Journal:

Article Title: Cancer-induced Expansion and Activation of CD11b + Gr-1 + Cells Predispose Mice to Adenoviral-triggered Anaphylactoid-type Reactions

doi: 10.1038/mt.2008.280

Figure Lengend Snippet: Myeloid cell depletion using clodronate-liposome prevents recombinant adenovirus (rAd)-induced death in 4T1 tumor-bearing animals. (a) Treatment with clodronate-liposome depletes splenic CD11b+Gr-1+ cells in 4T1 tumor-bearing animals. Phosphate-buffered saline (PBS)-liposome-treated animals serve as controls. Clodronate-liposome and PBS-liposome were given 24 hours before spleen isolation. Live cells expressing CD45 marker are gated to determine CD11b (horizontal axis) and Gr-1 (vertical axis) expression. Spleens from two and three animals are pooled from PBS-liposome and clodronate-liposome-treated 4T1 tumor-bearing animals, respectively, to generate this figure. (b) Myeloid cell depletion prevents rAd-induced death in 4T1 tumor-bearing animals. Tumors were implanted 30 days prior to treatment with clodronate-liposome or PBS-liposome. Animals received IV injection of rAd 24 hours after clodronate treatment. Number of animals is four per treatment, and the figure is representative of three independent experiments. Data are represented as mean ± SEM. Statistical analysis was done using nonparametric Mann–Whitney test.

Article Snippet: These cells were then labeled with phycoerythrin-conjugated rat anti-mouse Gr-1 mAb and sorted using FACS Vantage cell sorter (BD Biosciences, San Jose, CA).

Techniques: Recombinant, Isolation, Expressing, Marker, IV Injection, MANN-WHITNEY

Journal:

Article Title: Cancer-induced Expansion and Activation of CD11b + Gr-1 + Cells Predispose Mice to Adenoviral-triggered Anaphylactoid-type Reactions

doi: 10.1038/mt.2008.280

Figure Lengend Snippet: Passive transfer of CD11b+Gr-1+ splenocytes from 4T1 tumor-bearing animals conveys predisposition to anaphylactoid-type reactions in normal, non-tumor-bearing BALB/c mice. Normal, non-tumor-bearing BALB/c animals were retroorbitally injected with one of the following: 3 × 108 red blood cell (RBC)-depleted splenic cells from normal animals (group 1), 3 × 108 RBC-depleted splenic cells from 4T1 tumor-bearing animals (group 2), or 3 × 108 CD11b+Gr-1+ MACS sorted splenic cells from 4T1 tumor-bearing animals (group 3). Twenty-four hours following passive transfer, animals received IV injection of recombinant adenovirus (rAd). Data are pooled from experiments conducted on four separate days. Statistical analysis between groups 1 and 3 was done using stratified Wilcoxon exact rank sum test. Direct statistical comparison between groups 2 and 3 was not performed because the passive transfer experiments on respective experimental groups were conducted on separate days.

Article Snippet: These cells were then labeled with phycoerythrin-conjugated rat anti-mouse Gr-1 mAb and sorted using FACS Vantage cell sorter (BD Biosciences, San Jose, CA).

Techniques: Injection, IV Injection, Recombinant

Journal:

Article Title: Cancer-induced Expansion and Activation of CD11b + Gr-1 + Cells Predispose Mice to Adenoviral-triggered Anaphylactoid-type Reactions

doi: 10.1038/mt.2008.280

Figure Lengend Snippet: Differential modulation of myeloid cells by 4T1 and 66cl4 tumors correlates with their susceptibility to anaphylactoid-type reactions. (a) Several genes are differentially expressed in 4T1 and 66cl4 tumor cell lines. Expression values are determined by real-time PCR and the values are normalized relative to ubiquitin expression. Dark gray bars represent 4T1 tumor cells whereas gray bars represent 66cl4 tumor cells. (b) Relative spleen weights are increased in 4T1 tumor-bearing animals as compared with normal and 66cl4 tumor-bearing animals. Spleen weights were taken from animals 23 days post-tumor implantation and are represented as a percentage of body weights. Age-matched animals were used for naive, non-tumor-bearing animals. Groups not sharing a superscript letter differ significantly at P < 0.001 (ANOVA followed by Tukey's test). (c) Increased percentage of CD11b+Gr-1+ cells in spleens isolated from 4T1 tumor-bearing animals. Spleens were pooled from three respective tumor-bearing animals at day 20 post-tumor implant. CD45-positive splenic cells were gated to determine CD11b (horizontal axis) and Gr-1 (vertical axis) expression. (d) Increased susceptibility to recombinant adenovirus (rAd) (1 × 1010 particles)-induced anaphylactoid-type reactions was seen in 4T1 tumor-bearing animals but not in 66cl4 tumor-bearing animals. Animals were implanted with respective tumors 31 days prior to the experiment. The data are representative of three independent experiments. Data are represented as mean ± SEM. Statistical analysis was done using nonparametric Kruskal–Wallis test followed by Dunn's multiple comparison test. Groups not sharing a superscript letter differ significantly at P < 0.05. COX-2, cyclooxygenase 2; G-CSF, granulocyte colony–stimulating factor; GM-CSF, granulocyte–macrophage-colony–stimulating factor; NOS2, nitric oxide synthetase 2; TNFα, tumor necrosis factor α.

