Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate Increases the Population of Non-Classical Monocytes in Asymptomatic Chagas Disease Patients and Modulates Inflammatory Cytokines in PBMC

doi: 10.3389/fcimb.2021.785166

Figure Lengend Snippet: HLA-DR expression in monocyte. The mean fluorescence intensity (MFI) of HLA-DR was determined in basal CD14pos cells after 20 h of T. cruzi lysate (Tc) stimulation or fenofibrate (Tc + Fen) treatment. Monocytes were selected based on FSC and SSC. After excluding doublets and debris, live cells were selected, monocytes were classified by CD14 positive staining. The mean fluorescence intensity (MFI) of HLA-DR was calculated both in total monocytes (A) . It shows the mean fluorescence intensity (MFI) of CD14pos/HLA-DRpos monocytes in healthy (HI) (B) , asymptomatic (Asy) (C) and chronic Chagas disease (CHD) patients (D) , where each patient is represented by a circle. The results are shown as the mean of the experiments ± SEM. These data were analyzed by fitting a mixed effect model with a Tukey post-hoc test.

Article Snippet: PBMC were stained with LIVE/DEADTM fixable dye (Invitrogen) at room temperature for 15 min and labeled with the following antibodies at 4°C for 30 min: CD14 (#E-AB-F1209C, Elabscience), CD16 (#E-AB-F1005M, Elabscience), HLA-DR (#E-AB-F1111H, Elabscience), and CCR2 (#357209, Elabscience).

Techniques: Expressing, Fluorescence, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Fenofibrate Increases the Population of Non-Classical Monocytes in Asymptomatic Chagas Disease Patients and Modulates Inflammatory Cytokines in PBMC

doi: 10.3389/fcimb.2021.785166

Figure Lengend Snippet: HLA-DR expression in T. cruzi stimulated and fenofibrate treated monocyte subpopulations. The mean fluorescence intensity percentage of HLA-DR + cells was determined in PBMC stimulated or not with T. cruzi lysate (Tc) and treated or not with fenofibrate (Tc + Fen) after 20 h, according to CD14 and CD16 expression. It shows the mean fluorescence intensity (MFI) of classical (CD14high/CD16neg) (A) , intermediate (CD14high/CD16pos) (B) and non-classical (CD14low/CD16pos) (C) monocytes with HLA-DR + expression. The results are shown as the mean of the experiments ± SEM. These data were analyzed by fitting a mixed effect model with a Tukey post-hoc test.

Article Snippet: PBMC were stained with LIVE/DEADTM fixable dye (Invitrogen) at room temperature for 15 min and labeled with the following antibodies at 4°C for 30 min: CD14 (#E-AB-F1209C, Elabscience), CD16 (#E-AB-F1005M, Elabscience), HLA-DR (#E-AB-F1111H, Elabscience), and CCR2 (#357209, Elabscience).

Techniques: Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

doi: 10.1371/journal.pone.0006811

Figure Lengend Snippet: (A) Cell surface loading. HLA-DR4 expressing dendritic cells (DC) generated from the bone marrow of HLA-DR4 transgenic mice were incubated for 4 h with medium alone (left panel) or with 5 µg/ml biotinylated HA 306–318 peptide in the absence (middle panel) or presence of 250 µM AdEtOH, the model MLE compound used throughout this study (right panel). Contour plots are shown for DC after staining with anti-HLA-DR antibody (→ MHC expression) and streptavidin (→ peptide load). Mean peptide loading (MFI of streptavidin signal) is indicated. (B) CD4+ T cell response. DC from HLA-DR4tg mice (left panel) and from HLA-DR1tg mice (right panel) were pulsed for 4 h with indicated amounts of HA 306–318 peptide in the absence (open circle) and presence (closed circle) of 250 µM AdEtOH. The cells were used to challenge HA 306–318 specific, HLA-DR4-restricted 8475/94 cells and HLA-DR1-restricted EvHA/X5 T cell hybridoma cells, respectively. Background proliferation was measured in absence of peptide (dashed line).

