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  • ct26  (ATCC)
    99
    ATCC ct26
    Imaging of hypoxia within <t>CT26</t> tumour bearing mice ( n = 13), PET-MRI a – d Representative PET-MRI images showing the global co-localisation of FMISO uptake and BOLD MRI signal. Images were acquired 120 min post-injection of 10 MBq of [ 18 F]-Fluoromisonidazole (FMISO). PET. a Representative maximum-intensity-projection FMISO PET Image; b Transversal slice showing FMISO uptake within the tumour; c T2*-weighted MRI and d BOLD image derived from T2* mapping. White dashed lines: tumour limits, white arrows: oxygenated tumour area (increased BOLD signal), black circle: hypoxic tumour areas (decreased BOLD signal). CLI ( e ): Representative FMISO CLI image of the same mouse as for a to d acquired just after the PET-MRI scan. f Tumour-to-background ratio for PET, MRI and CLI following the injection of FMISO determined by the ratio of the signal from the tumour and a contralateral irrelevant region of interest (muscle); n = 13, *** p
    Ct26, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 331 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher wt expression kit
    Imaging of hypoxia within <t>CT26</t> tumour bearing mice ( n = 13), PET-MRI a – d Representative PET-MRI images showing the global co-localisation of FMISO uptake and BOLD MRI signal. Images were acquired 120 min post-injection of 10 MBq of [ 18 F]-Fluoromisonidazole (FMISO). PET. a Representative maximum-intensity-projection FMISO PET Image; b Transversal slice showing FMISO uptake within the tumour; c T2*-weighted MRI and d BOLD image derived from T2* mapping. White dashed lines: tumour limits, white arrows: oxygenated tumour area (increased BOLD signal), black circle: hypoxic tumour areas (decreased BOLD signal). CLI ( e ): Representative FMISO CLI image of the same mouse as for a to d acquired just after the PET-MRI scan. f Tumour-to-background ratio for PET, MRI and CLI following the injection of FMISO determined by the ratio of the signal from the tumour and a contralateral irrelevant region of interest (muscle); n = 13, *** p
    Wt Expression Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6307 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    The Jackson Laboratory wt mice
    Imaging of hypoxia within <t>CT26</t> tumour bearing mice ( n = 13), PET-MRI a – d Representative PET-MRI images showing the global co-localisation of FMISO uptake and BOLD MRI signal. Images were acquired 120 min post-injection of 10 MBq of [ 18 F]-Fluoromisonidazole (FMISO). PET. a Representative maximum-intensity-projection FMISO PET Image; b Transversal slice showing FMISO uptake within the tumour; c T2*-weighted MRI and d BOLD image derived from T2* mapping. White dashed lines: tumour limits, white arrows: oxygenated tumour area (increased BOLD signal), black circle: hypoxic tumour areas (decreased BOLD signal). CLI ( e ): Representative FMISO CLI image of the same mouse as for a to d acquired just after the PET-MRI scan. f Tumour-to-background ratio for PET, MRI and CLI following the injection of FMISO determined by the ratio of the signal from the tumour and a contralateral irrelevant region of interest (muscle); n = 13, *** p
    Wt Mice, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 94/100, based on 1338 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher genechip wt terminal labeling kit
    Imaging of hypoxia within <t>CT26</t> tumour bearing mice ( n = 13), PET-MRI a – d Representative PET-MRI images showing the global co-localisation of FMISO uptake and BOLD MRI signal. Images were acquired 120 min post-injection of 10 MBq of [ 18 F]-Fluoromisonidazole (FMISO). PET. a Representative maximum-intensity-projection FMISO PET Image; b Transversal slice showing FMISO uptake within the tumour; c T2*-weighted MRI and d BOLD image derived from T2* mapping. White dashed lines: tumour limits, white arrows: oxygenated tumour area (increased BOLD signal), black circle: hypoxic tumour areas (decreased BOLD signal). CLI ( e ): Representative FMISO CLI image of the same mouse as for a to d acquired just after the PET-MRI scan. f Tumour-to-background ratio for PET, MRI and CLI following the injection of FMISO determined by the ratio of the signal from the tumour and a contralateral irrelevant region of interest (muscle); n = 13, *** p
    Genechip Wt Terminal Labeling Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2839 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher genechip wt plus reagent kit
    Imaging of hypoxia within <t>CT26</t> tumour bearing mice ( n = 13), PET-MRI a – d Representative PET-MRI images showing the global co-localisation of FMISO uptake and BOLD MRI signal. Images were acquired 120 min post-injection of 10 MBq of [ 18 F]-Fluoromisonidazole (FMISO). PET. a Representative maximum-intensity-projection FMISO PET Image; b Transversal slice showing FMISO uptake within the tumour; c T2*-weighted MRI and d BOLD image derived from T2* mapping. White dashed lines: tumour limits, white arrows: oxygenated tumour area (increased BOLD signal), black circle: hypoxic tumour areas (decreased BOLD signal). CLI ( e ): Representative FMISO CLI image of the same mouse as for a to d acquired just after the PET-MRI scan. f Tumour-to-background ratio for PET, MRI and CLI following the injection of FMISO determined by the ratio of the signal from the tumour and a contralateral irrelevant region of interest (muscle); n = 13, *** p
    Genechip Wt Plus Reagent Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    The Jackson Laboratory wt c57bl 6 mice
    CD73 and extracellular adenosine are required for the ability of neutrophils to kill S. pneumoniae. (A) PMNs were isolated from the bone marrow of <t>C57BL/6</t> (WT) or CD73 -/- mice and treated with 100μM Adenosine or PBS (vehicle control) for 30 minutes at 37°C. (B) PMNs were isolated from the bone marrow of C57BL/6 (WT) mice and treated with 100μM of the CD37 inhibitor (α,β methylene ADP), 100μM of the CD39 inhibitor (POM-1) or PBS (vehicle control-VC) for 30 minutes at 37°C. The reactions were then infected with S. pneumoniae pre-opsonized with homologous sera for 45 minutes at 37°C. Reactions were then stopped by placing samples on ice and viable CFU were determined after serial dilution and plating. The percentage of bacteria killed upon incubation with PMNs was determined by comparing surviving CFU to a no PMN control. Positive percent killing indicates bacterial death while negative percent indicates bacterial growth. Data shown are pooled from three separate experiments (n=3 biological replicates or mice per strain) where each condition was tested in triplicate (n=3 technical replicates) per experiment. Asterisks indicate significance calculated by Student’s t-test. (C) PMNs were isolated from the blood of young healthy donors and pre-treated with the CD73 inhibitor (α,β methylene ADP) or vehicle control for 30 minutes at 37°C and then incubated for 45 minutes with complement-opsonized S. pneumoniae TIGR4. For each donor, the average percent bacterial killing compared to a no PMN control was calculated from triplicate wells per condition. Data from 7 donors are shown. Significant differences denoted by asterisk, were determined by paired t-test.
    Wt C57bl 6 Mice, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 93/100, based on 1015 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    The Jackson Laboratory wild type wt mice
    CD73 and extracellular adenosine are required for the ability of neutrophils to kill S. pneumoniae. (A) PMNs were isolated from the bone marrow of <t>C57BL/6</t> (WT) or CD73 -/- mice and treated with 100μM Adenosine or PBS (vehicle control) for 30 minutes at 37°C. (B) PMNs were isolated from the bone marrow of C57BL/6 (WT) mice and treated with 100μM of the CD37 inhibitor (α,β methylene ADP), 100μM of the CD39 inhibitor (POM-1) or PBS (vehicle control-VC) for 30 minutes at 37°C. The reactions were then infected with S. pneumoniae pre-opsonized with homologous sera for 45 minutes at 37°C. Reactions were then stopped by placing samples on ice and viable CFU were determined after serial dilution and plating. The percentage of bacteria killed upon incubation with PMNs was determined by comparing surviving CFU to a no PMN control. Positive percent killing indicates bacterial death while negative percent indicates bacterial growth. Data shown are pooled from three separate experiments (n=3 biological replicates or mice per strain) where each condition was tested in triplicate (n=3 technical replicates) per experiment. Asterisks indicate significance calculated by Student’s t-test. (C) PMNs were isolated from the blood of young healthy donors and pre-treated with the CD73 inhibitor (α,β methylene ADP) or vehicle control for 30 minutes at 37°C and then incubated for 45 minutes with complement-opsonized S. pneumoniae TIGR4. For each donor, the average percent bacterial killing compared to a no PMN control was calculated from triplicate wells per condition. Data from 7 donors are shown. Significant differences denoted by asterisk, were determined by paired t-test.
    Wild Type Wt Mice, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 94/100, based on 671 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Nugen wt ovation pico rna amplification system
    Reproducibility of expression data obtained using <t>Nugen</t> chemistry across amounts of input <t>RNA</t> . Pearson's correlation coefficients were computed and represented graphically for each pairwise comparison of assays. Lower correlations were observed for data obtained from smallest amounts of input RNA (50 pg and 100 pg).
    Wt Ovation Pico Rna Amplification System, supplied by Nugen, used in various techniques. Bioz Stars score: 92/100, based on 1500 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    The Jackson Laboratory wt c57bl 6j mice
    Blocking GRP blunts influenza-induced cytokine induction in vivo . WT <t>C57BL/6J</t> mice (5 mice/treatment group) were infected i.n. with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice received vehicle (saline+0.0096% DMSO) or GRP inhibitor (NSC77427; 20 μM/mouse; i.v.) daily from day 2 to day 6 post-infection. On day 7 post-infection, lungs were harvested and total RNA was extracted to measure gene expression by qRT-PCR. ** p
    Wt C57bl 6j Mice, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 92/100, based on 701 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher reverse transcription kit
    Blocking GRP blunts influenza-induced cytokine induction in vivo . WT <t>C57BL/6J</t> mice (5 mice/treatment group) were infected i.n. with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice received vehicle (saline+0.0096% DMSO) or GRP inhibitor (NSC77427; 20 μM/mouse; i.v.) daily from day 2 to day 6 post-infection. On day 7 post-infection, lungs were harvested and total RNA was extracted to measure gene expression by qRT-PCR. ** p
    Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6367 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher wt terminal labeling kit
    Blocking GRP blunts influenza-induced cytokine induction in vivo . WT <t>C57BL/6J</t> mice (5 mice/treatment group) were infected i.n. with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice received vehicle (saline+0.0096% DMSO) or GRP inhibitor (NSC77427; 20 μM/mouse; i.v.) daily from day 2 to day 6 post-infection. On day 7 post-infection, lungs were harvested and total RNA was extracted to measure gene expression by qRT-PCR. ** p
    Wt Terminal Labeling Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 922 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Charles River Laboratories wt mice
    Blocking GRP blunts influenza-induced cytokine induction in vivo . WT <t>C57BL/6J</t> mice (5 mice/treatment group) were infected i.n. with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice received vehicle (saline+0.0096% DMSO) or GRP inhibitor (NSC77427; 20 μM/mouse; i.v.) daily from day 2 to day 6 post-infection. On day 7 post-infection, lungs were harvested and total RNA was extracted to measure gene expression by qRT-PCR. ** p
    Wt Mice, supplied by Charles River Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 345 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    The Jackson Laboratory wt c57bl 6
    IL-25–elicited MPP type2 cells promote Th2 cytokine–dependent responses in vivo. <t>C57BL/6</t> WT mice (The Jackson Laboratory) were treated i.p. with 0.3 µg IL-25 daily for 7 d. MLNs were harvested, and MPP type2 cells were sort-purified and injected intradermally into naive C57BL/6 WT mice. (a) IL-4, IL-5, and IL-13 cytokine production from skin-draining LN cells from mice receiving intradermal injection of control or IL-25–elicited MPP type2 cells after 48-h αCD3/αCD28 stimulation measured by ELISA. Data in a are representative of two independent experiments. (b–f) C57BL/6 WT mice were treated i.p. with 0.3 µg IL-25 daily for 7 d. MLNs were harvested, and MPP type2 cells were sort-purified and injected into T. muris –infected (INF) Il17rb −/− mice (Charles River). (b) IFN-γ cytokine production by T. muris antigen–stimulated MLN cells. (c) Total serum IgE antibody titers measured by ELISA. (d) Periodic acid–Schiff/Alcian blue–stained colon sections of intestine tissue from naive or infected WT or Il17rb −/− mice ± MPP type2 cells. N, naive (inset). Bars, 100 µm. (e) Goblet cell counts from d. (f) Worm burdens from T. muris –infected mice were assessed at day 21 after infection. Data in b–f are representative of two independent experiments (WT naive, n = 2–3; WT INF, n = 6–8; Il17rb −/− naive, n = 2–3; Il17rb −/− INF, n = 6–7; Il17rb −/− INF + MPP type2 cells, n = 6). *, P
    Wt C57bl 6, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 91/100, based on 410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Charles River Laboratories c57bl 6 wt mice
    Neutrophils are the predominant peritoneal cellular subset that express pro-IL-1β + at early time points following P. aeruginosa infection. <t>C57BL/6</t> ( n = 15) and ASC −/− ( n = 15) mice were infected intraperitoneally with a sublethal dose (3 × 10 6 CFU) of WT PA14. Mice were euthanized 4 h postinfection and peritoneal lavage was collected. A : representative dot plots depicting the gating scheme for pro-IL-1β + cells at 4 h postinfection. Bacteria were excluded by gating. B : percentage of pro-IL-1β + Ly6G + ( left ) and pro-IL-1β + F4/80 + ( right ) cells out of the total peritoneal cells at 4 h postinfection. C : percentage of Ly6G + F4/80 − ( left ) and Ly6G − F4/80 + ( right ) within the gated CD45 + pro-IL-1β + population as depicted in A above. Data are expressed as means ± SD from 5 independent biological experiments.
    C57bl 6 Wt Mice, supplied by Charles River Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    The Jackson Laboratory wt littermates
    Neutrophils are the predominant peritoneal cellular subset that express pro-IL-1β + at early time points following P. aeruginosa infection. <t>C57BL/6</t> ( n = 15) and ASC −/− ( n = 15) mice were infected intraperitoneally with a sublethal dose (3 × 10 6 CFU) of WT PA14. Mice were euthanized 4 h postinfection and peritoneal lavage was collected. A : representative dot plots depicting the gating scheme for pro-IL-1β + cells at 4 h postinfection. Bacteria were excluded by gating. B : percentage of pro-IL-1β + Ly6G + ( left ) and pro-IL-1β + F4/80 + ( right ) cells out of the total peritoneal cells at 4 h postinfection. C : percentage of Ly6G + F4/80 − ( left ) and Ly6G − F4/80 + ( right ) within the gated CD45 + pro-IL-1β + population as depicted in A above. Data are expressed as means ± SD from 5 independent biological experiments.
    Wt Littermates, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 93/100, based on 250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher controls kit
    Neutrophils are the predominant peritoneal cellular subset that express pro-IL-1β + at early time points following P. aeruginosa infection. <t>C57BL/6</t> ( n = 15) and ASC −/− ( n = 15) mice were infected intraperitoneally with a sublethal dose (3 × 10 6 CFU) of WT PA14. Mice were euthanized 4 h postinfection and peritoneal lavage was collected. A : representative dot plots depicting the gating scheme for pro-IL-1β + cells at 4 h postinfection. Bacteria were excluded by gating. B : percentage of pro-IL-1β + Ly6G + ( left ) and pro-IL-1β + F4/80 + ( right ) cells out of the total peritoneal cells at 4 h postinfection. C : percentage of Ly6G + F4/80 − ( left ) and Ly6G − F4/80 + ( right ) within the gated CD45 + pro-IL-1β + population as depicted in A above. Data are expressed as means ± SD from 5 independent biological experiments.
    Controls Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 949 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Imaging of hypoxia within CT26 tumour bearing mice ( n = 13), PET-MRI a – d Representative PET-MRI images showing the global co-localisation of FMISO uptake and BOLD MRI signal. Images were acquired 120 min post-injection of 10 MBq of [ 18 F]-Fluoromisonidazole (FMISO). PET. a Representative maximum-intensity-projection FMISO PET Image; b Transversal slice showing FMISO uptake within the tumour; c T2*-weighted MRI and d BOLD image derived from T2* mapping. White dashed lines: tumour limits, white arrows: oxygenated tumour area (increased BOLD signal), black circle: hypoxic tumour areas (decreased BOLD signal). CLI ( e ): Representative FMISO CLI image of the same mouse as for a to d acquired just after the PET-MRI scan. f Tumour-to-background ratio for PET, MRI and CLI following the injection of FMISO determined by the ratio of the signal from the tumour and a contralateral irrelevant region of interest (muscle); n = 13, *** p

    Journal: EJNMMI Research

    Article Title: Cherenkov luminescence imaging is a fast and relevant preclinical tool to assess tumour hypoxia in vivo

    doi: 10.1186/s13550-018-0464-7

    Figure Lengend Snippet: Imaging of hypoxia within CT26 tumour bearing mice ( n = 13), PET-MRI a – d Representative PET-MRI images showing the global co-localisation of FMISO uptake and BOLD MRI signal. Images were acquired 120 min post-injection of 10 MBq of [ 18 F]-Fluoromisonidazole (FMISO). PET. a Representative maximum-intensity-projection FMISO PET Image; b Transversal slice showing FMISO uptake within the tumour; c T2*-weighted MRI and d BOLD image derived from T2* mapping. White dashed lines: tumour limits, white arrows: oxygenated tumour area (increased BOLD signal), black circle: hypoxic tumour areas (decreased BOLD signal). CLI ( e ): Representative FMISO CLI image of the same mouse as for a to d acquired just after the PET-MRI scan. f Tumour-to-background ratio for PET, MRI and CLI following the injection of FMISO determined by the ratio of the signal from the tumour and a contralateral irrelevant region of interest (muscle); n = 13, *** p

    Article Snippet: A total of 1 × 106 murine CT26 (ATCC, CRL-2638, USA) colon cancer cells were implanted subcutaneously at the right flank of depilated mice.

