Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) In uninfected skin, rare and faint nuclear HIF-1α staining occurred in the epidermis whereas the dermis was negative for HIF-1α (A1). (B to L) In all cutaneous infections, a strong nuclear HIF-1α signal was detected in keratinocytes, predominantly of the spinal cell layer (examplified in L). Moreover, endothelial cells of dermal capillaries frequently stained positive (arrows in B1, C1, D1, E1, G, I1). Arrowheads: (B2) border zone of the positive abscess; (C2): positive neutrophils; (F): positive dermal lympho-histiocytic infiltrates; (F1): large positive nuclei of a multi-nuclear giant cell; (F2): skin lesion surrounded by a HIF-1 positive inflammatory infiltrate; (H): positive sub-corneal neutrophils (higher power in H1); (I): positive intra-epidermal infiltrate; (K): intradermal nodules containing leishmaniae. (A) Control: uninfected sample; (B–E): bioptic samples of patients suffering from infections with humanpathogenic bacteria [(B) S. aureus , (C) S. agalactiae and S. aureus , (D) A. baumanii and E. coli and (E) B. burgdorferi ], (F, G) with humanpathogenic Herpes-viruses [(F) Varicella zoster virus (VZV) and (G) Human Herpes Virus-8 (HHV 8)], (H–J) with humanpathogenic fungi [(H+I) T. rubrum and (J) C. albicans ] or (K, L) with the protozoic pathogen L. donovani . Magnifications: all 250×, except for A1, B1, B2, C1, D1, E1, F2, I1 (all 400×), and C2, F1, H1, L (all 1,000×).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Staining, Control, Bacteria, Virus

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Four to six hours upon infection, HIF-1 activation was determined by Western blotting (loading control: actin). pO 2 levels were quantified in the medium of control and infected cells (A: S. aureus ATCC 33592, MOI 20, infection time: six hours; B: S. epidermidis ATCC 12228, MOI 20, infection time: six hours; C: E. coli ATCC 25992, MOI 10, infection time: four hours). Negative control: uninfected cells, positive control: hypoxia or DFO (200 µmol/L).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Four to six hours upon infection, HIF-1 activation was determined by Western blotting (loading control: actin). pO 2 levels were quantified in the medium of control and infected cells (D: P. aeruginosa ATCC 27853, MOI 10, infection time: four hours; E: S. agalactiae SK 43, MOI 200, infection time: five hours; F: C. albicans ATCC 90028, MOI 20, infection time: six hours). Negative control: uninfected cells, positive control: hypoxia or DFO (200 µmol/L).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: Six hours upon infection, HIF-1 activation was analyzed in (A) Western blots (loading control: actin) and by determining (B) the induction of the HIF-1-dependent luciferase activity. HeLa-229 cells were transfected with a 4xHRE-luc reporter construct 24 hours before infection (triplicate means given; for details see ). Induction was determined by chemiluminescence. Bacterial induced (C) VEGF and (D) HIF-1α transcription. Total mRNA was prepared six hours upon infection, transcribed into cDNA and quantified by real-time Light-Cycler-PCR (ratio: VEGF/actin or HIF-1α/actin transcripts; triplicate means given). Negative control: uninfected cells; S. aureus wt (8325-4); S. aureus Δ hemB ( S. aureus ermB Ω hemB 8325-4, SCV); S. aureus Δ hemB , hemB -complemented ( S. aureus pCX19Ω hemB 8325-4, complemented SCV mutant) ; positive control: DFO (200 µmol/L). Multiplicity of infection (MOI): 20. * significant difference to control cells (P<0.05).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Activation Assay, Western Blot, Control, Luciferase, Activity Assay, Transfection, Construct, Negative Control, Mutagenesis, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: The HIF-1α protein was analyzed in Western blots six hours upon infection (loading control: actin) of HeLa 229 cells with the following bacterial strains: (A) S. aureus 8325-4 (wt), S. aureus ermB Ω hemB 8325-4 (SCV), S. aureus SA113 (wt), S. aureus SA113 Δ ypf::ermB (reduced in lipoteichoic acid synthesis), S. aureus SA113 Δ tagO (defective in producing wall teichoic acid); (B) S. aureus ATCC 25923 (wt), S. epidermidis ATCC 12228 (wt), S. aureus Newman (wt), S. aureus Newman mAH12 (defective in producing the extracellular adherence protein EAP). Negative control: uninfected cells, positive control: DFO (200 µM). Multiplicity of infection (MOI): 20.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Western Blot, Infection, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: The HIF-1α protein was analyzed in Western blots six hours upon infection (loading control: actin). The following patient isolates were used: S. aureus patient isolate 1 (wt; S. aureus A22223 I), S. aureus patient isolate 2 (clinical SCV; S. aureus A22223 II), S. aureus patient isolate 3 (wt; S. aureus OM 1a), S. aureus patient isolate 4 (clinical SCV; S. aureus OM 1b). Negative control: uninfected cells, positive control: hypoxia. Multiplicity of infection (MOI): 20.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Western Blot, Infection, Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) Control and infected HeLa-229 cells were incubated with the hypoxic marker pimonidazole hydrochloride which was visualized by using specific Cy3 labelled antibodies (for details see ). Scale bare: 40 µm. (B) pO 2 levels were quantified in the medium of control and infected cells (triplicate means given). (C) Hela-229 cells were incubated in gas permeable (gas-perm) and conventional (conv) culture dishes and HIF 1α protein was analyzed in Western blots (loading control: actin). (D) pO 2 levels in the medium of control or S. aureus infected HeLa-229 cells (means of doubles). Negative control: uninfected cells; S. aureus wt (8325-4); S. aureus Δ hemB ( S. aureus ermB Ω hemB 8325-4, SCV); S. aureus Δ hemB , hemB -complemented ( S. aureus pCX19Ω hemB 8325-4, complemented SCV mutant). Multiplicity of infection (MOI): 20. * significant difference to control cells (P<0.05).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Control, Infection, Incubation, Marker, Western Blot, Negative Control, Mutagenesis