Article Snippet: For flow cytometry staining the following were used: α-CD4-PerCP-Cy5.5, α-CD11c-APC (both from BD PharMingen) α-CD154-PE, α-IFNγ-APC (both from Miltenyi), α-HLA-DR-PE (BD Bioscience), α-CD86-Biotin (produced in our laboratory, clone GL1), streptavidin-APC (Caltag).

Techniques: Expressing, Generated, Transgenic Assay, Incubation, Staining

Journal: PLoS ONE

Article Title: Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

doi: 10.1371/journal.pone.0006811

Figure Lengend Snippet: (A) Cell surface loading of MLE-susceptible and –non-susceptible MHC molecules. DC generated from non-susceptible BALB/c mice (left panel) and AdEtOH-susceptible HLA-DR1tg (right panel) were incubated for 4 h with the indicated amounts of biotinylated HA 306–318 peptide in the absence (open circles) or presence (closed circles) of 250 µM AdEtOH. Peptide loading was determined on CD11c+ cells by analyzing the mean fluorescence of the streptavidin-signal gated on a distinct expression of HLA-DR. Background fluorescence was detected in the absence of biotinylated peptide (dashed line). (B) Peptide priming of mice. BALB/c (upper panel) and DR1tg (lower panel) mice were s.c. primed with 100 µg and 3 µg HA 306–318, respectively in IFA/CpG supplemented without (left panel) or with AdEtOH (middle panel). Specific T cell response was determined by intracellular flow cytometry staining at day 12 after priming. Lymph node cells were incubated for 6 hrs in the presence of 10 µg HA 306–318 or 10 µg MOG 35–55 as irrelevant control peptide (right panel) and αCD28 antibody. 3 µg/ml Brefeldin A was added for the last 2 h. Intracellular IFNγ-production was analyzed on CD4+ CD154+ double positive T cells. Numbers indicate frequency of CD4+ CD154+ IFNγ+ cells among total CD4+ cells. Data representative of at least two independent experiments are shown.

Article Snippet: For flow cytometry staining the following were used: α-CD4-PerCP-Cy5.5, α-CD11c-APC (both from BD PharMingen) α-CD154-PE, α-IFNγ-APC (both from Miltenyi), α-HLA-DR-PE (BD Bioscience), α-CD86-Biotin (produced in our laboratory, clone GL1), streptavidin-APC (Caltag).

Techniques: Generated, Incubation, Fluorescence, Expressing, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

doi: 10.1371/journal.pone.0006811

Figure Lengend Snippet: (A) Cell surface loading of NY-ESO-1 epitopes. L929 fibroblasts transfected with HLA-DR1 (left panel) or HLA-DR4 (right panel) were incubated for 4 h with titrated amounts of NY-ESO-1 89–101 or NY-ESO-1 119–143, respectively. Loading was performed in the absence (closed circles) or presence (open circles) of 250 µM AdEtOH. Non-transfected L929 cells were used as a negative control (left side). Peptide loading was determined by analyzing the mean fluorescence of the streptavidin-signal gated on a distinct expression of HLA-DR. Background fluorescence was detected in the absence of biotinylated peptide (dashed line). (B) Detection of tumour-specific T cell response in vivo . Groups of HLA-DR1tg (left panel, n = 13) or HLA-DR4tg (right panel, n = 10) mice were s.c. primed with 5 µg of the respective NY-ESO-1 epitopes in IFA/CpG supplemented with or without AdEtOH. 12 days after vaccination, 1×10 6 Lymph node cells were incubated with titrated amounts of NY-ESO-1 89–101 or NY-ESO-1 119–143, respectively. IFNγ-detection was carried out 48 hrs later using an Elispot assay and summarized data were analyzed using student's t test.

Article Snippet: For flow cytometry staining the following were used: α-CD4-PerCP-Cy5.5, α-CD11c-APC (both from BD PharMingen) α-CD154-PE, α-IFNγ-APC (both from Miltenyi), α-HLA-DR-PE (BD Bioscience), α-CD86-Biotin (produced in our laboratory, clone GL1), streptavidin-APC (Caltag).

Techniques: Transfection, Incubation, Negative Control, Fluorescence, Expressing, In Vivo, Enzyme-linked Immunospot