    Techniques: Imaging, Mouse Assay, Positron Emission Tomography, Magnetic Resonance Imaging, Injection, Derivative Assay

    CD73 and extracellular adenosine are required for the ability of neutrophils to kill S. pneumoniae. (A) PMNs were isolated from the bone marrow of C57BL/6 (WT) or CD73 -/- mice and treated with 100μM Adenosine or PBS (vehicle control) for 30 minutes at 37°C. (B) PMNs were isolated from the bone marrow of C57BL/6 (WT) mice and treated with 100μM of the CD37 inhibitor (α,β methylene ADP), 100μM of the CD39 inhibitor (POM-1) or PBS (vehicle control-VC) for 30 minutes at 37°C. The reactions were then infected with S. pneumoniae pre-opsonized with homologous sera for 45 minutes at 37°C. Reactions were then stopped by placing samples on ice and viable CFU were determined after serial dilution and plating. The percentage of bacteria killed upon incubation with PMNs was determined by comparing surviving CFU to a no PMN control. Positive percent killing indicates bacterial death while negative percent indicates bacterial growth. Data shown are pooled from three separate experiments (n=3 biological replicates or mice per strain) where each condition was tested in triplicate (n=3 technical replicates) per experiment. Asterisks indicate significance calculated by Student’s t-test. (C) PMNs were isolated from the blood of young healthy donors and pre-treated with the CD73 inhibitor (α,β methylene ADP) or vehicle control for 30 minutes at 37°C and then incubated for 45 minutes with complement-opsonized S. pneumoniae TIGR4. For each donor, the average percent bacterial killing compared to a no PMN control was calculated from triplicate wells per condition. Data from 7 donors are shown. Significant differences denoted by asterisk, were determined by paired t-test.

    Journal: bioRxiv

    Article Title: Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production

    doi: 10.1101/716456

    Figure Lengend Snippet: CD73 and extracellular adenosine are required for the ability of neutrophils to kill S. pneumoniae. (A) PMNs were isolated from the bone marrow of C57BL/6 (WT) or CD73 -/- mice and treated with 100μM Adenosine or PBS (vehicle control) for 30 minutes at 37°C. (B) PMNs were isolated from the bone marrow of C57BL/6 (WT) mice and treated with 100μM of the CD37 inhibitor (α,β methylene ADP), 100μM of the CD39 inhibitor (POM-1) or PBS (vehicle control-VC) for 30 minutes at 37°C. The reactions were then infected with S. pneumoniae pre-opsonized with homologous sera for 45 minutes at 37°C. Reactions were then stopped by placing samples on ice and viable CFU were determined after serial dilution and plating. The percentage of bacteria killed upon incubation with PMNs was determined by comparing surviving CFU to a no PMN control. Positive percent killing indicates bacterial death while negative percent indicates bacterial growth. Data shown are pooled from three separate experiments (n=3 biological replicates or mice per strain) where each condition was tested in triplicate (n=3 technical replicates) per experiment. Asterisks indicate significance calculated by Student’s t-test. (C) PMNs were isolated from the blood of young healthy donors and pre-treated with the CD73 inhibitor (α,β methylene ADP) or vehicle control for 30 minutes at 37°C and then incubated for 45 minutes with complement-opsonized S. pneumoniae TIGR4. For each donor, the average percent bacterial killing compared to a no PMN control was calculated from triplicate wells per condition. Data from 7 donors are shown. Significant differences denoted by asterisk, were determined by paired t-test.

    Article Snippet: Wild type (WT) C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Isolation, Mouse Assay, Infection, Serial Dilution, Incubation

    Detoxification of ROS is important for S. pneumoniae to resist killing by wildtype, but not CD73 -/- PMNs. (A) PMNs isolated from the bone marrow of C57BL/6 (WT) mice were treated with PBS (VC), 10μM DPI, EUK 134 (25μM) or Ascorbic acid (100μM) for 30 minutes at 37°C. (B) PMNs isolated from the bone marrow of C57BL/6 (WT) or CD73 -/- mice were left unmanipulated. (C) PMNs isolated from the bone marrow of CD73 -/- mice were treated with 100μM adenosine in the absence or presence of EUK 134 or Ascorbic acid for 30 minutes at 37°C. (A-C) The PMNs were then infected with the indicated wild type or ΔsodA S. pneumoniae in the presence of homologous sera for 45 minutes at 37°C. Reactions were then stopped by placing samples on ice and viable CFU were determined after serial dilution and plating. The percentage of bacteria killed by comparing surviving CFU to a no PMN control under the same conditions. Data shown are pooled from four separate experiments (n=4 biological replicates or mice per strain) where each condition was tested in triplicate (n=3 technical replicates) per experiment. Asterisks indicate significance calculated by Student’s t-test.

    Journal: bioRxiv

    Article Title: Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production

    doi: 10.1101/716456

    Figure Lengend Snippet: Detoxification of ROS is important for S. pneumoniae to resist killing by wildtype, but not CD73 -/- PMNs. (A) PMNs isolated from the bone marrow of C57BL/6 (WT) mice were treated with PBS (VC), 10μM DPI, EUK 134 (25μM) or Ascorbic acid (100μM) for 30 minutes at 37°C. (B) PMNs isolated from the bone marrow of C57BL/6 (WT) or CD73 -/- mice were left unmanipulated. (C) PMNs isolated from the bone marrow of CD73 -/- mice were treated with 100μM adenosine in the absence or presence of EUK 134 or Ascorbic acid for 30 minutes at 37°C. (A-C) The PMNs were then infected with the indicated wild type or ΔsodA S. pneumoniae in the presence of homologous sera for 45 minutes at 37°C. Reactions were then stopped by placing samples on ice and viable CFU were determined after serial dilution and plating. The percentage of bacteria killed by comparing surviving CFU to a no PMN control under the same conditions. Data shown are pooled from four separate experiments (n=4 biological replicates or mice per strain) where each condition was tested in triplicate (n=3 technical replicates) per experiment. Asterisks indicate significance calculated by Student’s t-test.

    Article Snippet: Wild type (WT) C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Isolation, Mouse Assay, Infection, Serial Dilution

    Adoptive transfer of PMNs from wild type mice boosts resistance of CD73 -/- mice to S. pneumoniae. C57BL/6 (WT) or CD73 -/- mice were mock treated (no transfer) or injected i.p with 2.5×10 6 of the indicated PMNs isolated from the bone marrow of C57BL/6 or CD73 -/- mice. One hour post transfer, mice were infected i.t with 5×10 5 CFU of S. pneumoniae and bacterial numbers in the lung (A), blood (B) and brain (C) were determined 24 hours post infection. Significant differences, determined by Student’s t-test, are indicated by asterisks. Pooled data from n=5 mice per group are shown.

    Journal: bioRxiv

    Article Title: Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production

    doi: 10.1101/716456

    Figure Lengend Snippet: Adoptive transfer of PMNs from wild type mice boosts resistance of CD73 -/- mice to S. pneumoniae. C57BL/6 (WT) or CD73 -/- mice were mock treated (no transfer) or injected i.p with 2.5×10 6 of the indicated PMNs isolated from the bone marrow of C57BL/6 or CD73 -/- mice. One hour post transfer, mice were infected i.t with 5×10 5 CFU of S. pneumoniae and bacterial numbers in the lung (A), blood (B) and brain (C) were determined 24 hours post infection. Significant differences, determined by Student’s t-test, are indicated by asterisks. Pooled data from n=5 mice per group are shown.

    Article Snippet: Wild type (WT) C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Adoptive Transfer Assay, Mouse Assay, Injection, Isolation, Infection

    CD73 inhibits IL-10 production from PMNs following pneumococcal infection. (A) PMNs from the indicated mouse strains were incubated for 45 minutes at 37°C with S. pneumoniae pre-opsonized with homologous sera or mock treated with buffer and homologous sera (uninfected) in vitro . The supernatants were then collected and assayed for IL-10 production by ELISA. (B) PBS control or adenosine (100μM) were added to the PMNs 30 minutes prior to in vitro infection and the fold-change in IL-10 production was calculated by dividing the values of infected reactions by uninfected controls for each condition. Data were pooled from three separate experiments (n=3 mice) with each condition tested in triplicate per experiment. Asterisks indicate significant differences determined by Student’s t-test. (C-E) Wild-type C57BL/6 or CD73 -/- mice were mock-infected or i.t challenged with 5 x 10 5 CFU of S. pneumoniae . Six (grey bars) and 18 hours (black bars) following challenge, (C) the percentage of IL-10 producing PMNs (Ly6G+), (D) the mean florescent intensities (MFI) of IL-10 in PMNs (Ly6G+) and (E) the number of IL-10 producing PMNs (Ly6G+) recruited into the lungs were determined by intracellular cytokine staining and flow cytometry (see Materials and Methods). Pooled data are from three separate experiments (n=6-9 mice per strain per time point). Significant differences determined by Student’s t-test are indicated by asterisks.

    Journal: bioRxiv

    Article Title: Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production

    doi: 10.1101/716456

    Figure Lengend Snippet: CD73 inhibits IL-10 production from PMNs following pneumococcal infection. (A) PMNs from the indicated mouse strains were incubated for 45 minutes at 37°C with S. pneumoniae pre-opsonized with homologous sera or mock treated with buffer and homologous sera (uninfected) in vitro . The supernatants were then collected and assayed for IL-10 production by ELISA. (B) PBS control or adenosine (100μM) were added to the PMNs 30 minutes prior to in vitro infection and the fold-change in IL-10 production was calculated by dividing the values of infected reactions by uninfected controls for each condition. Data were pooled from three separate experiments (n=3 mice) with each condition tested in triplicate per experiment. Asterisks indicate significant differences determined by Student’s t-test. (C-E) Wild-type C57BL/6 or CD73 -/- mice were mock-infected or i.t challenged with 5 x 10 5 CFU of S. pneumoniae . Six (grey bars) and 18 hours (black bars) following challenge, (C) the percentage of IL-10 producing PMNs (Ly6G+), (D) the mean florescent intensities (MFI) of IL-10 in PMNs (Ly6G+) and (E) the number of IL-10 producing PMNs (Ly6G+) recruited into the lungs were determined by intracellular cytokine staining and flow cytometry (see Materials and Methods). Pooled data are from three separate experiments (n=6-9 mice per strain per time point). Significant differences determined by Student’s t-test are indicated by asterisks.

    Article Snippet: Wild type (WT) C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Infection, Incubation, In Vitro, Enzyme-linked Immunosorbent Assay, Mouse Assay, Staining, Flow Cytometry

    Expression of CD73 on pulmonary PMNs changes over the course of infection. C57BL/6 mice were inoculated i.t with 5×10 5 CFU of S. pneumoniae TIGR4. The blood, bone marrow and lungs were harvested, plated for bacterial enumeration and analyzed by flow cytometry. (A) Mice were euthanized at 0, 6, 12, 18, 24 and 48 hours following infection. We then compared bacterial numbers in the lungs to PMN numbers (Ly6G+ cells) for the first 0-12 and subsequent 18-72 hours post infection and performed Pearson correlation analysis where asterisks denote significant correlation. We gated on PMNs (Ly6G+ cells) and also monitored (B) the percentage of cells expressing CD73 and (B-C) the amounts of CD73 (mean fluorescent intensity or MFI) in the indicated organs, at the indicated time points. (D) We gated on all Ly6G+ cells and then gated on CD73 positive versus negative populations and compared the expression (MFI) of Ly6G. Data are pooled from two separate experiments with 4-5 mice per time point (A) and 4-7 mice per time point (B-D). Asterisks indicate significant differences from uninfected controls calculated by Student’s t-test.

    Journal: bioRxiv

    Article Title: Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production

    doi: 10.1101/716456

    Figure Lengend Snippet: Expression of CD73 on pulmonary PMNs changes over the course of infection. C57BL/6 mice were inoculated i.t with 5×10 5 CFU of S. pneumoniae TIGR4. The blood, bone marrow and lungs were harvested, plated for bacterial enumeration and analyzed by flow cytometry. (A) Mice were euthanized at 0, 6, 12, 18, 24 and 48 hours following infection. We then compared bacterial numbers in the lungs to PMN numbers (Ly6G+ cells) for the first 0-12 and subsequent 18-72 hours post infection and performed Pearson correlation analysis where asterisks denote significant correlation. We gated on PMNs (Ly6G+ cells) and also monitored (B) the percentage of cells expressing CD73 and (B-C) the amounts of CD73 (mean fluorescent intensity or MFI) in the indicated organs, at the indicated time points. (D) We gated on all Ly6G+ cells and then gated on CD73 positive versus negative populations and compared the expression (MFI) of Ly6G. Data are pooled from two separate experiments with 4-5 mice per time point (A) and 4-7 mice per time point (B-D). Asterisks indicate significant differences from uninfected controls calculated by Student’s t-test.

    Article Snippet: Wild type (WT) C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Expressing, Infection, Mouse Assay, Flow Cytometry

    Blocking IL-10 rescues the anti-microbial function of CD73 -/- PMNs and reverses the susceptibility of CD73 -/- mice to pneumococcal challenge. (A-B) Wild-type (WT) C57BL/6 or CD73 -/- mice were treated i.p with IL-10 blocking antibody JES5-2A5 (anti-IL-10) or isotype control two hours prior to pulmonary challenge with 5×10 5 CFU of S. pneumoniae . Pneumococcal burdens in the lungs (A) and blood (B) were determined 48 hours post-infection. Pooled data from three separate experiments with (CD73 -/- +/-anti-IL-10 n=8 mice; WT+ anti-IL-10 n=5 mice and WT + isotype n=13 mice per group) are shown. Values significantly different by Student’s t-test are indicated by asterisk. (C) PMNs from C57BL/6 or CD73 -/- mice were incubated for 20 minutes with the indicated anti-IL-10 (1μg/ml JES5-2A5), isotype control (1μg/ml), rIL-10 (50ng/ml) or adenosine (100μM) and then infected with pre-opsonized S. pneumoniae for 45 minutes at 37°C. Reactions were then plated on blood agar plates and the percentage of bacteria killed compared to a no PMN control under the same conditions was calculated. Data shown are pooled from three separate experiments (n=3 mice per strain) with each condition tested in triplicate. Asterisks indicate significant differences determined by Student’s t-test.

    Journal: bioRxiv

    Article Title: Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production

    doi: 10.1101/716456

    Figure Lengend Snippet: Blocking IL-10 rescues the anti-microbial function of CD73 -/- PMNs and reverses the susceptibility of CD73 -/- mice to pneumococcal challenge. (A-B) Wild-type (WT) C57BL/6 or CD73 -/- mice were treated i.p with IL-10 blocking antibody JES5-2A5 (anti-IL-10) or isotype control two hours prior to pulmonary challenge with 5×10 5 CFU of S. pneumoniae . Pneumococcal burdens in the lungs (A) and blood (B) were determined 48 hours post-infection. Pooled data from three separate experiments with (CD73 -/- +/-anti-IL-10 n=8 mice; WT+ anti-IL-10 n=5 mice and WT + isotype n=13 mice per group) are shown. Values significantly different by Student’s t-test are indicated by asterisk. (C) PMNs from C57BL/6 or CD73 -/- mice were incubated for 20 minutes with the indicated anti-IL-10 (1μg/ml JES5-2A5), isotype control (1μg/ml), rIL-10 (50ng/ml) or adenosine (100μM) and then infected with pre-opsonized S. pneumoniae for 45 minutes at 37°C. Reactions were then plated on blood agar plates and the percentage of bacteria killed compared to a no PMN control under the same conditions was calculated. Data shown are pooled from three separate experiments (n=3 mice per strain) with each condition tested in triplicate. Asterisks indicate significant differences determined by Student’s t-test.

