Journal: Infection and Immunity

Article Title: Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44

doi: 10.1128/iai.68.6.3394-3402.2000

Figure Lengend Snippet: FIG. 1. Cytokine mRNA expression in human PBLs exposed to the HGE agent or rP44. (A) Cytokine mRNA expression in human PBLs exposed to the HGE agent (100 bacteria/cell), rP44 (1 mg/ml), or E. coli LPS (1 mg/ml) for 2 h. Total RNA was extracted and subjected to RT-PCR. The cDNAs, in quantities normalized against G3PDH mRNA levels in corresponding samples, were amplified for 25 cycles (20 cycles for IL-8 mRNA), and PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes. The data presented (donor no. 2 in Table 1) are representative of six donors (Table 1) who had similar results for IL-1b, TNF-a, IL-6, and LPS. (B) Linearity of RT-PCR. Different amounts of cDNA from human PBLs (107 cells) incubated with E. coli LPS (1 mg/ml) for 2 h were amplified for 25 cycles. The PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes (M). The data presented (donor no. 1 in Table 1) are representative of two independent experiments (donor no. 1 and 2) that gave similar results. The lower panel shows a plot of the relative band densities of the PCR products, recorded by a gel video system and analyzed by an image analysis software, against the amounts of cDNA present in the PCR.

Article Snippet: IL-1b, TNF-a, and IL-6 levels were measured by using human cytokine immunoassay kits (Quantikine; R&D Systems, Minneapolis, Minn.) according to the manufacturer’s protocol.

Techniques: Expressing, Bacteria, Reverse Transcription Polymerase Chain Reaction, Incubation, Software

Journal: Infection and Immunity

Article Title: Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44

doi: 10.1128/iai.68.6.3394-3402.2000

Figure Lengend Snippet: FIG. 2. Dose-dependent induction of IL-1b, TNF-a, and IL-6 mRNA expression in human PBLs. (A) PBLs were incubated for 2 h with different amounts of the HGE agent. Total RNA was extracted and subjected to the competitive RT-PCR. The PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes (M). (B) Relative amounts of cytokine mRNAs expressed in human PBLs in response to the HGE agent. Band densities were recorded by a gel video system and analyzed by an image analysis software, and ratios of target to MIMIC PCR products were plotted against the estimated HGE agent numbers per cell. The amounts of cDNAs were normalized against G3PDH mRNA levels in corresponding samples. The data presented (donor no. 2 in Table 1) are representative of two independent experiments (donor no. 2 and 3 in Table 1) that gave similar results.

Article Snippet: IL-1b, TNF-a, and IL-6 levels were measured by using human cytokine immunoassay kits (Quantikine; R&D Systems, Minneapolis, Minn.) according to the manufacturer’s protocol.

Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Software

Journal: Infection and Immunity

Article Title: Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44

doi: 10.1128/iai.68.6.3394-3402.2000

Figure Lengend Snippet: FIG. 3. Time course analysis of IL-1b, TNF-a, and IL-6 mRNA expression in human PBLs in response to the HGE agent (100 bacteria/cell), rP44 (1 mg/ml), or E. coli LPS (1 mg/ml). (A) Human PBLs were incubated for the indicated time periods. Total RNA was extracted and subjected to the competitive RT-PCR. The PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes. (B) Relative amounts of cytokine mRNAs expressed. Band densities were recorded by a gel video system and analyzed by an image analysis software, and the ratios of target to MIMIC PCR products were plotted against the incubation time. The amounts of cDNAs were normalized against G3PDH mRNA levels in corresponding samples. The data presented (donor no. 3 in Table 1) are representative of two independent experiments (donor no. 2 and 3) that gave similar results.

Article Snippet: IL-1b, TNF-a, and IL-6 levels were measured by using human cytokine immunoassay kits (Quantikine; R&D Systems, Minneapolis, Minn.) according to the manufacturer’s protocol.

