Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Deciphering complement receptor type 1 interactions with recognition proteins of the lectin complement pathway.

doi: 10.4049/jimmunol.1202451

Figure Lengend Snippet: FIGURE 1. Characterization of the recom- binant CR1 CCP 22–30_His fragment. (A) SDS-PAGE analysis of 4 mg CR1 CCP22-30 under reducing (R) and nonreducing (NR) conditions. The positions of the m.w. mark- ers are indicated. (B) CD spectroscopy of CR1 CCP22–30. A spectrum was recorded in the far-UV (200–260 nm) and collected six times. The mean values for each wavelength were calculated. The maximal ellipticity is indicated by an arrowhead. (C) Electron microscopy analysis of CR1 CCP22–30 (375000) after negative staining with 2% sodium silicotungstate.

Article Snippet: Recombinant soluble human CR1 was purchased from R&D Systems Europe (Lille, France).

Techniques: SDS Page, Circular Dichroism, Electron Microscopy, Negative Staining

Journal: International Journal of Nanomedicine

Article Title: An Immunoreceptor-Targeting Strategy with Minimalistic C3b Peptide Fusion Enhances SARS-CoV-2 RBD mRNA Vaccine Immunogenicity

doi: 10.2147/IJN.S463546

Figure Lengend Snippet: Confirmation of targeting proficiency for mC3 and mFc ligands via immunofluorescence assay (IFA). ( A ) Illustration of the ligands-receptors interactions that were demonstrated by IFA. Following mRNA transfection into 293T cells, receptors were incubated with the cells. Then, RBD-mC3 bound CR1 while RBD-mFc bound FcγR. The RBD-mC3 and RBD-mFc were labeled by anti-RBD monoclonal antibodies. The CR1 and FcγR were marked using anti-CR1 polyclonal antibodies and anti-FcγR monoclonal antibodies, respectively. ( B ) Antibodies targeting CR1 (FITC, green) and RBD-mC3 (Cy5, purple) depicted the respective distributions of CR1 and RBD-mC3. ( C ) Colocalization analysis of RBD-mC3 and CR1. ( D ) Antibodies against FcγR (green) and RBD-mFc (purple) showed the distributions of FcγR and RBD-mFc respectively. The blue color (DAPI) represented the cell nuclei. ( E ) Colocalization analysis of RBD-mFc and FcγR. Three regions of interest (ROI) were analyzed. Pearson’s coefficient (R) was calculated in colocalization analysis. R > 0.8 suggests a very strong correlation.

Article Snippet: Then, the cells were blocked with 10% goat serum (Jackson ImmunoResearch) for 1 hr at room temperature, followed by one-hour incubation of receptor proteins, including recombinant human CD35 protein (CR1) (R&D Systems #5748-CD-050, NE Minneapolis, MN) and mouse CD64/FCGR1 protein (Sino Biological #50086-M08H).

Techniques: Immunofluorescence, Transfection, Incubation, Labeling, Bioprocessing

Journal: Oncogene

Article Title: Emergent Role of the Fractalkine Axis in Dissemination of Peritoneal Metastasis from Epithelial Ovarian Carcinoma