    Article Snippet: Wild type (WT) C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Blocking Assay, Mouse Assay, Infection, Incubation

    CD73 does not affect bacterial uptake or intracellular killing by PMNs. (A) Bone marrow PMNs from C57BL/6 mice were incubated for 40 minutes at 37°C with wild type or ΔPLY S. pneumoniae that were pre-opsonized with homologous sera at an MOI of 2. Gentamicin (50μg/ml) was then added for 30 minutes to kill extracellular bacteria. PMNs were washed and plated on blood agar plates to determine the amount of bacteria engulfed and the % of bacteria taken up (uptake) of the original infecting inoculum was calculated. (B) Wild type (S.p) or pneumolysin deficient ( ΔPLY S.p ) S. pneumoniae were incubated with 100 μg/ml Gentamycin for 30 minutes and the percentage of bacteria killed compared to a mock treatment control was then calculated by plating on blood agar plates. (C) C57BL/6 PMNs were incubated for 45 minutes at 37°C with the indicated strains of wild type (S.p) or pneumolysin deficient ( ΔPLY S.p ) S. pneumoniae pre-opsonized with sera. The percentage of bacteria killed compared to a no PMN control under the same conditions was then calculated by plating for viable bacteria on blood agar plates. (D-E) Bone marrow PMNs from the indicated strains of mice were incubated with S. pneumoniae ΔPLY pre-opsonized with homologous sera at an MOI of 2 for 10 minutes at 37°C and gentamicin (50μg/ml) was then added for 30 minutes to kill extracellular bacteria. PMNs were then washed and one set (D) immediately plated on blood agar plates to determine the amount of bacteria engulfed and the % of bacteria taken up (uptake) of the original infecting inoculum was calculated. The other sets of PMNs (E) were incubated for 30 and 90 more minutes and then plated to enumerate viable bacteria. The % of bacteria of the engulfed inoculum that was killed was then calculated (Intracellular Killing). Data shown are pooled from (A) two separate experiments, (B) two separate experiments and (C) four separate experiments (n=4 mice) and (D-E) two separate experiments (n=2 mice per strain) where each condition was tested in quadruplicates per experiment. Values significantly different by Student’s t-test are indicated by asterisk.

    Journal: bioRxiv

    Article Title: Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production

    doi: 10.1101/716456

    Figure Lengend Snippet: CD73 does not affect bacterial uptake or intracellular killing by PMNs. (A) Bone marrow PMNs from C57BL/6 mice were incubated for 40 minutes at 37°C with wild type or ΔPLY S. pneumoniae that were pre-opsonized with homologous sera at an MOI of 2. Gentamicin (50μg/ml) was then added for 30 minutes to kill extracellular bacteria. PMNs were washed and plated on blood agar plates to determine the amount of bacteria engulfed and the % of bacteria taken up (uptake) of the original infecting inoculum was calculated. (B) Wild type (S.p) or pneumolysin deficient ( ΔPLY S.p ) S. pneumoniae were incubated with 100 μg/ml Gentamycin for 30 minutes and the percentage of bacteria killed compared to a mock treatment control was then calculated by plating on blood agar plates. (C) C57BL/6 PMNs were incubated for 45 minutes at 37°C with the indicated strains of wild type (S.p) or pneumolysin deficient ( ΔPLY S.p ) S. pneumoniae pre-opsonized with sera. The percentage of bacteria killed compared to a no PMN control under the same conditions was then calculated by plating for viable bacteria on blood agar plates. (D-E) Bone marrow PMNs from the indicated strains of mice were incubated with S. pneumoniae ΔPLY pre-opsonized with homologous sera at an MOI of 2 for 10 minutes at 37°C and gentamicin (50μg/ml) was then added for 30 minutes to kill extracellular bacteria. PMNs were then washed and one set (D) immediately plated on blood agar plates to determine the amount of bacteria engulfed and the % of bacteria taken up (uptake) of the original infecting inoculum was calculated. The other sets of PMNs (E) were incubated for 30 and 90 more minutes and then plated to enumerate viable bacteria. The % of bacteria of the engulfed inoculum that was killed was then calculated (Intracellular Killing). Data shown are pooled from (A) two separate experiments, (B) two separate experiments and (C) four separate experiments (n=4 mice) and (D-E) two separate experiments (n=2 mice per strain) where each condition was tested in quadruplicates per experiment. Values significantly different by Student’s t-test are indicated by asterisk.

    Article Snippet: Wild type (WT) C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Mouse Assay, Incubation

    (A) Footpad swelling (compared to the uninfected contralateral footpad) (mm) of infected LTβR −/− , LTβ −/− , wt C57BL/6 (C57 +/+), and BALB/c mice (mean ± SE; n = 9 for C57BL/6 wt and LTβ −/− mice; n = 8 for LTβR −/− and BALB/c mice). 1, P

    Journal: Infection and Immunity

    Article Title: The Absence of Cutaneous Lymph Nodes Results in a Th2 Response and Increased Susceptibility to Leishmania major Infection in Mice ▿

    doi: 10.1128/IAI.01714-07

    Figure Lengend Snippet: (A) Footpad swelling (compared to the uninfected contralateral footpad) (mm) of infected LTβR −/− , LTβ −/− , wt C57BL/6 (C57 +/+), and BALB/c mice (mean ± SE; n = 9 for C57BL/6 wt and LTβ −/− mice; n = 8 for LTβR −/− and BALB/c mice). 1, P

    Article Snippet: C57BL/6 wt mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Infection, Mouse Assay

    (A) Footpad swelling (compared to the uninfected contralateral footpad) (mm) of wt mice lacking pLNs (pLN_null) generated by gestational treatment with anti-TNF and LTβR-IgG fusion protein (see Materials and Methods) and control C57BL/6 (C57 control) mice (mean ± SE; n = 13 for wt mice lacking pLNs; n = 7 for C57BL/6). (B to D) LDA of footpads (B), liver (C), or bone marrow (D) of wt mice lacking pLNs or control C57BL/6 mice (C57 control) 10 weeks after infection. The number of living parasites for single mice (diamonds) and the means for wt mice lacking pLN and C57BL/6 mice (horizontal bar) are indicated. (E and F) Cytokine secretion of CD4 + cells isolated from spleens of wt mice lacking pLNs or control C57BL/6 mice 10 weeks after infection. Cells were incubated for 48 h with syngeneic DC stimulated for 48 h with soluble Leishmania antigen (sLmAg) (filled bars) or with unstimulated syngeneic dendritic cells (w/o Ag) (open bars). IFN-γ (E) or IL-4 (F) secretion was measured by ELISA (mean ± SE; n = 5). For all panels: *, P

    Journal: Infection and Immunity

    Article Title: The Absence of Cutaneous Lymph Nodes Results in a Th2 Response and Increased Susceptibility to Leishmania major Infection in Mice ▿

    doi: 10.1128/IAI.01714-07

    Figure Lengend Snippet: (A) Footpad swelling (compared to the uninfected contralateral footpad) (mm) of wt mice lacking pLNs (pLN_null) generated by gestational treatment with anti-TNF and LTβR-IgG fusion protein (see Materials and Methods) and control C57BL/6 (C57 control) mice (mean ± SE; n = 13 for wt mice lacking pLNs; n = 7 for C57BL/6). (B to D) LDA of footpads (B), liver (C), or bone marrow (D) of wt mice lacking pLNs or control C57BL/6 mice (C57 control) 10 weeks after infection. The number of living parasites for single mice (diamonds) and the means for wt mice lacking pLN and C57BL/6 mice (horizontal bar) are indicated. (E and F) Cytokine secretion of CD4 + cells isolated from spleens of wt mice lacking pLNs or control C57BL/6 mice 10 weeks after infection. Cells were incubated for 48 h with syngeneic DC stimulated for 48 h with soluble Leishmania antigen (sLmAg) (filled bars) or with unstimulated syngeneic dendritic cells (w/o Ag) (open bars). IFN-γ (E) or IL-4 (F) secretion was measured by ELISA (mean ± SE; n = 5). For all panels: *, P

    Article Snippet: C57BL/6 wt mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Mouse Assay, Generated, Infection, Isolation, Incubation, Enzyme-linked Immunosorbent Assay

    (A) Footpad swelling (compared to the uninfected contralateral footpad) (mm) of LTβR −/− and wt C57BL/6 (C57+/+) mice infected with a small parasite inoculum (10 4 parasites) (mean ± SE; n = 4 for LTβR −/− mice and n = 5 for C57+/+ mice). *, P

    Journal: Infection and Immunity

    Article Title: The Absence of Cutaneous Lymph Nodes Results in a Th2 Response and Increased Susceptibility to Leishmania major Infection in Mice ▿

    doi: 10.1128/IAI.01714-07

    Figure Lengend Snippet: (A) Footpad swelling (compared to the uninfected contralateral footpad) (mm) of LTβR −/− and wt C57BL/6 (C57+/+) mice infected with a small parasite inoculum (10 4 parasites) (mean ± SE; n = 4 for LTβR −/− mice and n = 5 for C57+/+ mice). *, P

    Article Snippet: C57BL/6 wt mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Mouse Assay, Infection

    (A and B) Cytokine secretion of CD4 + cells isolated from spleens of LTβR −/− or wt C57BL/6 (C57 +/+) mice 2 weeks after L. major infection. Cells were incubated for 48 h with syngeneic DC stimulated for 48 h with soluble Leishmania antigen (filled bars) or with unstimulated syngeneic DC (open bars). IFN-γ (A) or IL-4 (B) secretion was measured by ELISA (mean ± SE; n = 3). (C and D) Cytokine secretion of CD4 + cells isolated from spleens of LTβR −/− or wt C57BL/6 (C57 +/+) mice 2 weeks after injection of 2 × 10 7 nonviable L. major parasites. Cells were incubated for 48 h with syngeneic DC stimulated for 48 h with soluble Leishmania antigen (filled bars) or with unstimulated syngeneic DC (open bars). IFN-γ (C) or IL-4 (D) secretion was measured by ELISA (mean ± SE; n = 3). For all panels: *, P

    Journal: Infection and Immunity

    Article Title: The Absence of Cutaneous Lymph Nodes Results in a Th2 Response and Increased Susceptibility to Leishmania major Infection in Mice ▿

    doi: 10.1128/IAI.01714-07

    Figure Lengend Snippet: (A and B) Cytokine secretion of CD4 + cells isolated from spleens of LTβR −/− or wt C57BL/6 (C57 +/+) mice 2 weeks after L. major infection. Cells were incubated for 48 h with syngeneic DC stimulated for 48 h with soluble Leishmania antigen (filled bars) or with unstimulated syngeneic DC (open bars). IFN-γ (A) or IL-4 (B) secretion was measured by ELISA (mean ± SE; n = 3). (C and D) Cytokine secretion of CD4 + cells isolated from spleens of LTβR −/− or wt C57BL/6 (C57 +/+) mice 2 weeks after injection of 2 × 10 7 nonviable L. major parasites. Cells were incubated for 48 h with syngeneic DC stimulated for 48 h with soluble Leishmania antigen (filled bars) or with unstimulated syngeneic DC (open bars). IFN-γ (C) or IL-4 (D) secretion was measured by ELISA (mean ± SE; n = 3). For all panels: *, P

    Article Snippet: C57BL/6 wt mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Isolation, Mouse Assay, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Injection

    (A) Footpad swelling (compared to the uninfected contralateral footpad) (mm) of LTβR-IgG fusion protein-treated (LTβRlgG) or human IgG-treated (HuIgG) C57BL/6 mice (mean ± SE; n = 5). (B) LDA of footpads of LTβR-IgG fusion protein-treated or huIgG-treated C57BL/6 mice 12 weeks after infection. The number of living parasites for single mice (diamonds) and the means (horizontal bars) are indicated.

    Journal: Infection and Immunity

    Article Title: The Absence of Cutaneous Lymph Nodes Results in a Th2 Response and Increased Susceptibility to Leishmania major Infection in Mice ▿

    doi: 10.1128/IAI.01714-07

    Figure Lengend Snippet: (A) Footpad swelling (compared to the uninfected contralateral footpad) (mm) of LTβR-IgG fusion protein-treated (LTβRlgG) or human IgG-treated (HuIgG) C57BL/6 mice (mean ± SE; n = 5). (B) LDA of footpads of LTβR-IgG fusion protein-treated or huIgG-treated C57BL/6 mice 12 weeks after infection. The number of living parasites for single mice (diamonds) and the means (horizontal bars) are indicated.

    Article Snippet: C57BL/6 wt mice were purchased from Jackson Laboratories (Bar Harbor, ME).

    Techniques: Mouse Assay, Infection

    Mean ± SEM of the estimated numbers of intact motor units (MUNE) in hindlimb muscles of C57BL/6‐ Smn +/− transgenic mice expressed as a percentage of corresponding muscles in C57BL/6‐wild type control mice The mean number of motor units was 30% lower ( P

    Journal: The Journal of Physiology

    Article Title: Compensatory axon sprouting for very slow axonal die‐back in a transgenic model of spinal muscular atrophy type III

    doi: 10.1113/JP273404

    Figure Lengend Snippet: Mean ± SEM of the estimated numbers of intact motor units (MUNE) in hindlimb muscles of C57BL/6‐ Smn +/− transgenic mice expressed as a percentage of corresponding muscles in C57BL/6‐wild type control mice The mean number of motor units was 30% lower ( P

    Article Snippet: Breeding pairs of C57BL/6‐ Smn +/− transgenic mice (Jablonka et al . ) and C57BL/6‐wild type (WT) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in the Health Sciences Laboratory Animal Services (HSLAS) at the University of Alberta.

    Techniques: Transgenic Assay, Mouse Assay

    Mean ± SEM of the wet weights of fast‐twitch TA, MG and EDL muscles and slow‐twitch SOL muscles of C57BL/6‐ Smn +/− transgenic and C57BL/6‐wild type mice Muscle weights of Smn +/− were 97 ± 2.7% ( P > 0.44) of control wild type.

    Journal: The Journal of Physiology

    Article Title: Compensatory axon sprouting for very slow axonal die‐back in a transgenic model of spinal muscular atrophy type III

    doi: 10.1113/JP273404

    Figure Lengend Snippet: Mean ± SEM of the wet weights of fast‐twitch TA, MG and EDL muscles and slow‐twitch SOL muscles of C57BL/6‐ Smn +/− transgenic and C57BL/6‐wild type mice Muscle weights of Smn +/− were 97 ± 2.7% ( P > 0.44) of control wild type.

    Article Snippet: Breeding pairs of C57BL/6‐ Smn +/− transgenic mice (Jablonka et al . ) and C57BL/6‐wild type (WT) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in the Health Sciences Laboratory Animal Services (HSLAS) at the University of Alberta.

    Techniques: Transgenic Assay, Mouse Assay

    Number of femoral as compared to sciatic motoneurons in the lumbosacral (L2–L4) spinal cord in 4‐month‐old Smn +/− transgenic mice and wild type control mice Motoneuron counts after backlabelling the femoral or sciatic nerves with either fluorogold or rubyred were completed on consecutive 40 μm thick longitudinal spinal cord sections between T11 and L2; motoneurons in all sections were counted. The data are corrected for thickness of the longitudinal sections. Each bar represents the mean ± SEM of data obtained from 5 (C57BL/6‐wild type) and 6 (C57BL/6‐ Smn +/− transgenic) mice. Note that the number of motoneurons in the Smn +/− transgenic mouse is not different from the wild type control for either femoral ( P > 0.91) or sciatic ( P > 0.83) nerves at 4 months of age.

    Journal: The Journal of Physiology

    Article Title: Compensatory axon sprouting for very slow axonal die‐back in a transgenic model of spinal muscular atrophy type III

    doi: 10.1113/JP273404

    Figure Lengend Snippet: Number of femoral as compared to sciatic motoneurons in the lumbosacral (L2–L4) spinal cord in 4‐month‐old Smn +/− transgenic mice and wild type control mice Motoneuron counts after backlabelling the femoral or sciatic nerves with either fluorogold or rubyred were completed on consecutive 40 μm thick longitudinal spinal cord sections between T11 and L2; motoneurons in all sections were counted. The data are corrected for thickness of the longitudinal sections. Each bar represents the mean ± SEM of data obtained from 5 (C57BL/6‐wild type) and 6 (C57BL/6‐ Smn +/− transgenic) mice. Note that the number of motoneurons in the Smn +/− transgenic mouse is not different from the wild type control for either femoral ( P > 0.91) or sciatic ( P > 0.83) nerves at 4 months of age.

