Journal: PLoS ONE

Article Title: Levels of Murine, but Not Human, CXCL13 Are Greatly Elevated in NOD-SCID Mice Bearing the AIDS-Associated Burkitt Lymphoma Cell Line, 2F7

doi: 10.1371/journal.pone.0072414

Figure Lengend Snippet: Immunohistochemistry was performed as described in the Materials and Methods , using an HRP/DAB color development system with hematoxylin counterstaining. Some sections were stained with both hematoxylin and eosin (H and E). Typical staining results for each marker are given in the figure as follows: ( A ) H and E, ( B ) human CXCR5, ( C ) a representative negative control, ( D ) human CXCL13, ( E ) murine CXCL13, and ( F ) F4/80. The results of the H&E staining in ( A ) show a tumor that in many respects resembles human BL, which typically contain significant numbers of infiltrating macrophages, giving a “starry sky” appearance (see arrows). The corner insert in ( A ) shows a 3-fold enlargement of the area in the image to which the upper arrow points, with such a macrophage in the center of the enlargement. The image in ( E ) shows that tumors appear to be infiltrated with scattered cells that stain positive for murine CXCL13. In an attempt to more precisely determine the cell type of these infiltrating cells serial tumor sections were stained separately for murine CXCL13 and for F4/80, and images of the resulting staining were enlarged 3-fold compared to the images in ( A – F ) to provide more detail ( G , H ). In ( G ) and ( H ), both mCXCL13(+) and F4/80(+) cells appear to be large cells that sometimes have dendritic processes. All images shown in this figure were taken at an original magnification of 200X. Each image contains an internal scale marker that is 100 microns in length.

Article Snippet: Levels of murine and human CXCL13 in serum and in tumor ascites fluid were determined by ELISA, using kits validated to be specific for either murine or human CXCL13 (QuantikineTM, R&D Systems), with no cross-reactivity.

Techniques: Immunohistochemistry, Staining, Marker, Negative Control

Journal: PLoS ONE

Article Title: Levels of Murine, but Not Human, CXCL13 Are Greatly Elevated in NOD-SCID Mice Bearing the AIDS-Associated Burkitt Lymphoma Cell Line, 2F7

doi: 10.1371/journal.pone.0072414

Figure Lengend Snippet: ( A ) Murine CXCL13 levels in the serum of control mice (n = 13) and in the serum of mice with tumors (n = 14), as determined by ELISA. ( B ) Murine CXCL13 levels in the ascites fluid of the mice with tumors. The solid square within each box plot represents the mean value for the data. In ( A ), there is an ~20-fold increase in mean mCXCL13 levels in the serum of mice with tumors compared to control mice (p < 0.0001; t-test). In ( B ), the long lower whisker in the box plot of the data reflects the presence of several low outliers; these lower values were generally seen in animals that developed smaller, more localized tumors at the site of injection as opposed to widespread metastatic disease.

Article Snippet: Levels of murine and human CXCL13 in serum and in tumor ascites fluid were determined by ELISA, using kits validated to be specific for either murine or human CXCL13 (QuantikineTM, R&D Systems), with no cross-reactivity.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Whisker Assay, Injection

Journal: BMC Immunology

Article Title: CXCL13 antibody for the treatment of autoimmune disorders

doi: 10.1186/s12865-015-0068-1

Figure Lengend Snippet: Effect on CXCL13- (A-D) and CXCL12- (negative control; E) induced migration of human and murine cells. Human pre-B-697-hCXCR5 cells (A, E) , human pre-B-697-hCXCR4 cells (E) , human tonsillar cells (B, E) , C57BL/6 splenocytes (C, E) , and SJL mouse splenocytes (D, E) were seeded into Transwell inserts. Diluted chemokines +/− antibodies were added into the lower chambers and plates were incubated for 2 h at 37°C (50 ug/ml of each antibody were used in CXCL12-induced migration assay). Inserts were removed and Alamar blue was added to the wells and incubated at 37°C overnight. Fluorescence was measured at wavelengths of 530 nm and 590 nm and the percent inhibition of chemokine-induced migration (A-D) or migration index (E) was calculated. Data represents an average of the measurements from at least three independent experiments + SEM. Statistical significance (relative to the isotype controls) was evaluated using one-way ANOVA followed by Bonferroni’s multiple comparison post test **** P < 0.0001; *** P < 0.0003; **P < 0.01.

Article Snippet: Chemokines/Cytokines: recombinant human CXCL13 (Peprotech; 300–47); recombinant murine CXCL13 (Peprotech; 250–24); recombinant murine CXCL12 (Peprotech; 250-20A); recombinant human CXCL12 (Peprotech; 300-28A); recombinant murine IL-12 (R&D Systems; 419-ML/CF); recombinant murine IL-2 (R&D Systems; 402-ML); recombinant murine IL-6 (R&D Systems; 406-ML/CF); recombinant human TGF-β (R&D Systems; 240-B); recombinant murine IL-23 (R&D Systems; 1887-ML).

