Journal: Molecular immunology

Article Title: Enhanced recognition of plasma proteins in a non-native state by complement C3b. A possible clearance mechanism for damaged proteins in blood

doi: 10.1016/j.molimm.2014.10.022

Figure Lengend Snippet: iC3b:plasma protein complexes bind to red blood cells (RBCs). Isolated human RBCs were treated for 30 minutes at 37°C with either with sham-treated NHS or serum activated with CVF. Serum aliquots were removed before (pre-RBC Tx) and after (post-RBC Tx) RBC treatment. RBCs were lysed with H2O and the membrane fraction (pellet) was separated from the supernatant. All samples were separated using SDS-PAGE on an 8% gel and immunoblotted for C3.

Article Snippet: Purified cobra venom factor (CVF), human serum depleted of individual complement components (C3, factor B and factor D), and purified human complement proteins (C3, factor B and factor D) were all purchased from Complement Technology, Inc. (Tyler, TX).

Techniques: Clinical Proteomics, Isolation, Membrane, SDS Page

Journal: Molecular immunology

Article Title: Enhanced recognition of plasma proteins in a non-native state by complement C3b. A possible clearance mechanism for damaged proteins in blood

doi: 10.1016/j.molimm.2014.10.022

Figure Lengend Snippet: Thermally denatured proteins activate the complement system via the alternative pathway. NHS was heated to 60°C for 1 hour (Δ serum) and then added to equal volume of either fresh NHS (lanes 2), factor B depleted serum (lanes 5), factor D depleted serum (lanes 6) or C3 depleted serum (lanes 7) and incubated for either 15 or 60 minutes at 37°C. The negative control was untreated NHS (lanes 1) or NHS that received equal amount of Δ serum in the presence of 10 mM EDTA (lanes 3) to prevent complement activation. The positive control was a mixture of Δ serum plus fresh serum treated with CVF (lanes 4) to activate the alternative pathway. Samples were separated using an 8% SDS-PAGE gel and blotted for factor B (alternative pathway activation and generation of the 33 kDa Ba fragment), C3 (110 kDa α-chain cleavage and 67 kDa iC3b generation) and α1PI (iC3b:α1PI complex formation).

Article Snippet: Purified cobra venom factor (CVF), human serum depleted of individual complement components (C3, factor B and factor D), and purified human complement proteins (C3, factor B and factor D) were all purchased from Complement Technology, Inc. (Tyler, TX).

Techniques: Incubation, Negative Control, Activation Assay, Positive Control, SDS Page

Journal: Molecular immunology

Article Title: Enhanced recognition of plasma proteins in a non-native state by complement C3b. A possible clearance mechanism for damaged proteins in blood

doi: 10.1016/j.molimm.2014.10.022

Figure Lengend Snippet: C3 dependent complement activators generate C5a in thermally denatured serum. Untreated NHS (Control) or NHS plus 60°C heated serum (Δ Serum) were treated with either CVF, zymosan, HAGG or buffer (Sham) for 60 minutes at 37°C and C5a levels were quantified by ELISA.

Article Snippet: Purified cobra venom factor (CVF), human serum depleted of individual complement components (C3, factor B and factor D), and purified human complement proteins (C3, factor B and factor D) were all purchased from Complement Technology, Inc. (Tyler, TX).

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Annals of surgical oncology

Article Title: Complement Inhibition: A Novel Form of Immunotherapy for Colon Cancer

doi: 10.1245/s10434-015-4778-7

Figure Lengend Snippet: Complement inhibition decreases tumor volume. a Tumor volumes of mice treated with PBS or CVF (500 μg/kg per mouse) measured at days 6 (78 vs. 44 mm3; p = 0.049), 12 (343 vs. 164 mm3; p = 0.019), and 16 (652 vs. 390 mm3; p = 0.03) after tumor establishment. Mice treated with CVF had significantly smaller tumors than those treated with PBS at all 3 time points. b Tumor volumes of mice treated with PBS or hCVF (1 mg/kg per mouse) measured at days 8 (54 vs. 29 mm3; p = 0.0015), 12 (82 vs. 42 mm3; p = 0.002), and 19 (222 vs. 99 mm3; p = 0.06) after tumor establishment. Mice treated with hCVF had significantly smaller tumors at days 8 and 12 after tumor establishment, but after this time point, significance was lost. c Tumor volumes of mice treated with PBS or CVF at a higher dose (1 mg/kg per mouse) measured at days 8 (282 vs. 90 mm3; p = 0.009) and 12 (613 vs. 152 mm3; p = 0.004) after tumor establishment. Mice treated with this dose of CVF had smaller tumors at days 8 and 12, but this difference was lost at later time points. d Tumor volumes of mice treated with PBS or SSL7 (100 μg IV day 2 and 12 after tumor establishment) measured at days 7 and 12 after tumor establishment. Mice treated with SSL7 had smaller tumors at days 7 (207 vs. 121 mm3; p = 0.001) and 12 (447 vs. 263 mm3; p = 0.005), but this difference was lost at later time points. *p < 0.05, **p < 0.01, ***p ≤ 0.001