    Article Snippet: Breeding pairs of C57BL/6‐ Smn +/− transgenic mice (Jablonka et al . ) and C57BL/6‐wild type (WT) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in the Health Sciences Laboratory Animal Services (HSLAS) at the University of Alberta.

    Techniques: Transgenic Assay, Mouse Assay

    Mean (±SEM) numbers of motoneurons of C57BL/6‐ Smn +/− transgenic and C57BL/6‐wild type control mice, back‐labelled from the sciatic nerve 2 months ( A ) and 4 months ( B ) after left sciatic nerve section and suture of the proximal nerve stump to an innervated muscle to prevent regeneration The corresponding proportions of surviving motoneurons after axotomy are shown in C and D . The left sciatic nerves served as intact internal controls. Two and 4 months after axotomy, the proximal stump of the sciatic nerve was cut and exposed to FR to back‐label the motoneurons that survived the injury. Mice were 5 and 7 months old at the end of experiments in A and B . Motoneuron counts were completed on consecutive 40 μm thick longitudinal spinal cord sections between T11 and L2; all sections were counted. The data are corrected for thickness of longitudinal sections. ( n = 7 for C57BL/6‐wild type and n = 8 for C57BL/6‐ Smn +/− transgenic mice).

    Journal: The Journal of Physiology

    Article Title: Compensatory axon sprouting for very slow axonal die‐back in a transgenic model of spinal muscular atrophy type III

    doi: 10.1113/JP273404

    Figure Lengend Snippet: Mean (±SEM) numbers of motoneurons of C57BL/6‐ Smn +/− transgenic and C57BL/6‐wild type control mice, back‐labelled from the sciatic nerve 2 months ( A ) and 4 months ( B ) after left sciatic nerve section and suture of the proximal nerve stump to an innervated muscle to prevent regeneration The corresponding proportions of surviving motoneurons after axotomy are shown in C and D . The left sciatic nerves served as intact internal controls. Two and 4 months after axotomy, the proximal stump of the sciatic nerve was cut and exposed to FR to back‐label the motoneurons that survived the injury. Mice were 5 and 7 months old at the end of experiments in A and B . Motoneuron counts were completed on consecutive 40 μm thick longitudinal spinal cord sections between T11 and L2; all sections were counted. The data are corrected for thickness of longitudinal sections. ( n = 7 for C57BL/6‐wild type and n = 8 for C57BL/6‐ Smn +/− transgenic mice).

    Article Snippet: Breeding pairs of C57BL/6‐ Smn +/− transgenic mice (Jablonka et al . ) and C57BL/6‐wild type (WT) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in the Health Sciences Laboratory Animal Services (HSLAS) at the University of Alberta.

    Techniques: Transgenic Assay, Mouse Assay

    Numbers of backlabelled common peroneal (CP) motoneurons that regenerated their nerves after sciatic nerve transection and resuture in C57BL/6‐ Smn +/− transgenic and C57BL/6‐wild type control mice Left CP nerves served as intact internal controls. The right sciatic nerves of 4‐month‐old mice were cut and subsequently repaired (coapted) by direct suture of the proximal and distal stumps. Three weeks later, left control and the right axotomized CP motoneurons were backlabelled via the CP nerve at sites that were distal to the repair site of the right CP nerve. Motoneuron counts were completed on 20 μm thick longitudinal spinal cord sections between T11 and L2; every second section was counted through the entire spinal cord. Data are corrected for thickness of the longitudinal sections. Each bar represents the mean (±SEM) of data obtained from 8 C57BL/6‐ Smn +/− transgenic and 7 C57BL/6‐wild type mice. The corrected motoneuron counts after right CP nerve regeneration were significantly different from counts from the left intact CP nerves but the counts were not different between the Smn +/− transgenic and the wild type mice.

    Journal: The Journal of Physiology

    Article Title: Compensatory axon sprouting for very slow axonal die‐back in a transgenic model of spinal muscular atrophy type III

    doi: 10.1113/JP273404

    Figure Lengend Snippet: Numbers of backlabelled common peroneal (CP) motoneurons that regenerated their nerves after sciatic nerve transection and resuture in C57BL/6‐ Smn +/− transgenic and C57BL/6‐wild type control mice Left CP nerves served as intact internal controls. The right sciatic nerves of 4‐month‐old mice were cut and subsequently repaired (coapted) by direct suture of the proximal and distal stumps. Three weeks later, left control and the right axotomized CP motoneurons were backlabelled via the CP nerve at sites that were distal to the repair site of the right CP nerve. Motoneuron counts were completed on 20 μm thick longitudinal spinal cord sections between T11 and L2; every second section was counted through the entire spinal cord. Data are corrected for thickness of the longitudinal sections. Each bar represents the mean (±SEM) of data obtained from 8 C57BL/6‐ Smn +/− transgenic and 7 C57BL/6‐wild type mice. The corrected motoneuron counts after right CP nerve regeneration were significantly different from counts from the left intact CP nerves but the counts were not different between the Smn +/− transgenic and the wild type mice.

    Article Snippet: Breeding pairs of C57BL/6‐ Smn +/− transgenic mice (Jablonka et al . ) and C57BL/6‐wild type (WT) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in the Health Sciences Laboratory Animal Services (HSLAS) at the University of Alberta.

    Techniques: Transgenic Assay, Mouse Assay

    Mean (±SEM) isometric contractile forces developed by fast‐twitch tibialis anterior (TA), medial gastrocnemius (MG) and extensor digitorum longus (EDL) muscles, and slow‐twitch soleus (SOL) muscles Maximal twitch forces in response to 2× threshold electrical stimulation of the sciatic nerve at 0.5 Hz. A and B , and, at 100 Hz, maximal tetanic forces ( C and D ) forces in C57BL/6‐ Smn +/− transgenic and C57BL/6‐wild type mice. The twitch forces of Smn +/− transgenic mouse muscles were 89.6 ± 17.8% ( P > 0.16) of those of the wild type controls; muscle tetanic forces of the Smn +/− transgenic mice were 93.6 ± 3.3% ( P > 0.20) of wild type controls.

    Journal: The Journal of Physiology

    Article Title: Compensatory axon sprouting for very slow axonal die‐back in a transgenic model of spinal muscular atrophy type III

    doi: 10.1113/JP273404

    Figure Lengend Snippet: Mean (±SEM) isometric contractile forces developed by fast‐twitch tibialis anterior (TA), medial gastrocnemius (MG) and extensor digitorum longus (EDL) muscles, and slow‐twitch soleus (SOL) muscles Maximal twitch forces in response to 2× threshold electrical stimulation of the sciatic nerve at 0.5 Hz. A and B , and, at 100 Hz, maximal tetanic forces ( C and D ) forces in C57BL/6‐ Smn +/− transgenic and C57BL/6‐wild type mice. The twitch forces of Smn +/− transgenic mouse muscles were 89.6 ± 17.8% ( P > 0.16) of those of the wild type controls; muscle tetanic forces of the Smn +/− transgenic mice were 93.6 ± 3.3% ( P > 0.20) of wild type controls.

    Article Snippet: Breeding pairs of C57BL/6‐ Smn +/− transgenic mice (Jablonka et al . ) and C57BL/6‐wild type (WT) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in the Health Sciences Laboratory Animal Services (HSLAS) at the University of Alberta.

    Techniques: Transgenic Assay, Mouse Assay

    Photomicrographs of longitudinal sections of the TA muscle from an 11‐month‐old C57BL/6‐ Smn +/− transgenic mouse stained with the combined silver/cholinesterase stain to visualize axons and motor endplates (EPs), respectively Examples of nodal ( A ), preterminal ( B ) and ultraterminal ( C ) sprouts observed in muscles are shown. D , mean (±SEM) proportions of endplates that were free of nerve endings (free EPs) or were innervated by axon sprouts (EPs innervated by sprouts) are plotted for C57BL/6‐ Smn +/− ]

    Journal: The Journal of Physiology

    Article Title: Compensatory axon sprouting for very slow axonal die‐back in a transgenic model of spinal muscular atrophy type III

    doi: 10.1113/JP273404

    Figure Lengend Snippet: Photomicrographs of longitudinal sections of the TA muscle from an 11‐month‐old C57BL/6‐ Smn +/− transgenic mouse stained with the combined silver/cholinesterase stain to visualize axons and motor endplates (EPs), respectively Examples of nodal ( A ), preterminal ( B ) and ultraterminal ( C ) sprouts observed in muscles are shown. D , mean (±SEM) proportions of endplates that were free of nerve endings (free EPs) or were innervated by axon sprouts (EPs innervated by sprouts) are plotted for C57BL/6‐ Smn +/− ]

    Article Snippet: Breeding pairs of C57BL/6‐ Smn +/− transgenic mice (Jablonka et al . ) and C57BL/6‐wild type (WT) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in the Health Sciences Laboratory Animal Services (HSLAS) at the University of Alberta.

    Techniques: Transgenic Assay, Staining

    Reproducibility of expression data obtained using Nugen chemistry across amounts of input RNA . Pearson's correlation coefficients were computed and represented graphically for each pairwise comparison of assays. Lower correlations were observed for data obtained from smallest amounts of input RNA (50 pg and 100 pg).

    Journal: BMC Genomics

    Article Title: Evaluation of methods for amplification of picogram amounts of total RNA for whole genome expression profiling

    doi: 10.1186/1471-2164-10-246

    Figure Lengend Snippet: Reproducibility of expression data obtained using Nugen chemistry across amounts of input RNA . Pearson's correlation coefficients were computed and represented graphically for each pairwise comparison of assays. Lower correlations were observed for data obtained from smallest amounts of input RNA (50 pg and 100 pg).

    Article Snippet: In contrast with other manufacturer protocols, the WT-Ovation™ Pico RNA Amplification System (Nugen) is not based on T7 polymerase cRNA synthesis.

    Techniques: Expressing

    Quality of cDNA targets synthesised following Nugen protocol from different amounts of input RNA . Bioanalyzer electrophoretic profiles of cDNA targets obtained after the amplification of 50 pg, 100 pg, 250 pg, 500 pg, and 1 ng of total RNA using Nugen chemistry.

    Journal: BMC Genomics

    Article Title: Evaluation of methods for amplification of picogram amounts of total RNA for whole genome expression profiling

    doi: 10.1186/1471-2164-10-246

    Figure Lengend Snippet: Quality of cDNA targets synthesised following Nugen protocol from different amounts of input RNA . Bioanalyzer electrophoretic profiles of cDNA targets obtained after the amplification of 50 pg, 100 pg, 250 pg, 500 pg, and 1 ng of total RNA using Nugen chemistry.

    Article Snippet: In contrast with other manufacturer protocols, the WT-Ovation™ Pico RNA Amplification System (Nugen) is not based on T7 polymerase cRNA synthesis.

    Techniques: Amplification

    Quality of input RNA and targets synthesised from different amounts of input RNA . A , Bioanalyzer electrophoretic profile of the diluted Universal Human Reference RNA used as input for all amplifications. This profile corresponds to a classical and non-degraded human RNA with two fine characteristic peaks corresponding to 18S and 28S RNAs. 8.4 corresponds to the RNA Integrity Number (RIN) and reflects the high quality of this RNA B , electrophoretic profiles of cRNA obtained after one- and two-round Affymetrix amplification using 2 μg, and 100 ng of input RNA respectively or water as negative control. C , D , E , F , electrophoretic profiles of cRNA or cDNA obtained using Ambion ( C ), Arcturus ( D ), Epicentre ( E ) and Nugen ( F ) amplification systems from 250 pg and 500 pg of input RNA, or water as negative control.

    Journal: BMC Genomics

    Article Title: Evaluation of methods for amplification of picogram amounts of total RNA for whole genome expression profiling

    doi: 10.1186/1471-2164-10-246

    Figure Lengend Snippet: Quality of input RNA and targets synthesised from different amounts of input RNA . A , Bioanalyzer electrophoretic profile of the diluted Universal Human Reference RNA used as input for all amplifications. This profile corresponds to a classical and non-degraded human RNA with two fine characteristic peaks corresponding to 18S and 28S RNAs. 8.4 corresponds to the RNA Integrity Number (RIN) and reflects the high quality of this RNA B , electrophoretic profiles of cRNA obtained after one- and two-round Affymetrix amplification using 2 μg, and 100 ng of input RNA respectively or water as negative control. C , D , E , F , electrophoretic profiles of cRNA or cDNA obtained using Ambion ( C ), Arcturus ( D ), Epicentre ( E ) and Nugen ( F ) amplification systems from 250 pg and 500 pg of input RNA, or water as negative control.

    Article Snippet: In contrast with other manufacturer protocols, the WT-Ovation™ Pico RNA Amplification System (Nugen) is not based on T7 polymerase cRNA synthesis.

    Techniques: Amplification, Negative Control

    Blocking GRP blunts influenza-induced cytokine induction in vivo . WT C57BL/6J mice (5 mice/treatment group) were infected i.n. with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice received vehicle (saline+0.0096% DMSO) or GRP inhibitor (NSC77427; 20 μM/mouse; i.v.) daily from day 2 to day 6 post-infection. On day 7 post-infection, lungs were harvested and total RNA was extracted to measure gene expression by qRT-PCR. ** p

    Journal: Mucosal immunology

    Article Title: Novel Role of Gastric Releasing Peptide (GRP)-Mediated Signaling in the Host Response to Influenza Infection

    doi: 10.1038/s41385-018-0081-9

    Figure Lengend Snippet: Blocking GRP blunts influenza-induced cytokine induction in vivo . WT C57BL/6J mice (5 mice/treatment group) were infected i.n. with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice received vehicle (saline+0.0096% DMSO) or GRP inhibitor (NSC77427; 20 μM/mouse; i.v.) daily from day 2 to day 6 post-infection. On day 7 post-infection, lungs were harvested and total RNA was extracted to measure gene expression by qRT-PCR. ** p

    Article Snippet: Mice and Cotton rats: Six to 8-week old, WT C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME).

    Techniques: Blocking Assay, In Vivo, Mouse Assay, Infection, Expressing, Quantitative RT-PCR

    Influenza infection of mice and cotton rats induces GRP in lungs and serum. (A) WT C57BL/6J mice were infected with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice were euthanized on days 0, 2, 4, 6, and 8 post-infection (3–5 mice/group; * p

    Journal: Mucosal immunology

    Article Title: Novel Role of Gastric Releasing Peptide (GRP)-Mediated Signaling in the Host Response to Influenza Infection

    doi: 10.1038/s41385-018-0081-9

    Figure Lengend Snippet: Influenza infection of mice and cotton rats induces GRP in lungs and serum. (A) WT C57BL/6J mice were infected with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice were euthanized on days 0, 2, 4, 6, and 8 post-infection (3–5 mice/group; * p

    Article Snippet: Mice and Cotton rats: Six to 8-week old, WT C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME).

    Techniques: Infection, Mouse Assay

    Blocking GRP and GRPR enhances survival after influenza infection. (A) WT C57BL/6J mice were infected with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice received vehicle (saline + 0.096% DMSO) or GRP inhibitor (NSC77427; 20 μM, 100 μl i.v./mouse) daily from day 2 to day 6 postinfection. Survival was monitored for 14 days. Data shown are combined results of 3 assays (5 mice/treatment group/experiment). (B) WT C57BL/6J mice were infected as described in (A). Mice received either control IgG or a highly specific anti-GRP IgG (100 μg; 100 μl i.v./mouse) on day 2 and day 4 post-PR8 infection. Survival was monitored as in (A). Data shown are combined results of 2 assays (5 mice/treatment group/experiment). (C) WT C57BL/6J mice were infected as described in (A). Mice received vehicle (saline) or GRPR antagonist (BW2258U89; 20 μM, 100 μl i.v./mouse). Survival was monitored as in (A). Data shown are combined results of 2 assays (5 mice/treatment group/experiment).

    Journal: Mucosal immunology

    Article Title: Novel Role of Gastric Releasing Peptide (GRP)-Mediated Signaling in the Host Response to Influenza Infection

    doi: 10.1038/s41385-018-0081-9

    Figure Lengend Snippet: Blocking GRP and GRPR enhances survival after influenza infection. (A) WT C57BL/6J mice were infected with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice received vehicle (saline + 0.096% DMSO) or GRP inhibitor (NSC77427; 20 μM, 100 μl i.v./mouse) daily from day 2 to day 6 postinfection. Survival was monitored for 14 days. Data shown are combined results of 3 assays (5 mice/treatment group/experiment). (B) WT C57BL/6J mice were infected as described in (A). Mice received either control IgG or a highly specific anti-GRP IgG (100 μg; 100 μl i.v./mouse) on day 2 and day 4 post-PR8 infection. Survival was monitored as in (A). Data shown are combined results of 2 assays (5 mice/treatment group/experiment). (C) WT C57BL/6J mice were infected as described in (A). Mice received vehicle (saline) or GRPR antagonist (BW2258U89; 20 μM, 100 μl i.v./mouse). Survival was monitored as in (A). Data shown are combined results of 2 assays (5 mice/treatment group/experiment).