Techniques: Negative Control, Migration, Incubation, Fluorescence, Inhibition

Journal: BMC Immunology

Article Title: CXCL13 antibody for the treatment of autoimmune disorders

doi: 10.1186/s12865-015-0068-1

Figure Lengend Snippet: Inhibition of human CXCL13-mediated internalization of human CXCR5 receptor. Human pre-B-697-huCXCR5 cells were pre-blocked with anti-human Fc for 15 min at 37°C, incubated with a pre-complexed huCXCL13/antibody mix for 2 h at 37°C, and subsequently stained with anti-human CXCR5 antibody for flow cytometric analysis. (A) Cell surface CXCR5 receptor expression. Geometric mean values were determined by FlowJo 7.6 software. (B) EC50 values were calculated from sigmoidal dose response curves (shown on the graph) with R 2 values equal to 0.9 (MAb 5261) and 0.98 (MAb 5261-muIg). Data points represent an average of measurements obtained from 3 independent experiments.

Article Snippet: Chemokines/Cytokines: recombinant human CXCL13 (Peprotech; 300–47); recombinant murine CXCL13 (Peprotech; 250–24); recombinant murine CXCL12 (Peprotech; 250-20A); recombinant human CXCL12 (Peprotech; 300-28A); recombinant murine IL-12 (R&D Systems; 419-ML/CF); recombinant murine IL-2 (R&D Systems; 402-ML); recombinant murine IL-6 (R&D Systems; 406-ML/CF); recombinant human TGF-β (R&D Systems; 240-B); recombinant murine IL-23 (R&D Systems; 1887-ML).

Techniques: Inhibition, Incubation, Staining, Expressing, Software

Journal: BMC Immunology

Article Title: CXCL13 antibody for the treatment of autoimmune disorders

doi: 10.1186/s12865-015-0068-1

Figure Lengend Snippet: Effect of murine anti-CXCL13 antibody on germinal center formation in C57BL/6 mice challenged with NP-KLH. Mice were treated with 30 mg/kg of MAb 5261-muIg antibody and corresponding mouse isotype control, on days −3, 0 (the day of the immunization), 4 and 7 (n = 9/group). (A) Total IgM and IgG1 anti-NP antibody-secreting cells were detected by ELISPOT on wells coated with NP14-BSA. (B) The frequencies of high affinity IgG1 anti-NP-secreting cells were determined by ELISPOT on wells coated with NP4-BSA. (C) Number of germinal centers (n = 9 mice/group). Follicles were hand counted. Number of PNA+ germinal centers in relation to total number of follicles per group was calculated using ImagePro software. (D) Mean germinal center area. Area of each PNA+ germinal center for each group was calculated using ImagePro software by PNA+ pixel area and then converted to μm. The numbers of GC analyzed were: 201 in Control group and 102 in MAb 5261-muIg-treated group. (E) Percentages of activated T cells (CD4+/CD62Llow/CD44+) in spleens and bone marrow. Statistical significance was evaluated by Student’s unpaired t -test. *P = 0.0125; **P = 0.0016; ****P < 0.0001; P = 0.2 and 0.71 for IgG1 and IgM portions of the graph, respectively (A) .

Article Snippet: Chemokines/Cytokines: recombinant human CXCL13 (Peprotech; 300–47); recombinant murine CXCL13 (Peprotech; 250–24); recombinant murine CXCL12 (Peprotech; 250-20A); recombinant human CXCL12 (Peprotech; 300-28A); recombinant murine IL-12 (R&D Systems; 419-ML/CF); recombinant murine IL-2 (R&D Systems; 402-ML); recombinant murine IL-6 (R&D Systems; 406-ML/CF); recombinant human TGF-β (R&D Systems; 240-B); recombinant murine IL-23 (R&D Systems; 1887-ML).

Techniques: Enzyme-linked Immunospot, Software

Journal: BMC Immunology

Article Title: CXCL13 antibody for the treatment of autoimmune disorders

doi: 10.1186/s12865-015-0068-1

Figure Lengend Snippet: Effect on CXCL13- (A-D) and CXCL12- (negative control; E) induced migration of human and murine cells. Human pre-B-697-hCXCR5 cells (A, E) , human pre-B-697-hCXCR4 cells (E) , human tonsillar cells (B, E) , C57BL/6 splenocytes (C, E) , and SJL mouse splenocytes (D, E) were seeded into Transwell inserts. Diluted chemokines +/− antibodies were added into the lower chambers and plates were incubated for 2 h at 37°C (50 ug/ml of each antibody were used in CXCL12-induced migration assay). Inserts were removed and Alamar blue was added to the wells and incubated at 37°C overnight. Fluorescence was measured at wavelengths of 530 nm and 590 nm and the percent inhibition of chemokine-induced migration (A-D) or migration index (E) was calculated. Data represents an average of the measurements from at least three independent experiments + SEM. Statistical significance (relative to the isotype controls) was evaluated using one-way ANOVA followed by Bonferroni’s multiple comparison post test **** P < 0.0001; *** P < 0.0003; **P < 0.01.

Article Snippet: Chemokines/Cytokines: recombinant human CXCL13 (Peprotech; 300–47); recombinant murine CXCL13 (Peprotech; 250–24); recombinant murine CXCL12 (Peprotech; 250-20A); recombinant human CXCL12 (Peprotech; 300-28A); recombinant murine IL-12 (R&D Systems; 419-ML/CF); recombinant murine IL-2 (R&D Systems; 402-ML); recombinant murine IL-6 (R&D Systems; 406-ML/CF); recombinant human TGF-β (R&D Systems; 240-B); recombinant murine IL-23 (R&D Systems; 1887-ML).

Techniques: Negative Control, Migration, Incubation, Fluorescence, Inhibition