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) HIF 1α immunohistochemistry and (B) hematoxilin-eosin staining of renal mouse abscesses induced by intravenous infection with S. aureus seven days upon infection (following sections). HIF 1α positive nuclei are most abundant within the abscesses borders, some interstitial staining also occurs within the renal parenchyma. In addition, renal tubules in the vicinity of the abscess stain also positively (halfed arrow in B). A: arcuate artery, G: glomerulus, V: arcuate vein; arrowheads: margin of abscesses. Magnifications: (A) 250×, (B) 100×.

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Immunohistochemistry, Staining, Infection

Journal: PLoS ONE

Article Title: Activation of Hypoxia Inducible Factor 1 Is a General Phenomenon in Infections with Human Pathogens

doi: 10.1371/journal.pone.0011576

Figure Lengend Snippet: (A) Survival of NMRI mice after intraperitoneal infection with S. aureus (wt; S. aureus 8325-4; n = 8) or S. aureus Δ hemB (SCV, S. aureus erm Ω hemB 8325-4; n = 8). Note the higher susceptibility of mice infected with S. aureus 8325-4 compared to the mice infected with S. aureus Δ hemB . *significant difference: P<0.001. (B) Inhibition of DFO induced HIF-1 activation in HeLa-229 cells by 17-DMAG. Cells were incubated with 17 DMAG (10 µmol/L) for 16 hours following induction of HIF 1 activation by the iron chelator DFO (200 µmol/L) for six hours. HIF 1α protein was analyzed in Western blots (loading control: actin). Negative control: uninfected cells. (C) Survival rate of NMRI mice after intraperitoneal infection with S. aureus ( S. aureus 8325-4). One group of mice was treated 24 h and 16 h before infection with the HIF-1 inhibiting compound 17-DMAG (25 mg/g body weight). Note the higher survival rate of 17-DMAG-treated mice (n = 45) compared with control mice (n = 45). *significant difference: P = 0.005 (Kaplan-Meier analysis).

Article Snippet: Peritoneal HIF-1α activation was analyzed by immunoblotting of the shock-frozen peritoneal specimens using the Nuclear Extract Kit (Active Motif; Rixensart, Belgium), a mouse monoclonal anti-HIF-1α (H1α67) antibody (Novus Biologicals, Littleton, USA) and horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibodies (Dako).

Techniques: Infection, Inhibition, Activation Assay, Incubation, Western Blot, Control, Negative Control

Journal: Frontiers in Immunology

Article Title: Hyper-Enriched Anti-RSV Immunoglobulins Nasally Administered: A Promising Approach for Respiratory Syncytial Virus Prophylaxis

doi: 10.3389/fimmu.2021.683902

Figure Lengend Snippet: Hyper-enriched anti-RSV Ig characterization. (A, B) The biological activity of anti-RSV Ig products was measured by in vitro neutralization test assessed on HEp-2 cells and using recombinant RSV-cherry. (A) The antiviral activity of the hyper-enriched anti-RSV Ig solution was compared to the starting solution (IVIg) and the antibody control, Synagis ® . (B) The neutralizing capacity of two batches of hyper-enriched anti-RSV Ig was compared to Synagis ® . Data are representative of 2 independent assays. (C) To determine the ability of the anti-RSV Ig product to neutralize a clinical isolate of RSV, the neutralization assay was performed by flow cytometry on HEp-2 infected cells after immunodetection of RSV-F protein. Results were expressed in MFI, 100 arbitrarily being the MFI obtained with RSV-infected HEp-2 cells without incubation with anti-RSV antibodies. Data are representative of 2 independent assays.

Article Snippet: After washing in RPMI + 5% FCS, RSV-infected cells were determined by flow cytometry using rabbit anti RSV-F (Sino Biologicals, 11049-R09) and secondary anti-rabbit PE (Cell Lab, 732751) to determine expression of F protein at the HEp-2 cell surface.

Techniques: Activity Assay, In Vitro, Neutralization, Recombinant, Flow Cytometry, Infection, Immunodetection, Incubation