doi: 10.1038/onc.2016.456

Figure Lengend Snippet: (A) High vs low CX 3 CR1 expression predicts an overall shorter survival in patients with serous ovarian carcinoma . Data from The Cancer Genome Atlas (TCGA) database for serous ovarian adenocarcinoma were analysed in Oncomine and plotted as CX 3 CR1 expression Log 2(median centered ratio) vs percent survival (green line; left panel). Data points with incomplete information were removed from the analysis; total number of specimens analysed was 557. Based on the value of median CX 3 CR1 expression, defined as CX 3 CR1 expression Log 2(median centered ratio) at 50% survival, the data set was divided in two (center panel), including specimens with CX 3 CR1 expression > 3.32, termed “high CX 3 CR1 expression” (n=143; red line), and specimens with CX 3 CR1 expression < 3.32, termed “low CX 3 CR1 expression” (n=414; blue line). Overall survival of patients expressing high and low levels of CX 3 CR1 was plotted and analysed with Kaplan-Meier plot using Prism software (center panel). Average overall survival of patients with high and low CX 3 CR1 expression is indicated in the Table (right panel). * p =0.0020, Log-rank (Mantel-Cox) test; * p =0.0027, Gehan-Breslow-Wilcoxon test. (B) High vs low CX 3 CR1 expression predicts an overall shorter survival in patients with serous ovarian carcinoma at FIGO Stages III and IV . The specimens in groups containing Stage III (n=429) and Stage IV (n=83) specimens were divided in two groups with “high” and “low” CX 3 CR1 expression (red and blue lines, respectively) corresponding to cases with higher than median CX 3 CR1 expression and lower than median CX 3 CR1 expression, respectively (3.23 for Stage III and 3.77 for Stage IV). Overall survival of patients with Stage III disease expressing high (n=100) and low (n=329) levels of CX 3 CR1 was plotted and analysed with Kaplan-Meier plot using Prism software (left panel); * p =0.0205, Log-rank (Mantel-Cox) test, * p =0.0170, Gehan-Breslow-Wilcoxon test. Overall survival of patients with Stage IV disease expressing high (n=23) and low (n=60) levels of CX 3 CR1 was plotted and analysed with Kaplan-Meier plot using Prism software (center panel);* p =0.0036, Log-rank (Mantel-Cox) test, * p =0.0197, Gehan-Breslow-Wilcoxon test. Average overall survival of patients at Stage III and IV disease with high and low CX 3 CR1 expression is indicated in the Table (right panel). (C) High vs low CX 3 CR1 expression predicts an overall shorter survival in postmenopausal patients with serous ovarian carcinoma . Overall survival of patients of pre-menopausal age with high (n=22) and low (n=108) CX 3 CR1 expression was plotted and analysed using Kaplan-Meier plots (left panel); p =0.8421, Log-rank (Mantel-Cox) test, p =0.5221, Gehan-Breslow-Wilcoxon test. Overall survival of patients of post-menopausal age with high (n=120) and low (n=307) CX 3 CR1 expression was plotted and analysed using Kaplan-Meier plots (center panel); * p =0.0046, Log-rank (Mantel-Cox) test, * p =0.0215, Gehan-Breslow-Wilcoxon test. Average overall survival of patients of both pre- and post-menopausal age with high and low CX 3 CR1 expression is indicated in the Table (right panel).

Article Snippet: Anti-CX 3 CL1, anti-CX 3 CR1, anti-rabbit, and anti-mouse biotin-conjugated antibodies were obtained from Abcam (Cambridge, MS).

Techniques: Expressing, Software

Journal: Oncogene

Article Title: Emergent Role of the Fractalkine Axis in Dissemination of Peritoneal Metastasis from Epithelial Ovarian Carcinoma

doi: 10.1038/onc.2016.456

Figure Lengend Snippet: (A) Short-term in vivo adhesion assay demonstrates that CX 3 CR1 is important for peritoneal adhesion . Immunofluorescence images (green fluorescence) demonstrate cells adherent to omentum and peritoneal wall, as indicated, 4 hours following i.p. injection of the GFP-labeled control (scr sh group) and experimental (cx2 clone) ID8 cells into abdomens of C57BL/6 (5/group). Adherent cells were visualized under the fluorescence microscope in tissues excised from sacrificed control (injected with ID8 and scr sh) and experimental (injected with cx2 and cx4 clones) animals, enumerated using Zeiss AxioVert software, and plotted. Red squares – mouse #1, purple checks – mouse #2, blue flakes – mouse #3, green triangles – mouse #4, blue diamonds – mouse #5. Bars show an average number of adherent cells in each group. The differences between groups (cx2 vs scr sh and cx4 vs scr sh) were statistically analysed using both Mann-Whitney U test and Students’ t-test; * p <0.05. Differences between ID8 and scr sh groups were not statistically significant using both Mann-Whitney U test and Students’ t-test. All groups were statistically analysed using one-way ANOVA; p =0.0026. (B) Downregulation of CX 3 CR1 leads to significant reduction of tumor formation . The number of animals bearing metastasis on the organs covered by the parietal peritoneum and the visceral peritoneum in both control (ID8 and scr sh combined to a total of 20 animals; designated “control”) and experimental (clones cx2, cx3, and cx6 combined to a total of 30 animals; designated “CX 3 CR1sh”) groups was calculated and plotted. Animals were considered positive for metastasis at each site if lesions visible with the naked eye were present upon dissection when animals reached humane endpoints. The data are shown as average ± standard deviation and were statistically analyzed using Students’ t-test.; * p <0.05. (C) Normal human mesothelium is strongly CX 3 CL1-positive . Expression of CX 3 CL1 in human mesothelium was determined using immunohistochemistry and anti-CX 3 CL1-specific antibodies. A typical image of the CX 3 CL1 immunostaining in normal mesothelium from intestine/colon of a 55 year old woman is shown (core B1, ). Black arrow points to the mesothelial monolayer. Brown, CX 3 CL1; blue, hematoxylin. Bar, 200 micron. Insert is a 5-fold magnification of the area outlined with dashed lines.