    Article Snippet: Mice and Cotton rats: Six to 8-week old, WT C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME).

    Techniques: Blocking Assay, Infection, Mouse Assay

    GRP inhibitor, NSC77427, reduces lung pathology after influenza PR8 challenge. WT C57BL/6J mice (5 mice/treatment group) were infected i.n. with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice received vehicle (saline+0.0096% DMSO) or GRP inhibitor (NSC77427; 20 μM/mouse; i.v.) daily from day 2 to day 6 post-infection. On day 7 post-infection, mice were euthanized and lungs were extracted, fixed, and stained for histopathology. Photomicrographs of representative sections were taken at 10x (A, B, and D), and at 40x (C). All scale bars are 100 microns. N = 4 mice/group. * indicates the pleural surface. (E) Quantitation of lung injury is based on the scoring system detailed in the Materials and Methods section. Data shown are mean ± SD. *, p = 0.002

    Journal: Mucosal immunology

    Article Title: Novel Role of Gastric Releasing Peptide (GRP)-Mediated Signaling in the Host Response to Influenza Infection

    doi: 10.1038/s41385-018-0081-9

    Figure Lengend Snippet: GRP inhibitor, NSC77427, reduces lung pathology after influenza PR8 challenge. WT C57BL/6J mice (5 mice/treatment group) were infected i.n. with mouse-adapted influenza strain PR8 (LD 90 ; ~7500 TCID 50 ). Mice received vehicle (saline+0.0096% DMSO) or GRP inhibitor (NSC77427; 20 μM/mouse; i.v.) daily from day 2 to day 6 post-infection. On day 7 post-infection, mice were euthanized and lungs were extracted, fixed, and stained for histopathology. Photomicrographs of representative sections were taken at 10x (A, B, and D), and at 40x (C). All scale bars are 100 microns. N = 4 mice/group. * indicates the pleural surface. (E) Quantitation of lung injury is based on the scoring system detailed in the Materials and Methods section. Data shown are mean ± SD. *, p = 0.002

    Article Snippet: Mice and Cotton rats: Six to 8-week old, WT C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME).

    Techniques: Mouse Assay, Infection, Staining, Histopathology, Quantitation Assay

    Hyaluronidase activity promotes GB37 virulence, and a CovS W297L substitution in GB37 contributes to the decreased hemolysis and increased hyaluronidase activity. (A) Twelve, 6- to 8-week-old wild-type (WT) C57BL/6J mice were given intraperitoneal (IP) injections with ~1 × 10 7 colony-forming units (CFU) of either GB37 or GB37Δ hylB . At approximately 48 hours postinfection, blood, spleens, lungs, and brains were harvested from the infected mice and CFU were enumerated. Pink symbols for GB37 indicate mice that succumbed to the infection within 48 hours. Note that mice infected with GB37 have increased CFU compared with GB37Δ hylB . *, P

    Journal: The Journal of Infectious Diseases

    Article Title: A Nonhemolytic Group B Streptococcus Strain Exhibits Hypervirulence

    doi: 10.1093/infdis/jix646

    Figure Lengend Snippet: Hyaluronidase activity promotes GB37 virulence, and a CovS W297L substitution in GB37 contributes to the decreased hemolysis and increased hyaluronidase activity. (A) Twelve, 6- to 8-week-old wild-type (WT) C57BL/6J mice were given intraperitoneal (IP) injections with ~1 × 10 7 colony-forming units (CFU) of either GB37 or GB37Δ hylB . At approximately 48 hours postinfection, blood, spleens, lungs, and brains were harvested from the infected mice and CFU were enumerated. Pink symbols for GB37 indicate mice that succumbed to the infection within 48 hours. Note that mice infected with GB37 have increased CFU compared with GB37Δ hylB . *, P

    Article Snippet: Wild-type (WT) C57BL/6J mice were purchased from Jackson Laboratories.

    Techniques: Activity Assay, Mouse Assay, Infection

    GB37 exhibits decreased hemolysis and pigmentation but increased virulence and hyaluronidase activity. (A) The zone of clearing around the colonies on sheep blood agar indicates hemolytic activity. GB37 exhibits little-to-no hemolysis compared with GB590, A909, or A909Δ covR . (B) Hemolytic group B streptococci (GBS) strains are pigmented, and nonhemolytic strains are nonpigmented on Granada Media. GB37 exhibits little-to-no pigmentation compared with GB590, A909, or A909Δ covR . (C) Ten, 6- to 8-week-old wild-type (WT) C57BL/6J mice were given intravenous injections with 1 × 10 8 colony-forming units of GB37, GB590, A909, COH1, or hyperhemolytic strains (A909Δ covR , COH1Δ covR ). Kalpan-Meier survival curve shows percentage survival of mice after the infection. Note that mice infected with GB37 succumbed to the infection within 4 days postinoculation in contrast those infected with A909, COH1, and GB590 ( P

    Journal: The Journal of Infectious Diseases

    Article Title: A Nonhemolytic Group B Streptococcus Strain Exhibits Hypervirulence

    doi: 10.1093/infdis/jix646

    Figure Lengend Snippet: GB37 exhibits decreased hemolysis and pigmentation but increased virulence and hyaluronidase activity. (A) The zone of clearing around the colonies on sheep blood agar indicates hemolytic activity. GB37 exhibits little-to-no hemolysis compared with GB590, A909, or A909Δ covR . (B) Hemolytic group B streptococci (GBS) strains are pigmented, and nonhemolytic strains are nonpigmented on Granada Media. GB37 exhibits little-to-no pigmentation compared with GB590, A909, or A909Δ covR . (C) Ten, 6- to 8-week-old wild-type (WT) C57BL/6J mice were given intravenous injections with 1 × 10 8 colony-forming units of GB37, GB590, A909, COH1, or hyperhemolytic strains (A909Δ covR , COH1Δ covR ). Kalpan-Meier survival curve shows percentage survival of mice after the infection. Note that mice infected with GB37 succumbed to the infection within 4 days postinoculation in contrast those infected with A909, COH1, and GB590 ( P

    Article Snippet: Wild-type (WT) C57BL/6J mice were purchased from Jackson Laboratories.

    Techniques: Activity Assay, Mouse Assay, Infection

    IL-25–elicited MPP type2 cells promote Th2 cytokine–dependent responses in vivo. C57BL/6 WT mice (The Jackson Laboratory) were treated i.p. with 0.3 µg IL-25 daily for 7 d. MLNs were harvested, and MPP type2 cells were sort-purified and injected intradermally into naive C57BL/6 WT mice. (a) IL-4, IL-5, and IL-13 cytokine production from skin-draining LN cells from mice receiving intradermal injection of control or IL-25–elicited MPP type2 cells after 48-h αCD3/αCD28 stimulation measured by ELISA. Data in a are representative of two independent experiments. (b–f) C57BL/6 WT mice were treated i.p. with 0.3 µg IL-25 daily for 7 d. MLNs were harvested, and MPP type2 cells were sort-purified and injected into T. muris –infected (INF) Il17rb −/− mice (Charles River). (b) IFN-γ cytokine production by T. muris antigen–stimulated MLN cells. (c) Total serum IgE antibody titers measured by ELISA. (d) Periodic acid–Schiff/Alcian blue–stained colon sections of intestine tissue from naive or infected WT or Il17rb −/− mice ± MPP type2 cells. N, naive (inset). Bars, 100 µm. (e) Goblet cell counts from d. (f) Worm burdens from T. muris –infected mice were assessed at day 21 after infection. Data in b–f are representative of two independent experiments (WT naive, n = 2–3; WT INF, n = 6–8; Il17rb −/− naive, n = 2–3; Il17rb −/− INF, n = 6–7; Il17rb −/− INF + MPP type2 cells, n = 6). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IL-25 simultaneously elicits distinct populations of innate lymphoid cells and multipotent progenitor type 2 (MPPtype2) cells

    doi: 10.1084/jem.20122332

    Figure Lengend Snippet: IL-25–elicited MPP type2 cells promote Th2 cytokine–dependent responses in vivo. C57BL/6 WT mice (The Jackson Laboratory) were treated i.p. with 0.3 µg IL-25 daily for 7 d. MLNs were harvested, and MPP type2 cells were sort-purified and injected intradermally into naive C57BL/6 WT mice. (a) IL-4, IL-5, and IL-13 cytokine production from skin-draining LN cells from mice receiving intradermal injection of control or IL-25–elicited MPP type2 cells after 48-h αCD3/αCD28 stimulation measured by ELISA. Data in a are representative of two independent experiments. (b–f) C57BL/6 WT mice were treated i.p. with 0.3 µg IL-25 daily for 7 d. MLNs were harvested, and MPP type2 cells were sort-purified and injected into T. muris –infected (INF) Il17rb −/− mice (Charles River). (b) IFN-γ cytokine production by T. muris antigen–stimulated MLN cells. (c) Total serum IgE antibody titers measured by ELISA. (d) Periodic acid–Schiff/Alcian blue–stained colon sections of intestine tissue from naive or infected WT or Il17rb −/− mice ± MPP type2 cells. N, naive (inset). Bars, 100 µm. (e) Goblet cell counts from d. (f) Worm burdens from T. muris –infected mice were assessed at day 21 after infection. Data in b–f are representative of two independent experiments (WT naive, n = 2–3; WT INF, n = 6–8; Il17rb −/− naive, n = 2–3; Il17rb −/− INF, n = 6–7; Il17rb −/− INF + MPP type2 cells, n = 6). *, P

    Article Snippet: WT BALB/c and WT C57BL/6 and Rag1−/− mice were obtained from Jackson Laboratory, and C57BL/6 Ly5.2/Cr (CD45.1) mice were obtained from the National Cancer Institute.

    Techniques: In Vivo, Mouse Assay, Purification, Injection, Enzyme-linked Immunosorbent Assay, Infection, Staining

    IL-25 elicits ILC2 and MPP type2 cells independent of IL-33 signaling. (a–d) C57BL/6 WT mice and C57BL/6 Il33 −/− mice (Taconic) were treated i.p. with PBS (control) or 0.3 µg of recombinant IL-25 daily for 7 d. MLNs were harvested at day 7, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a and c) Frequency of ILC2 in control or IL-25–treated WT (a) or Il33 −/− mice (c). (b and d) Total cell numbers of ILC2 in control or IL-25–treated WT (b) or Il33 −/− mice (d). (e and g) Frequency of MPP type2 cells in control or IL-25–treated WT (e) or Il33 −/− mice (g). (f and h) Total cell numbers of MPP type2 cells in control or IL-25–treated WT (f) or Il33 −/− mice (h). Data in a–h are representative of two independent experiments (control, n = 4; IL-25–treated WT, n = 6; IL-25–treated Il33 −/− , n = 6). (i–l) BALB/c WT mice (The Jackson Laboratory) were treated with PBS (control) or 0.3 µg of recombinant IL-25 plus isotype (IgG) or anti-T1/ST2 mAbs. MLNs were harvested at day 7, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. Frequency and total numbers of T1/ST2 pos IL-7Rα pos ILC2 (i and j) and T1/ST2 neg IL-7Rα neg c-kit pos MPP type2 cells (k and l). Plots are gated on live, Lin neg (CD4, CD8α, CD11b, CD11c, and CD19) cells. Data in i–l are representative of two independent experiments (control, n = 6; IL-25 treated, n = 9; anti-T1/ST2 IL-25 treated, n = 9). Error bars indicate SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: IL-25 simultaneously elicits distinct populations of innate lymphoid cells and multipotent progenitor type 2 (MPPtype2) cells

    doi: 10.1084/jem.20122332

    Figure Lengend Snippet: IL-25 elicits ILC2 and MPP type2 cells independent of IL-33 signaling. (a–d) C57BL/6 WT mice and C57BL/6 Il33 −/− mice (Taconic) were treated i.p. with PBS (control) or 0.3 µg of recombinant IL-25 daily for 7 d. MLNs were harvested at day 7, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a and c) Frequency of ILC2 in control or IL-25–treated WT (a) or Il33 −/− mice (c). (b and d) Total cell numbers of ILC2 in control or IL-25–treated WT (b) or Il33 −/− mice (d). (e and g) Frequency of MPP type2 cells in control or IL-25–treated WT (e) or Il33 −/− mice (g). (f and h) Total cell numbers of MPP type2 cells in control or IL-25–treated WT (f) or Il33 −/− mice (h). Data in a–h are representative of two independent experiments (control, n = 4; IL-25–treated WT, n = 6; IL-25–treated Il33 −/− , n = 6). (i–l) BALB/c WT mice (The Jackson Laboratory) were treated with PBS (control) or 0.3 µg of recombinant IL-25 plus isotype (IgG) or anti-T1/ST2 mAbs. MLNs were harvested at day 7, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. Frequency and total numbers of T1/ST2 pos IL-7Rα pos ILC2 (i and j) and T1/ST2 neg IL-7Rα neg c-kit pos MPP type2 cells (k and l). Plots are gated on live, Lin neg (CD4, CD8α, CD11b, CD11c, and CD19) cells. Data in i–l are representative of two independent experiments (control, n = 6; IL-25 treated, n = 9; anti-T1/ST2 IL-25 treated, n = 9). Error bars indicate SEM.

    Article Snippet: WT BALB/c and WT C57BL/6 and Rag1−/− mice were obtained from Jackson Laboratory, and C57BL/6 Ly5.2/Cr (CD45.1) mice were obtained from the National Cancer Institute.

    Techniques: Mouse Assay, Recombinant, Flow Cytometry, Cytometry

    IL-25–mediated induction of MPP type2 cells and type 2 inflammation occurs independently of ILC2. (a–i) C57BL/6 Rag1 −/− mice (The Jackson Laboratory) were treated i.p. with PBS (control) or 0.3 µg of IL-25 plus isotype mAb (Iso.) or anti-CD90 mAb. (a and b) Frequencies (a) and total cell numbers (b) of CD90 pos T1/ST2 pos IL-7Rα pos ILC2 in the MLNs of control or IL-25–treated mice given either isotype or anti-CD90 mAb. (c and d) Frequencies (c) and total cell numbers (d) of CD90 neg T1/ST2 neg IL-7Rα neg MPP type2 cells in the MLNs of control or IL-25–treated mice given either isotype or anti-CD90 mAb. Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) cells or as indicated. (e and f) Quantitative real-time PCR of Il4 , Il5 , and Il13 gene expression levels from lung (e) or small intestinal tissue (f) of control or IL-25–treated mice receiving either isotype or anti-CD90 mAb. RQ, relative quantification. (g) Periodic acid–Schiff/Alcian blue staining of lung and small intestine (SI) tissue from control or IL-25–treated mice receiving either isotype or anti-CD90 mAb. Bars: (top) 50 µm; (bottom) 100 µm. (h) Goblet cell (GC) counts from g. nd, not detected. (i) Frequency and total cell numbers of eosinophils in the lung from control or IL-25–treated mice given either isotype or anti-CD90 mAb. Frequency is percentage of SSC hi SiglecF pos cells gated on live, Lin neg CD11b pos cells. Data in a–i are representative of three independent experiments (control + Iso., n = 6; control + anti-CD90, n = 6; IL-25 treated + Iso., n = 6; IL-25 treated + anti-CD90, n = 6). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IL-25 simultaneously elicits distinct populations of innate lymphoid cells and multipotent progenitor type 2 (MPPtype2) cells

    doi: 10.1084/jem.20122332

    Figure Lengend Snippet: IL-25–mediated induction of MPP type2 cells and type 2 inflammation occurs independently of ILC2. (a–i) C57BL/6 Rag1 −/− mice (The Jackson Laboratory) were treated i.p. with PBS (control) or 0.3 µg of IL-25 plus isotype mAb (Iso.) or anti-CD90 mAb. (a and b) Frequencies (a) and total cell numbers (b) of CD90 pos T1/ST2 pos IL-7Rα pos ILC2 in the MLNs of control or IL-25–treated mice given either isotype or anti-CD90 mAb. (c and d) Frequencies (c) and total cell numbers (d) of CD90 neg T1/ST2 neg IL-7Rα neg MPP type2 cells in the MLNs of control or IL-25–treated mice given either isotype or anti-CD90 mAb. Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) cells or as indicated. (e and f) Quantitative real-time PCR of Il4 , Il5 , and Il13 gene expression levels from lung (e) or small intestinal tissue (f) of control or IL-25–treated mice receiving either isotype or anti-CD90 mAb. RQ, relative quantification. (g) Periodic acid–Schiff/Alcian blue staining of lung and small intestine (SI) tissue from control or IL-25–treated mice receiving either isotype or anti-CD90 mAb. Bars: (top) 50 µm; (bottom) 100 µm. (h) Goblet cell (GC) counts from g. nd, not detected. (i) Frequency and total cell numbers of eosinophils in the lung from control or IL-25–treated mice given either isotype or anti-CD90 mAb. Frequency is percentage of SSC hi SiglecF pos cells gated on live, Lin neg CD11b pos cells. Data in a–i are representative of three independent experiments (control + Iso., n = 6; control + anti-CD90, n = 6; IL-25 treated + Iso., n = 6; IL-25 treated + anti-CD90, n = 6). *, P

    Article Snippet: WT BALB/c and WT C57BL/6 and Rag1−/− mice were obtained from Jackson Laboratory, and C57BL/6 Ly5.2/Cr (CD45.1) mice were obtained from the National Cancer Institute.