Techniques: Expressing, Bacteria, Incubation, Reverse Transcription Polymerase Chain Reaction, Software

Journal: Infection and Immunity

Article Title: Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44

doi: 10.1128/iai.68.6.3394-3402.2000

Figure Lengend Snippet: FIG. 4. Examination of rP44 preparation for contamination of endotoxin and other cytokine-inducing components. rP44 preparation was incubated with a nitrocellulose membrane coated with a MAb against P44 (5C11) (1) or an uncoated membrane (2). The solutions were then added to PBLs and incubated at 37°C for 2 h, and TNF-a mRNA was examined by RT-PCR. The PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes. The data presented (donor no. 5 in Table 1) are representative of two independent ex- periments (donor no. 5 and 6) that gave similar results.

Article Snippet: IL-1b, TNF-a, and IL-6 levels were measured by using human cytokine immunoassay kits (Quantikine; R&D Systems, Minneapolis, Minn.) according to the manufacturer’s protocol.

Techniques: Incubation, Membrane, Reverse Transcription Polymerase Chain Reaction

Journal: Infection and Immunity

Article Title: Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44

doi: 10.1128/iai.68.6.3394-3402.2000

Figure Lengend Snippet: FIG. 5. Influences of various treatments on expression of IL-1b, TNF-a, and IL-6 mRNAs in human PBLs exposed to the HGE agent (100 bacteria/cell), rP44 (1 mg/ml), or E. coli LPS (1 mg/ml). To examine which components of the HGE agent are responsible for expression of the three proinflammatory-cytokine mRNAs, PBLs (107 cells) were incubated for 2 h with HGE agent that had been subjected to different treatments as described in Materials and Methods. Total RNA was extracted and subjected to RT-PCR. The amounts of cDNAs used were normalized against G3PDH mRNA levels in corresponding samples. The PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes. The data presented (donor no. 4 in Table 1) are representative of three independent experiments (donor no. 3 to 5) that gave similar results.

Article Snippet: IL-1b, TNF-a, and IL-6 levels were measured by using human cytokine immunoassay kits (Quantikine; R&D Systems, Minneapolis, Minn.) according to the manufacturer’s protocol.

Techniques: Expressing, Bacteria, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Infection and Immunity

Article Title: Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44

doi: 10.1128/iai.68.6.3394-3402.2000

Figure Lengend Snippet: FIG. 6. Cytokine mRNA expression in human neutrophils and monocytes. (A) Human neutrophils purified (.95%) by Ficoll-Paque and Percoll gradient centrifugation were used for IL-1b, TNF-a, and IL-6 mRNA expression. Neutrophils (107 cells) were incubated for 2 h with the HGE agent (100 bacteria/cell), rP44 (1 mg/ml), or E. coli LPS (1 mg/ml). The data presented (donor ID 4 in Table 1) are representative of two independent experiments (donor no. 4 and 5) that gave similar results. (B) Purified (.95%) monocytes (107 cells) were incubated for 2 h under the stimulation conditions used for the neutrophils. Total RNA was extracted and subjected to RT-PCR. The amounts of cDNAs used were normalized against G3PDH mRNA levels in corresponding samples. The PCR products were resolved on agarose gels containing EtBr. DNA size markers (HaeIII fragments of fX174 RF DNA) were run in the leftmost lanes. The data presented (donor ID 5 in Table 1) are representative of two independent experiments (donor no. 5 and 6) that gave similar results.

Article Snippet: IL-1b, TNF-a, and IL-6 levels were measured by using human cytokine immunoassay kits (Quantikine; R&D Systems, Minneapolis, Minn.) according to the manufacturer’s protocol.

