were determined by one-way ANOVA, * p < 0.05, ** p < 0.005.

Article Snippet: Purified exosomes from OSP2 cells (without or with inhibitor treatment, Manumycin A (BioViotica, Liestal, Switzerland; 250 nM in DMSO) or from patient sera were mixed (1:1 (~20 μg)) with positively selected (StemCell, Seattle, WA, USA) CD3+ T cells activated with a CD3/CD28 activation cocktail (1×, BioLegend, San Diego, CA, USA) from healthy donors (HumanCellsBio, Milpitas, CA, USA) for 24 h. Where indicated, exosomes were pre-incubated with an ICP blockade at 1:100 dilution (BTLA (Cat. 7473, ProSci, Poway, CA, USA); PD-L2 (Cat. 4063, ProSci); LAG-3 (Cat. 13485, Raybiotech, Peachtree Corners, GA, USA)).

Techniques: Standard Deviation, Expressing, Derivative Assay

Journal: Viruses

Article Title: Dendritic Cells Pulsed with HAM/TSP Exosomes Sensitize CD4 T Cells to Enhance HTLV-1 Infection, Induce Helper T-Cell Polarization, and Decrease Cytotoxic T-Cell Response

doi: 10.3390/v16091443

Figure Lengend Snippet: Dendritic cell-pulsed exosomes polarize T cells. ( A ) Exosome stimulation schematic and representative gating strategy. Donor CD3+ T cells and matched total DCs were isolated ( n = 4). ( B ) DCs were exposed to OSP2-derived exosomes for 24 h and subsequently co-incubated with donor-matched T cells for another 24 h. Absolute count of CD4+ T-cells expressing subtype-associated markers: Th1 (IFN-γ, CCR5), Th2 (IL-4, CCR4), Th17 (IL-17, CCR6), and Treg (CD25, FoxP3). Quantification (pg/mL) of IFN-γ, TGF-β, and TNF-α cytokine levels in exosome-stimulated DC–T-cell co-culture ( n = 3). ( C ) Representative flow plots and quantification ( n = 4) of percent positive CD4+ T cells expressing functional markers of IFN-γ, IL-4, IL-17a, CD25, CCR5, and CCR6 after stimulation with OSP2 cell-derived exosomes. Bars represent mean ± standard deviation (SD). Statistical differences were determined by one-way ANOVA, * p < 0.05, ** p < 0.005, *** p < 0.001.

Article Snippet: Purified exosomes from OSP2 cells (without or with inhibitor treatment, Manumycin A (BioViotica, Liestal, Switzerland; 250 nM in DMSO) or from patient sera were mixed (1:1 (~20 μg)) with positively selected (StemCell, Seattle, WA, USA) CD3+ T cells activated with a CD3/CD28 activation cocktail (1×, BioLegend, San Diego, CA, USA) from healthy donors (HumanCellsBio, Milpitas, CA, USA) for 24 h. Where indicated, exosomes were pre-incubated with an ICP blockade at 1:100 dilution (BTLA (Cat. 7473, ProSci, Poway, CA, USA); PD-L2 (Cat. 4063, ProSci); LAG-3 (Cat. 13485, Raybiotech, Peachtree Corners, GA, USA)).

Techniques: Isolation, Derivative Assay, Incubation, Expressing, Co-Culture Assay, Functional Assay, Standard Deviation

Journal: Viruses

Article Title: Dendritic Cells Pulsed with HAM/TSP Exosomes Sensitize CD4 T Cells to Enhance HTLV-1 Infection, Induce Helper T-Cell Polarization, and Decrease Cytotoxic T-Cell Response

