Journal:

Article Title: Differential Early Interactions between Salmonella enterica Serovar Typhi and Two Other Pathogenic Salmonella Serovars with Intestinal Epithelial Cells

doi:

Figure Lengend Snippet: Cytokine gene expression by Int407 stimulated with S. typhi ISP1820, S. typhimurium TML, and S. dublin Lane. RNA was prepared from 2.6 × 106 uninfected Int407 cells or cells stimulated with S. typhi ISP1820, S. typhimurium TML, or S. dublin Lane at MOIs of approximately 20 bacteria per cell in a standard invasion assay. cDNA was prepared from 1 μg of each mRNA sample, and RT-PCR was performed with primers specific for human IL-6 and the housekeeping gene HPRT. Blots were hybridized with enhanced chemiluminescence-labeled oligonucleotide probes specific for the corresponding genes. Autoradiographs were scanned, and the fold increase of the IL-6-specific cytokine gene was calculated by comparison of the signals elicited by bacteria with those elicited by medium alone. Each lane of the autoradiograph represents the RT-PCR product from a single well. These results are representative of three separate experiments.

Article Snippet: Recombinant murine IL-6 was also purchased from R&D Systems.

Techniques: Expressing, Bacteria, Invasion Assay, Reverse Transcription Polymerase Chain Reaction, Labeling, Comparison, Autoradiography

Journal:

Article Title: Differential Early Interactions between Salmonella enterica Serovar Typhi and Two Other Pathogenic Salmonella Serovars with Intestinal Epithelial Cells

doi:

Figure Lengend Snippet: The effects of extending the incubation periods postinfection on invasion and IL-6 induction of Salmonella-infected Int407 cells. Monolayers of Int407 cells (2.5 × 105 cells per well of a 24-well tissue culture plate) were infected with an MOI of 20 S. typhi ISP1820 (squares), S. typhimurium TML (diamonds), or S. dublin Lane (circles) bacteria per cell and incubated for 90 min to allow the bacteria to adhere and invade. After removal of the extracellular bacteria by washing, the cultures were further incubated in the presence of gentamicin for an additional 90, 120, 180, or 240 min. At the end of the culture period, the supernatants were removed, and the concentration of IL-6 in each supernatant was determined by bioassay. The Int407 cells were lysed, and the number of bacteria surviving gentamicin treatment was determined. These results are representative of two independent experiments.

Article Snippet: Recombinant murine IL-6 was also purchased from R&D Systems.

Techniques: Incubation, Infection, Bacteria, Concentration Assay, Bioassay

Journal:

Article Title: Differential Early Interactions between Salmonella enterica Serovar Typhi and Two Other Pathogenic Salmonella Serovars with Intestinal Epithelial Cells

doi:

Figure Lengend Snippet: Effects of increasing the MOI on invasion and IL-6 induction of Salmonella-infected Int407 cells. Monolayers of Int407 cells (2.5 × 105 cells per well of a 24-well tissue culture plate) were infected with various doses of S. typhi ISP1820 (squares), S. typhimurium TML (circles), or S. dublin Lane (diamonds) and incubated for 90 min to allow the bacteria to adhere and invade. After removal of the extracellular bacteria by washing, the cultures were further incubated in the presence of gentamicin for an additional 90 min. At the end of the culture period, the supernatants were removed, and the concentration of IL-6 in each supernatant was determined by bioassay. The Int407 cells were lysed, and the number of bacteria surviving gentamicin treatment was determined. These results are representative of four independent experiments. The values plotted for S. typhi ISP1820 are taken from the work of Weinstein et al. (47).

Article Snippet: Recombinant murine IL-6 was also purchased from R&D Systems.