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Staining

    IL-25–elicited MPP type2 cells possess a unique transcriptional profile from ILC2 cells. (a and b) C57BL/6 WT mice (The Jackson Laboratory) were treated with 0.3 µg of recombinant IL-25 daily for 7 d. MLNs and PECs were harvested from IL-25–treated mice and MPP type2 cells (Lin neg T1/ST2 neg IL-7Rα neg CD4 neg CD90 neg CD25 neg c-kit pos ; a) were FACS purified to ≥95% purity (b). Three biological replicates of MPP type2 cells were collected, and mRNA was isolated, amplified, and hybridized to Affymetrix gene chips for microarray analysis. The previously published microarray gene expression profile of lung-resident ILC2 was used for comparison (GEO series no. GSE46468 ; Monticelli et al., 2011 ). (c) Heat map representing gene expression profiles of the top 100 differentially expressed genes between MPP type2 cells and ILC2. Red indicates high expression, and blue indicates low expression. (d) GSEA comparing the gene expression signatures of MPP type2 cells and ILC2. (e) List of leading edge genes from GSEA analysis from d. (f) PCA plot comparing transcriptional profiles for MPP type2 cells (1), ex vivo nuocytes (2; GSE25890 ), NHCs (3; GSE18752 ), lung-resident ILC2 (4; GSE46468 ), unstimulated lung NHCs (5; GSE36057 ), splenic LTi cells (6, GSE46468 ; and 7, GSE18752 ), and BM-GMPs (8). Categories of ILC2 (green shaded area), ILC3 (red shaded area), and progenitors (blue shaded area) were grouped (as indicated in f, dashed lines), and Euclidean distance measurements between MPP type2 cells and BM-GMP versus ILC2 or ILC3 were calculated (g). ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: IL-25 simultaneously elicits distinct populations of innate lymphoid cells and multipotent progenitor type 2 (MPPtype2) cells

    doi: 10.1084/jem.20122332

    Figure Lengend Snippet: IL-25–elicited MPP type2 cells possess a unique transcriptional profile from ILC2 cells. (a and b) C57BL/6 WT mice (The Jackson Laboratory) were treated with 0.3 µg of recombinant IL-25 daily for 7 d. MLNs and PECs were harvested from IL-25–treated mice and MPP type2 cells (Lin neg T1/ST2 neg IL-7Rα neg CD4 neg CD90 neg CD25 neg c-kit pos ; a) were FACS purified to ≥95% purity (b). Three biological replicates of MPP type2 cells were collected, and mRNA was isolated, amplified, and hybridized to Affymetrix gene chips for microarray analysis. The previously published microarray gene expression profile of lung-resident ILC2 was used for comparison (GEO series no. GSE46468 ; Monticelli et al., 2011 ). (c) Heat map representing gene expression profiles of the top 100 differentially expressed genes between MPP type2 cells and ILC2. Red indicates high expression, and blue indicates low expression. (d) GSEA comparing the gene expression signatures of MPP type2 cells and ILC2. (e) List of leading edge genes from GSEA analysis from d. (f) PCA plot comparing transcriptional profiles for MPP type2 cells (1), ex vivo nuocytes (2; GSE25890 ), NHCs (3; GSE18752 ), lung-resident ILC2 (4; GSE46468 ), unstimulated lung NHCs (5; GSE36057 ), splenic LTi cells (6, GSE46468 ; and 7, GSE18752 ), and BM-GMPs (8). Categories of ILC2 (green shaded area), ILC3 (red shaded area), and progenitors (blue shaded area) were grouped (as indicated in f, dashed lines), and Euclidean distance measurements between MPP type2 cells and BM-GMP versus ILC2 or ILC3 were calculated (g). ***, P

    Article Snippet: WT BALB/c and WT C57BL/6 and Rag1−/− mice were obtained from Jackson Laboratory, and C57BL/6 Ly5.2/Cr (CD45.1) mice were obtained from the National Cancer Institute.

    Techniques: Mouse Assay, Recombinant, FACS, Purification, Isolation, Amplification, Microarray, Expressing, Ex Vivo

    IL-25 simultaneously elicits phenotypically distinct populations of MPP type2 cells and ILC2. (a–r) C57BL/6 WT mice (The Jackson Laboratory) were treated i.p. with PBS (control), 0.3 µg of recombinant IL-33, or 0.3 µg of recombinant IL-25 daily for 2, 4, or 7 d. Indicated tissues were harvested at day 7 (or as indicated), and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a) Frequency of T1/ST2 pos IL-7Rα pos ILC2 in the Lin neg cell compartment from the MLNs of control or IL-33–treated mice. (b) CD90.2 (Thy1.2) and CD25 expression on ILC2 (black histograms) from IL-33–treated mice. (c) Total cell numbers of ILC2 from control or IL-33–treated mice. (d) Expression of c-kit on gated T1/ST2 neg IL-7Rα neg cells from control or IL-33–treated mice. (e) CD90 and CD25 expression on c-kit pos MPP type2 cells (black histograms) from IL-33–treated mice. (f) Total cell numbers of MPP type2 cells from control or IL-33–treated mice. (g) Frequency of ILC2 in the Lin neg cell compartment from the MLN of control or IL-25–treated mice. (h) CD90 and CD25 expression on ILC2 (black histograms) from IL-25–treated mice. (i) Total cell numbers of ILC2 from control or IL-25–treated mice. (j) Expression of c-kit on gated T1/ST2 neg IL-7Rα neg cells from control or IL-25–treated mice. (k) CD90 and CD25 expression on c-kit pos MPP type2 cells (black histograms) from IL-25–treated mice. (l) Total cell numbers of MPP type2 cells from control or IL-25–treated mice. Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) cells or as indicated. Gray shaded histograms represent CD90 or CD25 expression on Lin neg cells from control mice. (m and n) Frequencies of ILC2 (m) or MPP type2 cells (n) as gated in a, g, d, and i from the MLNs of control (open bars), IL-33–treated, or IL-25–treated mice at days 2, 4, and 6. (o–r) Frequencies of ILC2 and MPP type2 cells as gated in a and g and d and i from the blood (o), caudal LN (p), lung (q), and PEC (r) of control (open bars), IL-33–treated (black bars), or IL-25–treated (gray bars) mice at day 6. Data in a–n are representative of two or more independent experiments (control, n = 4; IL-33 treated, n = 8; IL-25 treated, n = 8). Data in o–r are representative of two independent experiments (control, n = 4; IL-25 treated, n = 8; IL-33 treated, n = 8). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: IL-25 simultaneously elicits distinct populations of innate lymphoid cells and multipotent progenitor type 2 (MPPtype2) cells

    doi: 10.1084/jem.20122332

    Figure Lengend Snippet: IL-25 simultaneously elicits phenotypically distinct populations of MPP type2 cells and ILC2. (a–r) C57BL/6 WT mice (The Jackson Laboratory) were treated i.p. with PBS (control), 0.3 µg of recombinant IL-33, or 0.3 µg of recombinant IL-25 daily for 2, 4, or 7 d. Indicated tissues were harvested at day 7 (or as indicated), and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a) Frequency of T1/ST2 pos IL-7Rα pos ILC2 in the Lin neg cell compartment from the MLNs of control or IL-33–treated mice. (b) CD90.2 (Thy1.2) and CD25 expression on ILC2 (black histograms) from IL-33–treated mice. (c) Total cell numbers of ILC2 from control or IL-33–treated mice. (d) Expression of c-kit on gated T1/ST2 neg IL-7Rα neg cells from control or IL-33–treated mice. (e) CD90 and CD25 expression on c-kit pos MPP type2 cells (black histograms) from IL-33–treated mice. (f) Total cell numbers of MPP type2 cells from control or IL-33–treated mice. (g) Frequency of ILC2 in the Lin neg cell compartment from the MLN of control or IL-25–treated mice. (h) CD90 and CD25 expression on ILC2 (black histograms) from IL-25–treated mice. (i) Total cell numbers of ILC2 from control or IL-25–treated mice. (j) Expression of c-kit on gated T1/ST2 neg IL-7Rα neg cells from control or IL-25–treated mice. (k) CD90 and CD25 expression on c-kit pos MPP type2 cells (black histograms) from IL-25–treated mice. (l) Total cell numbers of MPP type2 cells from control or IL-25–treated mice. Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) cells or as indicated. Gray shaded histograms represent CD90 or CD25 expression on Lin neg cells from control mice. (m and n) Frequencies of ILC2 (m) or MPP type2 cells (n) as gated in a, g, d, and i from the MLNs of control (open bars), IL-33–treated, or IL-25–treated mice at days 2, 4, and 6. (o–r) Frequencies of ILC2 and MPP type2 cells as gated in a and g and d and i from the blood (o), caudal LN (p), lung (q), and PEC (r) of control (open bars), IL-33–treated (black bars), or IL-25–treated (gray bars) mice at day 6. Data in a–n are representative of two or more independent experiments (control, n = 4; IL-33 treated, n = 8; IL-25 treated, n = 8). Data in o–r are representative of two independent experiments (control, n = 4; IL-25 treated, n = 8; IL-33 treated, n = 8). *, P

    Article Snippet: WT BALB/c and WT C57BL/6 and Rag1−/− mice were obtained from Jackson Laboratory, and C57BL/6 Ly5.2/Cr (CD45.1) mice were obtained from the National Cancer Institute.

    Techniques: Mouse Assay, Recombinant, Flow Cytometry, Cytometry, Expressing

    IL-25–mediated induction of MPP type2 cells occurs independently of Id2. (a–p) CD5/B220-depleted donor BM (WT, CD45.1; Id2 −/− , CD45.1.2) was transferred into lethally irradiated C57BL/6 WT (CD45.2) mice. After reconstitution for 8 wk after transplant, mice were treated i.p. with PBS (control), 0.3 µg of recombinant IL-33, or 0.3 µg of recombinant IL-25 daily for 7 d. MLNs were harvested, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a and c) Frequency of T1/ST2 pos IL-7Rα pos ILC2 from control or IL-33–treated WT BM chimera mice (a) or Id2-deficient BM chimera mice (c). (b and d) Total cell numbers of ILC2 from control or IL-33–treated WT BM chimera mice (b) or Id2-deficient BM chimera mice (d). (e and g) Frequency of c-kit pos MPP type2 cells from control or IL-33–treated WT BM chimera mice (e) or Id2-deficient BM chimera mice (g). (f and h) Total cell numbers of MPP type2 cells from control or IL-33–treated WT BM chimera mice (f) or Id2-deficient BM chimera mice (h). (i and k) Frequency of T1/ST2 pos IL-7Rα pos ILC2 cells from control or IL-25–treated WT BM chimera mice (i) or Id2-deficient BM chimera mice (k). (j and l) Total cell numbers of ILC2 from control or IL-25–treated WT BM chimera mice (j) or Id2-deficient BM chimera mice (l). (m and o) Frequency of c-kit pos MPP type2 cells from control or IL-25–treated WT BM chimera mice (m) or Id2-deficient BM chimera mice (o). (n and p) Total cell numbers of MPP type2 cells from control or IL-25–treated WT BM chimera mice (n) or Id2-deficient BM chimera mice (p). Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) donor-derived cells. Data in a–p are representative of two or more independent experiments (control, n = 4; IL-33 treated, n = 6; IL-25 treated, n = 6). Error bars indicate SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: IL-25 simultaneously elicits distinct populations of innate lymphoid cells and multipotent progenitor type 2 (MPPtype2) cells

    doi: 10.1084/jem.20122332

    Figure Lengend Snippet: IL-25–mediated induction of MPP type2 cells occurs independently of Id2. (a–p) CD5/B220-depleted donor BM (WT, CD45.1; Id2 −/− , CD45.1.2) was transferred into lethally irradiated C57BL/6 WT (CD45.2) mice. After reconstitution for 8 wk after transplant, mice were treated i.p. with PBS (control), 0.3 µg of recombinant IL-33, or 0.3 µg of recombinant IL-25 daily for 7 d. MLNs were harvested, and the frequency and total cell numbers of ILC2 and MPP type2 cells were assessed by flow cytometry. (a and c) Frequency of T1/ST2 pos IL-7Rα pos ILC2 from control or IL-33–treated WT BM chimera mice (a) or Id2-deficient BM chimera mice (c). (b and d) Total cell numbers of ILC2 from control or IL-33–treated WT BM chimera mice (b) or Id2-deficient BM chimera mice (d). (e and g) Frequency of c-kit pos MPP type2 cells from control or IL-33–treated WT BM chimera mice (e) or Id2-deficient BM chimera mice (g). (f and h) Total cell numbers of MPP type2 cells from control or IL-33–treated WT BM chimera mice (f) or Id2-deficient BM chimera mice (h). (i and k) Frequency of T1/ST2 pos IL-7Rα pos ILC2 cells from control or IL-25–treated WT BM chimera mice (i) or Id2-deficient BM chimera mice (k). (j and l) Total cell numbers of ILC2 from control or IL-25–treated WT BM chimera mice (j) or Id2-deficient BM chimera mice (l). (m and o) Frequency of c-kit pos MPP type2 cells from control or IL-25–treated WT BM chimera mice (m) or Id2-deficient BM chimera mice (o). (n and p) Total cell numbers of MPP type2 cells from control or IL-25–treated WT BM chimera mice (n) or Id2-deficient BM chimera mice (p). Plots are gated on live, Lin neg (CD4, CD8, CD11b, CD11c, and CD19) donor-derived cells. Data in a–p are representative of two or more independent experiments (control, n = 4; IL-33 treated, n = 6; IL-25 treated, n = 6). Error bars indicate SEM.

    Article Snippet: WT BALB/c and WT C57BL/6 and Rag1−/− mice were obtained from Jackson Laboratory, and C57BL/6 Ly5.2/Cr (CD45.1) mice were obtained from the National Cancer Institute.

    Techniques: Irradiation, Mouse Assay, Recombinant, Flow Cytometry, Cytometry, Derivative Assay

    Anti-CD49d antibody injections did not affect plaque load in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed, serially sectioned, and immunostained using anti-Aβ (4G8) antibody. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets. Quantitation of immunostaining was performed and O.D. values were averaged and plotted ± SD.

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Anti-CD49d antibody injections did not affect plaque load in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed, serially sectioned, and immunostained using anti-Aβ (4G8) antibody. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets. Quantitation of immunostaining was performed and O.D. values were averaged and plotted ± SD.

    Article Snippet: Cg-Tg (APPswe, PSEN1dE9)85Dbo/ Mmjax and wild-type mouse line, C57BL/6 (WT) were originally obtained from the Jackson Laboratory (Bar Harbor, Maine).

    Techniques: Mouse Assay, Injection, Purification, Quantitation Assay, Immunostaining

    Anti-CD49d antibody injections altered post-synaptic proteins APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenous (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Parietal cortex from left brain hemispheres was dissected out, lysed and western blotted using anti-PSD95, anti-synaptophysin and anti-Cox-2 antibodies with anti-α-tubulin as loading control. Optical densities for each antibody were normalized, averaged and graphed ± SD from 3-6 animal per group (*p

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Anti-CD49d antibody injections altered post-synaptic proteins APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenous (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Parietal cortex from left brain hemispheres was dissected out, lysed and western blotted using anti-PSD95, anti-synaptophysin and anti-Cox-2 antibodies with anti-α-tubulin as loading control. Optical densities for each antibody were normalized, averaged and graphed ± SD from 3-6 animal per group (*p

    Article Snippet: Cg-Tg (APPswe, PSEN1dE9)85Dbo/ Mmjax and wild-type mouse line, C57BL/6 (WT) were originally obtained from the Jackson Laboratory (Bar Harbor, Maine).