Article Snippet: These cells were then labeled with phycoerythrin-conjugated rat anti-mouse Gr-1 mAb and sorted using FACS Vantage cell sorter (BD Biosciences, San Jose, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Tumor Implantation, Isolation, Recombinant

Journal:

Article Title: Cancer-induced Expansion and Activation of CD11b + Gr-1 + Cells Predispose Mice to Adenoviral-triggered Anaphylactoid-type Reactions

doi: 10.1038/mt.2008.280

Figure Lengend Snippet: Splenic CD11b+Gr-1+ cells isolated from 4T1 tumor-bearing animals show altered expression of several genes when compared with corresponding cells isolated from naive and 66cl4 tumor-bearing animals. Spleens were isolated between days 18 and 22 post-tumor implant from 4T1 and 66cl4 tumor-bearing animals. Splenic cells were positively selected for CD11b expression using magnetic beads. These CD11b-positive cells were further sorted using fluorescence-activated cell sorting into three fractions depending on Gr-1 expression (Gr-1high, Gr-1medium, and Gr-1negative). Expression values are determined by real-time PCR, and the values are normalized relative to ubiquitin expression. Expression values from three independent experiments are averaged with error bars showing SE. White bars represent cells isolated from naive animals, black bars represent cells isolated from 4T1 tumor-bearing animals, and gray bars represent cells isolated from 66cl4 tumor-bearing animals. Data are represented as mean ± SEM. Statistical analysis was done using ANOVA followed by Tukey's test. Asterisk represents differences with P < 0.05. COX-2, cyclooxygenase 2; M-CSF, macrophage colony–stimulating factor; NOS2, nitric oxide synthetase 2; rAd, recombinant adenovirus; TNFα, tumor necrosis factor α.

Article Snippet: These cells were then labeled with phycoerythrin-conjugated rat anti-mouse Gr-1 mAb and sorted using FACS Vantage cell sorter (BD Biosciences, San Jose, CA).

Techniques: Isolation, Expressing, Magnetic Beads, Fluorescence, FACS, Real-time Polymerase Chain Reaction, Recombinant

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Thymic Deletion and Regulatory T Cells Prevent Antimyeloperoxidase GN

doi: 10.1681/ASN.2012090898

Figure Lengend Snippet: MPO immunostaining of murine thymii demonstrated that thymic MPO expression was predominantly in neutrophils. Immunohistochemical staining for MPO protein on murine thymic tissue sections from Mpo+/+ mice (A), Mpo−/− mice (B), and Aire−/− mice (C). The immunohistochemical staining is visualized by light microscopy. The higher cell density region constitutes the cortex, whereas the lower cell density region constitutes the medulla. The MPO protein distribution is in the corticomedullary junction in the normal Mpo+/+ and Aire−/− thymi. MPO protein is not detected in Mpo−/− mice. Colocalization of thymic neutrophils (D–F) and macrophages (G–I) with MPO in Mpo+/+ mice with MPO. The immunofluorescence is visualized by laser scanning confocal microscopy. (D) Neutrophils (Gr-1 green) are almost all positive for MPO (dark blue) (E), as observed by the merged image MPO and Gr-1 (light blue) (F). Fewer macrophages are present as demonstrated by F4/80 (green) (G), but most are MPO positive (dark blue) (H) as observed by the merged image MPO and F4/80 (light blue) (I). Original magnification, ×400 in A–C; ×800 in D–I.

Article Snippet: Frozen tissues were cut at 6 µm and stained using rabbit anti-cow Cytokeratin (Dako, Glostrup, Denmark), mouse anti-mouse MPO FITC (Hycult Biotech, Uden, The Netherlands), rat anti-mouse Gr-1 647 (BD Biosciences), or rat anti-mouse F4/80 647 (BD Biosciences).

Techniques: Immunostaining, Expressing, Immunohistochemical staining, Staining, Light Microscopy, Immunofluorescence, Confocal Microscopy