Techniques: Infection, Incubation, Bacteria, Concentration Assay, Bioassay

Journal:

Article Title: Differential Early Interactions between Salmonella enterica Serovar Typhi and Two Other Pathogenic Salmonella Serovars with Intestinal Epithelial Cells

doi:

Figure Lengend Snippet: Effects of invA and invE mutations on the adherence, invasion, and IL-6 induction of S. typhi ISP1820 and S. typhimurium TML on Int407 cells. Monolayers of Int407 cells (2.5 × 105 cells per well of a 24-well tissue culture plate) were overlaid with medium (uninfected control) or infected at a bacterium-to-cell ratio of 20:1 with S. typhi ISP1820, S. typhi SB130 (ISP1820 invA), S. typhi H553 (ISP1820 invE), S. typhimurium SR-11, S. typhimurium SB147 (SR-11 invA), or S. typhimurium SB109 (SR-11 invE) and incubated for 90 min to allow the bacteria to adhere and invade. Cultures were washed and incubated in the presence of gentamicin for an additional 90 min to eliminate extracellular bacteria. At the end of the culture period, the supernatants were removed, and the concentration of IL-6 in each supernatant was determined by B9 bioassay. The IECs were lysed, and the number of viable intracellular bacteria was determined. These results are representative of two independent experiments. Top and bottom bars for each strain represent percent cell association and percent invasion, respectively.

Article Snippet: Recombinant murine IL-6 was also purchased from R&D Systems.

Techniques: Control, Infection, Incubation, Bacteria, Concentration Assay, Bioassay

Journal: PLoS ONE

Article Title: Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis

doi: 10.1371/journal.pone.0028513

Figure Lengend Snippet: DiO-labeled HL-1 cells were cultured in a 6-well plate and infected 24 h later with CVB3 at an m.o.i. of 5 for 1 h. Four hours afterwards, CAPs were added at a ratio of 1 to 10 HL-1 cells. After 24 hours, cells were collected for Annexin V/7AAD FACS analysis. A. Panel shows representative pictures of uninfected HL-1, infected HL-1, uninfected HL-1 co-cultured with CAPs and infected HL-1 co-cultured with CAPs. B. Panel demonstrates representative pictures of Annexin V/7AAD dot plots on preselected DiO+ HL-1 cells. Bar graphs represent DiO+ Annexin V+/7AAD- HL-1 cells in cultures of uninfected (open bars) or CVB3-infected (closed bars) HL-1 with or without untreated or L-NAME treated CAPs or CAPs in the presence or absence of 1 µg/ml of anti-human IL-10 antibody (ab), or 1 µg/ml of anti-murine IFN–γ ab; n = 4/group. C. Representative pictures of TUNEL-stained heart sections (from left to right) of control and CVB3-infected mice receiving PBS (control+PBS and CVB3+PBS, respectively) and of control or CVB3-infected mice receiving CAPs (control+CAPs and CVB3+CAPs, respectively), at 200× magnification. D. Bar graphs represent the mean ± SEM of caspase 3/7 activity in LV homogenates of control mice (open bars) and CVB3-infected mice (closed bars) injected with PBS or CAPs; n = 5–7/group.

Article Snippet: Four hours later, recombinant murine IFN-γ (R&D Systems) was added at a final concentration of 4 pg/ml, corresponding to the concentration of murine IFN-γ present in medium of HL-1 cells and HL-1/CAPs co-cultures (data not shown).

Techniques: Labeling, Cell Culture, Infection, TUNEL Assay, Staining, Activity Assay, Injection

Journal: PLoS ONE

Article Title: Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis

doi: 10.1371/journal.pone.0028513

Figure Lengend Snippet: HeLa cells were incubated for 30 minutes with 1 ml of diluted supernatant of CVB3-infected HL-1 cells or HL-1 cells co-cultured with CAPs, in the presence or absence of L-NAME, anti-human IL-10 antibody, or anti-murine IFN-γ antibody, or with diluted LV homogenates from CVB3 or CVB3+CAPs mice. Then, cells were washed with PBS and covered with agar consisting of 50% 1.3% noble agar and 50% 2× MEM, supplemented with 4% FBS. A. Bar graphs represent the mean ± SEM of plaques counted in 6 wells/condition, 72 h after incubation with diluted medium (all at dilution 10 −4 ) of CVB3-infected HL-1 cells or HL-1 cells co-cultured with CAPs, in the presence or absence of L-NAME, 1 µg/ml of anti-human IL-10 antibody (Ab), or 1 µg/ml of anti-murine IFN-γ Ab. B. Bar graphs represent the mean ± SEM of plaques counted in 12 wells/group (3 wells/mouse and n = 4 mice/group), 72 h after incubation with diluted medium (all at dilution 10 −4 ) of CVB3+PBS or CVB3+CAPs mice as indicated.