Techniques: Expressing, Gradient Centrifugation, Incubation, Bacteria, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Transplantation

Article Title: Comparing Scaffold-Free and Fibrin-Based Adipose-Derived Stromal Cell Constructs for Adipose Tissue Engineering: An In Vitro and in Vivo Study

doi: 10.3727/096368912x653129

Figure Lengend Snippet: Figure 4. ASCs in fibrin-based constructs secrete higher levels of the angiogenic factors VEGF and HGF than ASC in spheroids. The secretion of the angiogenic factors fibroblast growth factor 2 (FGF2), angiopoietin (ANG2), platelet-derived growth factor (PDGF), tissue inhibitors of matrix metalloproteinases 2 (TIMP2), TIMP1, hepatocyte growth factor (HGF), and vascular endothe- lial growth factor (VEGF) by ASCs in spheroids and fibrin-based constructs over 48 h was measured by multiplex chemiluminescent ELISA at day 7. Unconditioned adipogenic medium was used as control condition. Values are presented as median (interquartile range); n = 5, 3 ASC donors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (fibrin-based construct conditioned medium vs. spheroid conditioned medium and control).

Article Snippet: A human cytokine inflammation multiplex chemiluminescent ELISA kit (Cat. No. 110451HU, Quansys Biosciences, West Logan, UT, USA) and a human cytokine inflammation multiplex chemiluminescent ELISA kit (cat. no.150251HU; Quansys Biosciences) were used to assay conditioned and unconditioned medium for the release of the following inflammatory cytokines and angiogenic factors: interleukin-1a (IL-1a), IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor-a (TNF-a), interferon-g (IFN-g), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), angiopoietin 2 (ANG2), fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and the tissue inhibitors of metalloproteinases (TIMP): TIMP1, and TIMP2.

Techniques: Construct, Derivative Assay, Multiplex Assay, Chemiluminescent ELISA, Control

Journal: Cell Transplantation

Article Title: Comparing Scaffold-Free and Fibrin-Based Adipose-Derived Stromal Cell Constructs for Adipose Tissue Engineering: An In Vitro and in Vivo Study

doi: 10.3727/096368912x653129

Figure Lengend Snippet: Figure 6. ASCs in fibrin-based constructs display increased adipogenic differentiation when compared to ASCs in spheroids. (A) ASCs in spheroids and fibrin-based constructs were cultured for 7 days in vitro in adipogenic medium. Cross-sections were stained with Oil Red O (showing intracellular lipid) and counterstained with haematoxylin. (A.1) Spheroid. (A.2) Fibrin-based construct. Scale bars: 100 µm. (B) Q-PCR was used to measure the expression levels of the adipocyte-specific markers peroxisome proliferator-activated receptor g (PPARG), perilipin 1 (PLIN 1), fatty acid binding protein 4 (FABP4), and leptin (LEP). Expression levels are relative to b-2- microglobulin-positive control housekeeping gene (dCt). Values are presented as medians (interquartile range). For each of the three ASC donors, nine spheroids, nine fibrin-based constructs, and 0.5 ´ 106 undifferentiated ASCs (=control) were prepared and assayed. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 (relative expression in fibrin-based constructs compared to spheroids and control). (C) The secre- tion of leptin by ASCs in conditioned medium of spheroids and fibrin-based constructs over 48 h was measured by sandwich ELISA at day 7. Unconditioned adipogenic medium was used as control condition. Values are presented as median (interquartile range); n = 9, 3 ASC donors. **p ≤ 0.01, ***p ≤ 0.001 (fibrin-based construct conditioned medium vs. spheroid conditioned medium and control).

Article Snippet: A human cytokine inflammation multiplex chemiluminescent ELISA kit (Cat. No. 110451HU, Quansys Biosciences, West Logan, UT, USA) and a human cytokine inflammation multiplex chemiluminescent ELISA kit (cat. no.150251HU; Quansys Biosciences) were used to assay conditioned and unconditioned medium for the release of the following inflammatory cytokines and angiogenic factors: interleukin-1a (IL-1a), IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor-a (TNF-a), interferon-g (IFN-g), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), angiopoietin 2 (ANG2), fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and the tissue inhibitors of metalloproteinases (TIMP): TIMP1, and TIMP2.

Techniques: Construct, Cell Culture, In Vitro, Staining, Expressing, Binding Assay, Positive Control, Control, Sandwich ELISA