doi: 10.3390/v16091443

Figure Lengend Snippet: Exosomes diminish CD8+ T-cell activity. ( A ) Schematic of treatment and gating strategy of CD8+ T cells. CD3+ T cells and total DCs were isolated from matched donors ( n = 4). DCs were stimulated with OSP2-derived exosomes for 24 h and subsequently co-incubated with donor-matched T cells for another 24 h. Absolute counts of CD8+ T cells expressing MIP-1a, Granzyme B, Perforin, IFN-γ, PD-1, and Ki67 after co-incubation with exosome-pulsed DCs. ( B ) Absolute counts of CD8+ T cells after CD3+/CD28+ activation and stimulation with OSP2 exosomes alone, or exosomes incubated with a cocktail of anti-PD-L2, anti-BTLA, anti-LAG-3, and anti-PD-1 blocking antibodies. ( C ) Absolute counts of CD8+ T cells expressing MIP-1a, Granzyme B, Perforin, IFN-γ, PD-1, and Ki67 in HTLV-1-infected patient T cells ( n = 4) stimulated with patient sera exosomes alone or exosomes incubated with a cocktail of anti-PD-L2, anti-BTLA, anti-LAG-3, and anti-PD-1 blocking antibodies. Counts were determined by experiments with n = 3/4 healthy donors, and error bars represent SEM of donor variation. Bars represent mean ± standard deviation (SD). Statistical differences were determined by one-way ANOVA. ( D ) Western blot and densitometry of CD3/CD28-stimulated T cells treated with Jurkat and OSP2 exosomes alone, or exosomes incubated with a cocktail of anti-PD-L2, anti-BTLA, anti-LAG-3, and anti-PD-1 blocking antibodies. Blot was probed for phosphorylated AKT or ERK. Error bars represent SEM of blot variation; statistical differences were determined by paired two-tailed T-test, * p < 0.05.

Article Snippet: Purified exosomes from OSP2 cells (without or with inhibitor treatment, Manumycin A (BioViotica, Liestal, Switzerland; 250 nM in DMSO) or from patient sera were mixed (1:1 (~20 μg)) with positively selected (StemCell, Seattle, WA, USA) CD3+ T cells activated with a CD3/CD28 activation cocktail (1×, BioLegend, San Diego, CA, USA) from healthy donors (HumanCellsBio, Milpitas, CA, USA) for 24 h. Where indicated, exosomes were pre-incubated with an ICP blockade at 1:100 dilution (BTLA (Cat. 7473, ProSci, Poway, CA, USA); PD-L2 (Cat. 4063, ProSci); LAG-3 (Cat. 13485, Raybiotech, Peachtree Corners, GA, USA)).

Techniques: Activity Assay, Isolation, Derivative Assay, Incubation, Expressing, Activation Assay, Blocking Assay, Infection, Standard Deviation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting the translational machinery in gastrointestinal stromal tumors (GIST) – a new therapeutic vulnerability

doi: 10.1101/2021.09.01.458633

Figure Lengend Snippet: Homoharringtonine inhibits nascent protein synthesis and reduces KIT protein expression in GIST cells. (A) Nascent protein synthesis of GIST cells treated with homoharringtonine (HHT, 0.1 μM; green), cycloheximide (CHX, 100 μg/ml; blue), KIT inhibitor (IM, 1μM for GIST882 and GIST-T1; sunitinib, SU, 1μM for GIST430 and GIST48; orange) or 0.1% DMSO control (red) for 1 h or 8 h. Cells were labelled with HPG during the last 30 min of drug treatment and incorporated cellular HPG linked to azide-modified Alexa488 was quantitated by flow cytometry. Unstained control cells are shown in black in the histogram. A change in nascent protein synthesis is indicated as a relative mean fluorescence value to the DMSO control in the bar graphs. Columns, mean + SE; *, p<0.05 in comparison to DMSO control; **, p<0.01 in comparison to control; ***, p<0.001 in comparison to control (Student’s t-test, 2-tailed). (B) Immunoblot analysis for KIT protein expression of imatinib (IM)-sensitive (GIST882, GIST-T1) and IM-resistant (GIST430, GIST48) GIST cells after treatment with the protein translation inhibitor cycloheximide (CHX; 30 μg/ml for 3 h). (C) Immunoblotting for KIT protein expression in IM-sensitive (GIST882, GIST-T1) and IM-resistant (GIST430, GIST48) GIST cells after treatment with HHT for 72 h at the indicated concentrations. Abbreviations: 882 (GIST882), T1 (GIST-T1), 430 (GIST430), 48 (GIST48). (B, C) Grouped immunoblot images are either cropped from different parts of the same gel or from a separate gel run with another aliquot of the same protein lysate

Article Snippet: Homoharringtonine (HHT; Santa Cruz) treatments were performed at the indicated concentrations (in DMSO) compared to 0.1% DMSO for up to 72 h. Treatment with imatinib mesylate (1 µM in DMSO; LC Laboratories) or sunitinib (1 µM in DMSO; LC Laboratories) served as control.

Techniques: Expressing, Modification, Flow Cytometry, Fluorescence, Western Blot