Article Snippet: Four hours later, recombinant murine IFN-γ (R&D Systems) was added at a final concentration of 4 pg/ml, corresponding to the concentration of murine IFN-γ present in medium of HL-1 cells and HL-1/CAPs co-cultures (data not shown).

Techniques: Incubation, Infection, Cell Culture

Journal: PLoS ONE

Article Title: Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis

doi: 10.1371/journal.pone.0028513

Figure Lengend Snippet: A. Cardiac MNCs were isolated from control mice (open bars) and CVB3-infected mice (closed bars) injected with PBS or CAPs. Next, cardiac MNCs were labeled with 10 µM of carboxyfluorescein succinimidyl ester to be able to measure cell proliferation, and stimulated with phorbol myristate acetate (PMA) and ionomycin at a final concentration of 50 ng/ml and 500 ng/ml, respectively, for 72 h followed by flow cytometry and analysis with FlowJo 8.7. software. Bar graphs represent the division index of cardiac MNCs with n = 4/group. B. Representative hematoxylin and eosin stained heart sections of control mice receiving PBS (control+PBS; upper left panel) or CAPs (control+CAPs; lower left panel), or of CVB3-infected mice receiving PBS (CVB3+PBS; upper right panel) or CAPs (CVB3+CAPs; lower right panel), at 200× magnification. MNCs were isolated from the spleen of control mice (open bar graph) or CVB3-infected (closed bar graph) mice. Next, carboxyfluorescein succinimidyl ester-labeled MNCs were directly stimulated with PMA and ionomycin and cultured with or without CAPs (untreated or 24 h pre-treated with L-NAME), in the presence or absence of 1 µg/ml of anti-human IL-10 or anti-murine IFN-γ antibody for 72 h. Then, cells were stained with monoclonal anti-CD4 or anti-CD8 antibodies, followed by flow cytometry and analysis with FlowJo 8.7. software. Bar graphs represent the division index of C. CD4+ (upper panel) and of D. CD8+ T cells (lower panel) with n = 4/group.

Article Snippet: Four hours later, recombinant murine IFN-γ (R&D Systems) was added at a final concentration of 4 pg/ml, corresponding to the concentration of murine IFN-γ present in medium of HL-1 cells and HL-1/CAPs co-cultures (data not shown).

Techniques: Isolation, Infection, Injection, Labeling, Concentration Assay, Flow Cytometry, Software, Staining, Cell Culture

Journal: PLoS ONE

Article Title: Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis

doi: 10.1371/journal.pone.0028513

Figure Lengend Snippet: Bar graphs represent the mean ± SEM of LV A. IL-10 and B. IFN-γ mRNA expression of control mice (open bars) and CVB3-infected mice (closed bars) injected with PBS or CAPs, as indicated, with n = 8/group.

Article Snippet: Four hours later, recombinant murine IFN-γ (R&D Systems) was added at a final concentration of 4 pg/ml, corresponding to the concentration of murine IFN-γ present in medium of HL-1 cells and HL-1/CAPs co-cultures (data not shown).

Techniques: Expressing, Infection, Injection

Journal: PLoS ONE

Article Title: Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis

doi: 10.1371/journal.pone.0028513

Figure Lengend Snippet: Bar graphs represent the mean ± SEM of A. NO (µM) or B. IL-10 (pg/ml) production in non-infected or CVB3-infected CAPs with or without supplementation of 4 pg/ml of recombinant murine IFN-γ, with n = 6/condition.

Article Snippet: Four hours later, recombinant murine IFN-γ (R&D Systems) was added at a final concentration of 4 pg/ml, corresponding to the concentration of murine IFN-γ present in medium of HL-1 cells and HL-1/CAPs co-cultures (data not shown).

Techniques: Infection, Recombinant