    Techniques: Mouse Assay, Injection, Purification, Western Blot

    Both the isotype control and anti-CD49d antibody injections affected brain CD4 immunoreactivity. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. The presence of IgG2b antibody in the brain was assessed by immunostaining using biotinylated anti-mouse IgG2b antibody (8A) . The presence of T cells in the brain was visualized using anti-CD4 antibody and immunoreactivity observed in the striatum (8B) and parietal cortex (8C) was imaged. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets.

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Both the isotype control and anti-CD49d antibody injections affected brain CD4 immunoreactivity. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. The presence of IgG2b antibody in the brain was assessed by immunostaining using biotinylated anti-mouse IgG2b antibody (8A) . The presence of T cells in the brain was visualized using anti-CD4 antibody and immunoreactivity observed in the striatum (8B) and parietal cortex (8C) was imaged. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets.

    Article Snippet: Cg-Tg (APPswe, PSEN1dE9)85Dbo/ Mmjax and wild-type mouse line, C57BL/6 (WT) were originally obtained from the Jackson Laboratory (Bar Harbor, Maine).

    Techniques: Mouse Assay, Injection, Purification, Immunostaining

    Both the isotype control (I.C.) and anti-CD49d antibody injections reduced microgliosis in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. Microgliosis was assessed by immunostaining using anti-CD68 (3A) and anti-Iba-1 (3B) antibodies and total number of resting microglia were assessed using anti-TMEM119 antibody (3C) . Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets. Quantitation of immunostaining was performed and O.D. values were averaged and plotted ± SD.

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Both the isotype control (I.C.) and anti-CD49d antibody injections reduced microgliosis in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. Microgliosis was assessed by immunostaining using anti-CD68 (3A) and anti-Iba-1 (3B) antibodies and total number of resting microglia were assessed using anti-TMEM119 antibody (3C) . Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets. Quantitation of immunostaining was performed and O.D. values were averaged and plotted ± SD.

    Article Snippet: Cg-Tg (APPswe, PSEN1dE9)85Dbo/ Mmjax and wild-type mouse line, C57BL/6 (WT) were originally obtained from the Jackson Laboratory (Bar Harbor, Maine).

    Techniques: Mouse Assay, Injection, Purification, Immunostaining, Quantitation Assay

    Anti-CD49d injections reduced IL-12 and IFN-γ levels in the brain. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. A portion of parietal cortex from the left brain hemispheres was dissected out, lysed, and used for multiple cytokine measurements using ELISA. Cytokine levels were determined from 3-6 animals per group ± SD (*p

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Anti-CD49d injections reduced IL-12 and IFN-γ levels in the brain. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. A portion of parietal cortex from the left brain hemispheres was dissected out, lysed, and used for multiple cytokine measurements using ELISA. Cytokine levels were determined from 3-6 animals per group ± SD (*p

    Article Snippet: Cg-Tg (APPswe, PSEN1dE9)85Dbo/ Mmjax and wild-type mouse line, C57BL/6 (WT) were originally obtained from the Jackson Laboratory (Bar Harbor, Maine).

    Techniques: Mouse Assay, Injection, Purification, Enzyme-linked Immunosorbent Assay

    IgG and anti-CD49d injections reduced multiple cytokine levels in the spleen. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Spleens were dissected out, lysed, and used for multiple cytokine measurements using ELISAs. Cytokine levels were determined from 3-6 animals per group ± SD (*p

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: IgG and anti-CD49d injections reduced multiple cytokine levels in the spleen. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Spleens were dissected out, lysed, and used for multiple cytokine measurements using ELISAs. Cytokine levels were determined from 3-6 animals per group ± SD (*p

    Article Snippet: Cg-Tg (APPswe, PSEN1dE9)85Dbo/ Mmjax and wild-type mouse line, C57BL/6 (WT) were originally obtained from the Jackson Laboratory (Bar Harbor, Maine).

    Techniques: Mouse Assay, Injection, Purification

    Anti-CD49d injections did not alter Aβ 1-40 and Aβ 1-42 levels in the brain. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Part of the parietal cortex from left brain hemispheres was dissected out, lysed and used for soluble and insoluble Aβ 1-42 and Aβ 1-40 measurements using ELISA. Aβ levels were determined from 3-6 animals per group ± SD (*p

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Anti-CD49d injections did not alter Aβ 1-40 and Aβ 1-42 levels in the brain. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Part of the parietal cortex from left brain hemispheres was dissected out, lysed and used for soluble and insoluble Aβ 1-42 and Aβ 1-40 measurements using ELISA. Aβ levels were determined from 3-6 animals per group ± SD (*p

    Article Snippet: Cg-Tg (APPswe, PSEN1dE9)85Dbo/ Mmjax and wild-type mouse line, C57BL/6 (WT) were originally obtained from the Jackson Laboratory (Bar Harbor, Maine).

    Techniques: Mouse Assay, Injection, Purification, Enzyme-linked Immunosorbent Assay

    Anti-CD49d injections reduced astrogliosis in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. Astrogliosis was assessed by immunostaining using anti-GFAP antibody (4A) . Representative images from 3-6 animals per group are shown at 20X magnification. Parietal cortex from left brain hemispheres was dissected out, lysed and western blotted using anti-GFAP antibody (4B) with anti-α-tubulin as loading control. Optical densities for each antibody were normalized, averaged and graphed ± SD from 3-6 animal per group (*p

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Anti-CD49d injections reduced astrogliosis in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. Astrogliosis was assessed by immunostaining using anti-GFAP antibody (4A) . Representative images from 3-6 animals per group are shown at 20X magnification. Parietal cortex from left brain hemispheres was dissected out, lysed and western blotted using anti-GFAP antibody (4B) with anti-α-tubulin as loading control. Optical densities for each antibody were normalized, averaged and graphed ± SD from 3-6 animal per group (*p

    Article Snippet: Cg-Tg (APPswe, PSEN1dE9)85Dbo/ Mmjax and wild-type mouse line, C57BL/6 (WT) were originally obtained from the Jackson Laboratory (Bar Harbor, Maine).

    Techniques: Mouse Assay, Injection, Purification, Immunostaining, Western Blot

    Neutrophils are the predominant peritoneal cellular subset that express pro-IL-1β + at early time points following P. aeruginosa infection. C57BL/6 ( n = 15) and ASC −/− ( n = 15) mice were infected intraperitoneally with a sublethal dose (3 × 10 6 CFU) of WT PA14. Mice were euthanized 4 h postinfection and peritoneal lavage was collected. A : representative dot plots depicting the gating scheme for pro-IL-1β + cells at 4 h postinfection. Bacteria were excluded by gating. B : percentage of pro-IL-1β + Ly6G + ( left ) and pro-IL-1β + F4/80 + ( right ) cells out of the total peritoneal cells at 4 h postinfection. C : percentage of Ly6G + F4/80 − ( left ) and Ly6G − F4/80 + ( right ) within the gated CD45 + pro-IL-1β + population as depicted in A above. Data are expressed as means ± SD from 5 independent biological experiments.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Differential ASC requirements reveal a key role for neutrophils and a noncanonical IL-1β response to Pseudomonas aeruginosa

    doi: 10.1152/ajplung.00228.2015

    Figure Lengend Snippet: Neutrophils are the predominant peritoneal cellular subset that express pro-IL-1β + at early time points following P. aeruginosa infection. C57BL/6 ( n = 15) and ASC −/− ( n = 15) mice were infected intraperitoneally with a sublethal dose (3 × 10 6 CFU) of WT PA14. Mice were euthanized 4 h postinfection and peritoneal lavage was collected. A : representative dot plots depicting the gating scheme for pro-IL-1β + cells at 4 h postinfection. Bacteria were excluded by gating. B : percentage of pro-IL-1β + Ly6G + ( left ) and pro-IL-1β + F4/80 + ( right ) cells out of the total peritoneal cells at 4 h postinfection. C : percentage of Ly6G + F4/80 − ( left ) and Ly6G − F4/80 + ( right ) within the gated CD45 + pro-IL-1β + population as depicted in A above. Data are expressed as means ± SD from 5 independent biological experiments.

    Article Snippet: C57BL/6 WT mice were obtained from the National Cancer Institute (Bethesda, MD) or Charles River Laboratories (Frederick, MD).

    Techniques: Infection, Mouse Assay

    ASC −/− mice are not protected from P. aeruginosa lethal challenge and produce comparable levels of elicited in vivo IL-1β. A : C57BL/6 and ASC −/− ( n = 7/group) were infected intratracheally with 5 × 10 6 colony-forming units (CFU) of WT PA14 and survival was monitored up to 96 h postinfection. Survival curves were compared using the log-rank test. B : C57BL/6 and ASC −/− ( n = 7/group) mice were infected intratracheally with a sublethal dose (3 × 10 6 CFU) of WT PA14. C57BL/6 mice instilled with PBS ( n = 5) were used as a control. Mice were euthanized 4 h postinfection and bronchoalveolar lavage (BAL) samples were collected. BAL samples were analyzed by ELISA for IL-1β production. C : C57BL/6 ( n = 14) and ASC −/− ( n = 9) mice were infected intraperitoneally with 5 × 10 6 CFU of WT PA14 and survival was monitored up to 96 h postinfection. C57BL/6 mice were challenged with 5 × 10 6 CFU of popB mutant PA14 ( n = 8) as a control. Survival curves were compared using the log-rank test. D : C57BL/6 and ASC −/− mice were infected intraperitoneally (IP) with a sublethal dose (3 × 10 6 CFU) of WT PA14. Mice were euthanized 4 h postinfection and peritoneal lavage and blood samples were collected. Peritoneal lavage ( top ; n = 15/group) and blood serum ( bottom ; n = 11/group) samples were analyzed by ELISA for IL-1β production. Data are expressed as means ± SD. E : representative Western blot for pro- and the biologically active cleaved mature IL-1β released in the peritoneum at 4 h postinfection. Results are representative of, or accumulated from, 2–5 independent experiments.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Differential ASC requirements reveal a key role for neutrophils and a noncanonical IL-1β response to Pseudomonas aeruginosa

    doi: 10.1152/ajplung.00228.2015

    Figure Lengend Snippet: ASC −/− mice are not protected from P. aeruginosa lethal challenge and produce comparable levels of elicited in vivo IL-1β. A : C57BL/6 and ASC −/− ( n = 7/group) were infected intratracheally with 5 × 10 6 colony-forming units (CFU) of WT PA14 and survival was monitored up to 96 h postinfection. Survival curves were compared using the log-rank test. B : C57BL/6 and ASC −/− ( n = 7/group) mice were infected intratracheally with a sublethal dose (3 × 10 6 CFU) of WT PA14. C57BL/6 mice instilled with PBS ( n = 5) were used as a control. Mice were euthanized 4 h postinfection and bronchoalveolar lavage (BAL) samples were collected. BAL samples were analyzed by ELISA for IL-1β production. C : C57BL/6 ( n = 14) and ASC −/− ( n = 9) mice were infected intraperitoneally with 5 × 10 6 CFU of WT PA14 and survival was monitored up to 96 h postinfection. C57BL/6 mice were challenged with 5 × 10 6 CFU of popB mutant PA14 ( n = 8) as a control. Survival curves were compared using the log-rank test. D : C57BL/6 and ASC −/− mice were infected intraperitoneally (IP) with a sublethal dose (3 × 10 6 CFU) of WT PA14. Mice were euthanized 4 h postinfection and peritoneal lavage and blood samples were collected. Peritoneal lavage ( top ; n = 15/group) and blood serum ( bottom ; n = 11/group) samples were analyzed by ELISA for IL-1β production. Data are expressed as means ± SD. E : representative Western blot for pro- and the biologically active cleaved mature IL-1β released in the peritoneum at 4 h postinfection. Results are representative of, or accumulated from, 2–5 independent experiments.

    Article Snippet: C57BL/6 WT mice were obtained from the National Cancer Institute (Bethesda, MD) or Charles River Laboratories (Frederick, MD).

    Techniques: Mouse Assay, In Vivo, Infection, Enzyme-linked Immunosorbent Assay, Mutagenesis, Western Blot

    Neutropenia leads to reduced peritoneal IL-1β production in response to P. aeruginosa . C57BL/6 and ASC −/− mice were depleted of neutrophils with anti-Ly6G IgG or treated with isotype control antibody ( A , C , and D ). A : representative analyses of efficacy of neutrophil depletion in circulating blood and spleens from the treatment groups. B : C57BL/6 mice were infected intraperitoneally with the indicated CFU of WT PA14. IL-1β content within the peritoneal lavage samples were subsequently assayed by ELISA( n = 2/group). C and D : C57BL/6 and ASC −/− mice were treated with isotype control antibody or depleted of neutrophils with anti-Ly6G antibody as described above. Mice were subsequently infected intraperitoneally with 3 × 10 6 CFU of WT PA14, euthanized 4 h postinfection, and peritoneal lavage and blood samples were collected for IL-1β analysis by ELISA. The peritoneal lavage fluid was analyzed for the total number of recovered CFU normalized to CFU of input bacteria. C : IL-1β from peritoneal lavage ( left ), blood serum ( middle ; BL/6, n = 10; ASC −/− , n = 7) samples and recovered peritoneal CFU from isotype IgG treated mice ( right ; n = 7/group). D : IL-1β from peritoneal lavage ( left ), blood serum ( right ; n = 10/group) and recovered peritoneal CFU from anti-Ly6G treated mice ( right ; n = 7/group). Data in A , C , and D are expressed as means ± SD and are derived from at least 3 independent biological experiments. ** P ≤ 0.05.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Differential ASC requirements reveal a key role for neutrophils and a noncanonical IL-1β response to Pseudomonas aeruginosa

    doi: 10.1152/ajplung.00228.2015

    Figure Lengend Snippet: Neutropenia leads to reduced peritoneal IL-1β production in response to P. aeruginosa . C57BL/6 and ASC −/− mice were depleted of neutrophils with anti-Ly6G IgG or treated with isotype control antibody ( A , C , and D ). A : representative analyses of efficacy of neutrophil depletion in circulating blood and spleens from the treatment groups. B : C57BL/6 mice were infected intraperitoneally with the indicated CFU of WT PA14. IL-1β content within the peritoneal lavage samples were subsequently assayed by ELISA( n = 2/group). C and D : C57BL/6 and ASC −/− mice were treated with isotype control antibody or depleted of neutrophils with anti-Ly6G antibody as described above. Mice were subsequently infected intraperitoneally with 3 × 10 6 CFU of WT PA14, euthanized 4 h postinfection, and peritoneal lavage and blood samples were collected for IL-1β analysis by ELISA. The peritoneal lavage fluid was analyzed for the total number of recovered CFU normalized to CFU of input bacteria. C : IL-1β from peritoneal lavage ( left ), blood serum ( middle ; BL/6, n = 10; ASC −/− , n = 7) samples and recovered peritoneal CFU from isotype IgG treated mice ( right ; n = 7/group). D : IL-1β from peritoneal lavage ( left ), blood serum ( right ; n = 10/group) and recovered peritoneal CFU from anti-Ly6G treated mice ( right ; n = 7/group). Data in A , C , and D are expressed as means ± SD and are derived from at least 3 independent biological experiments. ** P ≤ 0.05.

    Article Snippet: C57BL/6 WT mice were obtained from the National Cancer Institute (Bethesda, MD) or Charles River Laboratories (Frederick, MD).

    Techniques: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Derivative Assay

    ASC expression is required for IL-1β production by macrophages and dendritic cells in response to Pseudomonas aeruginosa infection. Peritoneal macrophages ( A ) or bone marrow-derived dendritic cells (BMDCs; B ) from C57BL/6 or ASC −/− mice were infected with PA14 [wild-type (WT)] or with the popB mutant of PA14 at the indicated multiplicity of infection (MOI). Culture supernatants were collected 3 h postinfection and analyzed by ELISA for IL-1β production. Data are expressed as means ± SD accumulated from 2 independent biological experiments ( n = 4 for all samples except uninfected; n = 3). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.005.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Differential ASC requirements reveal a key role for neutrophils and a noncanonical IL-1β response to Pseudomonas aeruginosa

    doi: 10.1152/ajplung.00228.2015

    Figure Lengend Snippet: ASC expression is required for IL-1β production by macrophages and dendritic cells in response to Pseudomonas aeruginosa infection. Peritoneal macrophages ( A ) or bone marrow-derived dendritic cells (BMDCs; B ) from C57BL/6 or ASC −/− mice were infected with PA14 [wild-type (WT)] or with the popB mutant of PA14 at the indicated multiplicity of infection (MOI). Culture supernatants were collected 3 h postinfection and analyzed by ELISA for IL-1β production. Data are expressed as means ± SD accumulated from 2 independent biological experiments ( n = 4 for all samples except uninfected; n = 3). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.005.