Article Snippet: Complement Protein Inhibitors Purified CVF ( Naja naja kaouthia ) was obtained from Quidel Corporation. hCVF was obtained from Dr. Carl Wilhelm Vogel (Cancer Research Center, Honolulu, HI).

Techniques: Inhibition

Journal: Annals of surgical oncology

Article Title: Complement Inhibition: A Novel Form of Immunotherapy for Colon Cancer

doi: 10.1245/s10434-015-4778-7

Figure Lengend Snippet: Complement depletion with CVF reduces immunosuppressive MDSCs and results in increased tumor infiltration of effector T cells via increased production of chemoattractive mediators. a FACS analysis shows increased percentage of CD11b+Gr1+ cells in splenocytes of PBS-treated mice vs. CVF-treated mice (p = 0.04563). b FACS analysis of tumor-infiltrating lymphocytes shows a higher percentage of CD8+ T cells in CVF-treated mice compared to control mice (p = 0.033) and c decreased percentage of CD4+ T cells infiltrating tumors in CVF-treated mice (p = 0.04). *p < 0.05, **p < 0.01, ***p ≤ 0.001

Article Snippet: Complement Protein Inhibitors Purified CVF ( Naja naja kaouthia ) was obtained from Quidel Corporation. hCVF was obtained from Dr. Carl Wilhelm Vogel (Cancer Research Center, Honolulu, HI).

Techniques: Control

Journal: Annals of surgical oncology

Article Title: Complement Inhibition: A Novel Form of Immunotherapy for Colon Cancer

doi: 10.1245/s10434-015-4778-7

Figure Lengend Snippet: Complement depletion with CVF results in increased production of chemoattractive chemokines in tumor microenvironment. qRT-PCR performed on tumor tissues of mice treated with PBS or CVF showed significantly higher expression [fold increase over HPRT (hypoxanthine guanine phosphoribosyl transferase) (murine housekeeping gene)] of chemokines CCL5 (0.03-fold vs. 4.3-fold; p = 0.001), CXCL10 (0.004 vs. 0.42; p = 0.002), and CXCL11 (0.001 vs. 0.023; p = 0.001) in CVF-treated mice 12 days after tumor establishment. *p < 0.05, **p < 0.01, ***p ≤ 0.001

Article Snippet: Complement Protein Inhibitors Purified CVF ( Naja naja kaouthia ) was obtained from Quidel Corporation. hCVF was obtained from Dr. Carl Wilhelm Vogel (Cancer Research Center, Honolulu, HI).

Techniques: Quantitative RT-PCR, Expressing

Journal: Annals of surgical oncology

Article Title: Complement Inhibition: A Novel Form of Immunotherapy for Colon Cancer

doi: 10.1245/s10434-015-4778-7

Figure Lengend Snippet: Complement inhibition with SSL7 results in increased production of chemoattractive chemokines in tumor microenvironment. qRT-PCR performed on tumor tissues of mice treated with PBS or CVF showed significantly higher expression [fold increase over HPRT (hypoxanthine guanine phosphoribosyl transferase) (murine housekeeping gene)] of chemokines: CCL5 (2.42 vs. 3.61; p = 0.001), CXCL10 (4.1 vs. 6.7; p = 0.001), and CXCL11 (0.038 vs. 0.06; p = 0.008). *p < 0.05, **p < 0.01, ***p ≤ 0.001

Article Snippet: Complement Protein Inhibitors Purified CVF ( Naja naja kaouthia ) was obtained from Quidel Corporation. hCVF was obtained from Dr. Carl Wilhelm Vogel (Cancer Research Center, Honolulu, HI).

Techniques: Inhibition, Quantitative RT-PCR, Expressing