Article Snippet: Anti-CX 3 CL1, anti-CX 3 CR1, anti-rabbit, and anti-mouse biotin-conjugated antibodies were obtained from Abcam (Cambridge, MS).

Techniques: In Vivo, Cell Adhesion Assay, Immunofluorescence, Fluorescence, Injection, Labeling, Control, Microscopy, Clone Assay, Software, MANN-WHITNEY, Dissection, Standard Deviation, Expressing, Immunohistochemistry, Immunostaining

Journal: Oncogene

Article Title: Emergent Role of the Fractalkine Axis in Dissemination of Peritoneal Metastasis from Epithelial Ovarian Carcinoma

doi: 10.1038/onc.2016.456

Figure Lengend Snippet: ( A ) An overall tumor burden was calculated by adding the weights of individual tumor nodules at all sites (omentum, peritoneal wall, diaphragm, liver, mesenthery, kidney, ovary&uterus, and pancreas) and plotted. Orange circles – mouse #1, red squares – mouse #2, purple checks – mouse #3, lime dashes – mouse #4, blue flakes – mouse #5, green triangles – mouse #6, green crosses – mouse #7, blue diamonds – mouse #8, orange dashes – mouse #9, lilac diamonds – mouse #10. Color coding for the control and experimental animal groups: red – ID8, green – scr sh, purple – cx2, orange – cx3, black– cx6. Bars show an average tumor burden in each group. Differences between two groups were analysed using both Mann-Whitney U test and Students’ t-test; * p <0.05. All groups were statistically analysed using one-way ANOVA; p =0.0030. ( B ) Typical images of gross tumor dissemination throughout the abdomen for ID8 and CX2 groups are shown (bottom panels). Yellow dashed lines outline tumor nodules. Specimens of tumors generated by ID8 and CX2 groups at the time of sacrifice were immunohistochemically stained for CX 3 CR1 ( C ) as well as Ki67, PCNA, and CD95 ( D ). Primary antibody omission was used as negative control; sections were incubated with secondary anti-mouse and anti-rabit antibodies only, as indicated. Bar, 200 micron. Brown: CX 3 CR1, Ki67, PCNA, or CD95, as indicated. Blue: hematoxylin.

Article Snippet: Anti-CX 3 CL1, anti-CX 3 CR1, anti-rabbit, and anti-mouse biotin-conjugated antibodies were obtained from Abcam (Cambridge, MS).

Techniques: Control, MANN-WHITNEY, Generated, Staining, Negative Control, Incubation

Journal: Oncogene

Article Title: Emergent Role of the Fractalkine Axis in Dissemination of Peritoneal Metastasis from Epithelial Ovarian Carcinoma

doi: 10.1038/onc.2016.456

Figure Lengend Snippet: (A) Expression of CX 3 CL1 in specimens of tumor-naive mouse omentum, pancreas, peritoneal wall, and diaphragm . Expression of CX 3 CL1 in total cell lysates (10 µg protein/lane) of peritoneal wall, diaphragm, omentum, and pancreas obtained from tumor-naive C57BL/6 mice (n=2) was detected using Western blot. Left lane shows positions of the molecular weight marker proteins. Mouse recombinant CX 3 CL1 (10 ng) served as a positive control. Actin served as a loading control. (B,C) Tumoral and stromal CX 3 CL1 expression . CX 3 CL1 in tumor and invaded stroma, as specified for each host site, was visualized with immunostaining in organ sites dissemination to which correlated with CX 3 CR1 expression in tumor cells (diaphragm, kidney, ( B )) and where it did not depend on CX 3 CR1 expression in the tumor cells (pancreas, omentum, ( C )). Images were generated using Aperio ScanScope; bar, 100 micron. Dashed yellow lines separate tumors from stroma. Brown, CX 3 CL1; blue, hematoxylin.

Article Snippet: Anti-CX 3 CL1, anti-CX 3 CR1, anti-rabbit, and anti-mouse biotin-conjugated antibodies were obtained from Abcam (Cambridge, MS).

Techniques: Expressing, Western Blot, Molecular Weight, Marker, Recombinant, Positive Control, Control, Immunostaining, Generated