    Article Snippet: C57BL/6 WT mice were obtained from the National Cancer Institute (Bethesda, MD) or Charles River Laboratories (Frederick, MD).

    Techniques: Expressing, Infection, Derivative Assay, Mouse Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay

    Macrophages and neutrophils temporally recruited to the peritoneum produce pro-IL-1β upon ex vivo infection. C57BL/6 mice were injected with thioglycollate and peritoneal cells were collected by lavage at 0 h (naïve uninjected), 4 h, or 3 days. The harvested cells were infected with WT or popB PA14 at MOI = 0.2 and analyzed 3 h postinfection. Bacteria were excluded from analyses by gating based on scatter controls, and the percentage of pro-IL-1β + F4/80 + and pro-IL-1β + Ly6G + cells was determined within the populations of peritoneal cells. A : representative dot plot for pro-IL-1β + cells at 0 or 4 h. B–D : percentage of pro-IL-1β + F4/80 + and pro-IL-1β + Ly6G + peritoneal cells from 0 h ( B ), 4 h ( C ), or 3 days ( D ) upon ex vivo infection. Data are expressed as means ± SD. Results are representative A , or compiled from independent biological experiments ( A–D ; n = 3).

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Differential ASC requirements reveal a key role for neutrophils and a noncanonical IL-1β response to Pseudomonas aeruginosa

    doi: 10.1152/ajplung.00228.2015

    Figure Lengend Snippet: Macrophages and neutrophils temporally recruited to the peritoneum produce pro-IL-1β upon ex vivo infection. C57BL/6 mice were injected with thioglycollate and peritoneal cells were collected by lavage at 0 h (naïve uninjected), 4 h, or 3 days. The harvested cells were infected with WT or popB PA14 at MOI = 0.2 and analyzed 3 h postinfection. Bacteria were excluded from analyses by gating based on scatter controls, and the percentage of pro-IL-1β + F4/80 + and pro-IL-1β + Ly6G + cells was determined within the populations of peritoneal cells. A : representative dot plot for pro-IL-1β + cells at 0 or 4 h. B–D : percentage of pro-IL-1β + F4/80 + and pro-IL-1β + Ly6G + peritoneal cells from 0 h ( B ), 4 h ( C ), or 3 days ( D ) upon ex vivo infection. Data are expressed as means ± SD. Results are representative A , or compiled from independent biological experiments ( A–D ; n = 3).

    Article Snippet: C57BL/6 WT mice were obtained from the National Cancer Institute (Bethesda, MD) or Charles River Laboratories (Frederick, MD).

    Techniques: Ex Vivo, Infection, Mouse Assay, Injection

    ASC is not required for pulmonary IL-1β production in response to an acute PA14 infection. C57BL/6 ( n = 7) and ASC −/− ( n = 7) mice were infected intratracheally with a sublethal dose (3 × 10 6 CFU) of WT PA14. C57BL/6 mice instilled with PBS ( n = 5) were used as a control. Mice were euthanized 4 h postinfection and BAL samples were collected. A : representative dot plot quantifying Ly6G + and CD11c + cells within a CD45 + pro-IL-1β + gate that excludes bacteria at 4 h postinfection. B : percentage of pro-IL-1β + Ly6G + ( left ) and pro-IL-1β + CD11c + ( right ) cells out of the total BAL cells at 4 h postinfection. C : percentage of Ly6G + CD11c − ( left ) and Ly6G − CD11c + ( right ) within the gated CD45 + pro-IL-1β + population as depicted in A . Data are expressed as means ± SD from 4 independent biological experiments.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Differential ASC requirements reveal a key role for neutrophils and a noncanonical IL-1β response to Pseudomonas aeruginosa

    doi: 10.1152/ajplung.00228.2015

    Figure Lengend Snippet: ASC is not required for pulmonary IL-1β production in response to an acute PA14 infection. C57BL/6 ( n = 7) and ASC −/− ( n = 7) mice were infected intratracheally with a sublethal dose (3 × 10 6 CFU) of WT PA14. C57BL/6 mice instilled with PBS ( n = 5) were used as a control. Mice were euthanized 4 h postinfection and BAL samples were collected. A : representative dot plot quantifying Ly6G + and CD11c + cells within a CD45 + pro-IL-1β + gate that excludes bacteria at 4 h postinfection. B : percentage of pro-IL-1β + Ly6G + ( left ) and pro-IL-1β + CD11c + ( right ) cells out of the total BAL cells at 4 h postinfection. C : percentage of Ly6G + CD11c − ( left ) and Ly6G − CD11c + ( right ) within the gated CD45 + pro-IL-1β + population as depicted in A . Data are expressed as means ± SD from 4 independent biological experiments.

    Article Snippet: C57BL/6 WT mice were obtained from the National Cancer Institute (Bethesda, MD) or Charles River Laboratories (Frederick, MD).

    Techniques: Infection, Mouse Assay

    Caspase-1 dependence for neutrophil secretion of IL-1β in response to P. aeruginosa . Neutrophils were enriched by negative selection, treated with either 20 μM Z-VAD or 20 μM DCIC where indicated, and infected with WT PA14, popB PA14 or Salmonella Typhimurium at the indicated MOI. Culture supernatants were collected 3 h postinfection and analyzed by ELISA for IL-1β production ( A , B , D , E , G , and H ). A : C57BL/6 neutrophils were infected with WT PA14 or popB PA14 at a MOI = 1 in the presence or absence of Z-VAD. B : C57BL/6 and caspase-1 −/− neutrophils were infected with WT PA14, popB PA14, or S . Typhimurium at the indicated MOI and IL-1β was analyzed by ELISA. Inset : WT C57BL/6 ( lanes 1 and 2 ) or caspase-1 −/− ( lanes 3 and 4 ) neutrophils were infected with popB PA14 at a MOI = 2 ( lanes 1 and 3 ) or uninfected ( lanes 2 and 4 ), and cells were collected at 5 h postinfection and lysates analyzed for pro-IL-1β by Western analyses. C : C57BL/6 and caspase-1 −/− mice were infected intraperitoneally with a sublethal dose (3 × 10 6 CFU) of WT PA14. Mice were euthanized 4 h postinfection and peritoneal lavage and blood samples were collected. Peritoneal lavage ( top ) and blood serum ( middle ) samples were analyzed by ELISA for IL-1β production ( n ≥ 9). Pro-IL-1β was assayed from whole cell lysates of recruited peritoneal cells at 4 h postinfection ( bottom ). D : murine neutrophils or macrophages of the indicated genotype were infected with PA14 at a MOI of 1 in the presence or absence of DCIC. Data for neutrophils were compared using one-way ANOVA with the Tukey-Kramer posttest. E : C57BL/6 and caspase-1 −/− neutrophils were infected with WT PA14 at MOI = 1 in the presence or absence of DCIC or Z-VAD. Data were compared using one-way ANOVA with the Tukey-Kramer posttest. F : C57BL/6 neutrophils were infected with popB mutant PA14 at MOI = 2 in the presence or absence of Z-VAD or DCIC, and subsequently analyzed by Western blot with the same protocol used in B , inset . G : C57BL/6 and neutrophil elastase −/− neutrophils were infected with WT PA14 or popB mutant PA14 at the indicated MOI. H : C57BL/6 and neutrophil elastase −/− neutrophils were infected with WT PA14 at MOI = 1 in the presence or absence of Z-VAD. Data were compared using one-way ANOVA with the Tukey-Kramer posttest. Results are representative of 2 ( A , n = 4; D , n = 6; E , n = 6; F and G , n = 6; H , n = 6) and 3 independent biological experiments ( B , n = 6; C , n ≥ 9). Data are expressed as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.005.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Differential ASC requirements reveal a key role for neutrophils and a noncanonical IL-1β response to Pseudomonas aeruginosa

    doi: 10.1152/ajplung.00228.2015

    Figure Lengend Snippet: Caspase-1 dependence for neutrophil secretion of IL-1β in response to P. aeruginosa . Neutrophils were enriched by negative selection, treated with either 20 μM Z-VAD or 20 μM DCIC where indicated, and infected with WT PA14, popB PA14 or Salmonella Typhimurium at the indicated MOI. Culture supernatants were collected 3 h postinfection and analyzed by ELISA for IL-1β production ( A , B , D , E , G , and H ). A : C57BL/6 neutrophils were infected with WT PA14 or popB PA14 at a MOI = 1 in the presence or absence of Z-VAD. B : C57BL/6 and caspase-1 −/− neutrophils were infected with WT PA14, popB PA14, or S . Typhimurium at the indicated MOI and IL-1β was analyzed by ELISA. Inset : WT C57BL/6 ( lanes 1 and 2 ) or caspase-1 −/− ( lanes 3 and 4 ) neutrophils were infected with popB PA14 at a MOI = 2 ( lanes 1 and 3 ) or uninfected ( lanes 2 and 4 ), and cells were collected at 5 h postinfection and lysates analyzed for pro-IL-1β by Western analyses. C : C57BL/6 and caspase-1 −/− mice were infected intraperitoneally with a sublethal dose (3 × 10 6 CFU) of WT PA14. Mice were euthanized 4 h postinfection and peritoneal lavage and blood samples were collected. Peritoneal lavage ( top ) and blood serum ( middle ) samples were analyzed by ELISA for IL-1β production ( n ≥ 9). Pro-IL-1β was assayed from whole cell lysates of recruited peritoneal cells at 4 h postinfection ( bottom ). D : murine neutrophils or macrophages of the indicated genotype were infected with PA14 at a MOI of 1 in the presence or absence of DCIC. Data for neutrophils were compared using one-way ANOVA with the Tukey-Kramer posttest. E : C57BL/6 and caspase-1 −/− neutrophils were infected with WT PA14 at MOI = 1 in the presence or absence of DCIC or Z-VAD. Data were compared using one-way ANOVA with the Tukey-Kramer posttest. F : C57BL/6 neutrophils were infected with popB mutant PA14 at MOI = 2 in the presence or absence of Z-VAD or DCIC, and subsequently analyzed by Western blot with the same protocol used in B , inset . G : C57BL/6 and neutrophil elastase −/− neutrophils were infected with WT PA14 or popB mutant PA14 at the indicated MOI. H : C57BL/6 and neutrophil elastase −/− neutrophils were infected with WT PA14 at MOI = 1 in the presence or absence of Z-VAD. Data were compared using one-way ANOVA with the Tukey-Kramer posttest. Results are representative of 2 ( A , n = 4; D , n = 6; E , n = 6; F and G , n = 6; H , n = 6) and 3 independent biological experiments ( B , n = 6; C , n ≥ 9). Data are expressed as means ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.005.

    Article Snippet: C57BL/6 WT mice were obtained from the National Cancer Institute (Bethesda, MD) or Charles River Laboratories (Frederick, MD).

    Techniques: Selection, Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Mouse Assay, Mutagenesis

    Higher proliferation of NK cells is associated with enhanced expression of nutrient receptors upon IL-18 treatment in vitro and LPS treatment in vivo . A , the number of expanded NK cells upon ex vivo stimulation with various concentrations of IL-18 relative to IL-2 alone was quantified. B , NK cells from the spleen of naïve C57BL/6 mice were ex vivo stimulated with IL-2 and IL-18. NK cells were prelabeled with cell proliferation dye and cultured with 300 units/ml IL-2 and 3 ng/ml IL-18 for 3 days. The dot plot depicts the dilution of cell proliferation dye on NK cells, and the histograms depict the MFI of CD98 expression on NK cells gated in regard to cell proliferation. C and D , C57BL/6 mice were either left untreated or challenged with 100 μg of LPS intraperitoneally and sacrificed on the indicated days for further analysis. C , NK cell proliferation upon treatment with LPS as measured by Ki-67 expression and BrdU incorporation. D , representative plots depicting the MFI of CD98 and CD71 expression on NK cells and T cells in the spleens of naïve ( D0 ) or LPS-treated mice at day 2 postinjection ( D2 ). For A and B , data are from one experiment representative of two independent experiments, with three replicates per group. For C and D , data are from one experiment representative of three independent experiments, with two to three mice per group. Data represent mean ± S.D. ( error bars ). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Interleukin-18 up-regulates amino acid transporters and facilitates amino acid–induced mTORC1 activation in natural killer cells

    doi: 10.1074/jbc.RA118.005892

    Figure Lengend Snippet: Higher proliferation of NK cells is associated with enhanced expression of nutrient receptors upon IL-18 treatment in vitro and LPS treatment in vivo . A , the number of expanded NK cells upon ex vivo stimulation with various concentrations of IL-18 relative to IL-2 alone was quantified. B , NK cells from the spleen of naïve C57BL/6 mice were ex vivo stimulated with IL-2 and IL-18. NK cells were prelabeled with cell proliferation dye and cultured with 300 units/ml IL-2 and 3 ng/ml IL-18 for 3 days. The dot plot depicts the dilution of cell proliferation dye on NK cells, and the histograms depict the MFI of CD98 expression on NK cells gated in regard to cell proliferation. C and D , C57BL/6 mice were either left untreated or challenged with 100 μg of LPS intraperitoneally and sacrificed on the indicated days for further analysis. C , NK cell proliferation upon treatment with LPS as measured by Ki-67 expression and BrdU incorporation. D , representative plots depicting the MFI of CD98 and CD71 expression on NK cells and T cells in the spleens of naïve ( D0 ) or LPS-treated mice at day 2 postinjection ( D2 ). For A and B , data are from one experiment representative of two independent experiments, with three replicates per group. For C and D , data are from one experiment representative of three independent experiments, with two to three mice per group. Data represent mean ± S.D. ( error bars ). *, p

    Article Snippet: WT C57BL/6 mice from Charles River were housed in a specific pathogen-free environment.

    Techniques: Expressing, In Vitro, In Vivo, Ex Vivo, Mouse Assay, Cell Culture, BrdU Incorporation Assay

    IL-18 induces the expression of nutrient receptors independently of mTORC1 pathway. NK cells from the spleen of naïve C57BL/6 mice were stimulated with different cytokines for 24 h in the presence or absence of rapamycin. A , representative plots depict the MFI of CD98 expression on NK cells. B , representative plots depict the percentage of CD71-expressing NK cells. C , representative plots depict the MFI of pS6 in NK cells. Data are from one experiment representative of three independent experiments, with two replicates per group. Data represent mean ± S.D. ( error bars ). NS , not significant; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Interleukin-18 up-regulates amino acid transporters and facilitates amino acid–induced mTORC1 activation in natural killer cells

    doi: 10.1074/jbc.RA118.005892

    Figure Lengend Snippet: IL-18 induces the expression of nutrient receptors independently of mTORC1 pathway. NK cells from the spleen of naïve C57BL/6 mice were stimulated with different cytokines for 24 h in the presence or absence of rapamycin. A , representative plots depict the MFI of CD98 expression on NK cells. B , representative plots depict the percentage of CD71-expressing NK cells. C , representative plots depict the MFI of pS6 in NK cells. Data are from one experiment representative of three independent experiments, with two replicates per group. Data represent mean ± S.D. ( error bars ). NS , not significant; *, p

    Article Snippet: WT C57BL/6 mice from Charles River were housed in a specific pathogen-free environment.

    Techniques: Expressing, Mouse Assay

    NK cells show increased glycolysis (ECAR) upon IL-18 stimulation. A , NK cells isolated by flow sorting were stimulated with IL-2 and either IL-12 or IL-18 for 24 h. The graph depicts the transcript levels of several nutrient receptors as quantified by quantitative real-time PCR. B , the ECAR levels of NK cells stimulated with IL-2 or IL-2/18 are shown. C , representative histograms depict the MFI of pS6 in NK cells upon stimulation with the indicated cytokines for 24 h. D , the ECAR levels of stimulated NK cells upon rapamycin treatment are shown. E , NK cell functions are reduced upon inhibition of the amino acid transporter LAT1. NK cells from the spleen of naïve C57BL/6 mice were stimulated with IL-2 and IL-18 cytokines for 18 h in the presence or absence of BCH. Representative histograms depict the proportion of NK cells expressing IFN-γ, granzyme B ( GzymB ), and pS6. Data are from one experiment representative of three independent experiments, with three to five replicates per group. Data represent mean ± S.D. ( error bars ). NS , not significant; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Interleukin-18 up-regulates amino acid transporters and facilitates amino acid–induced mTORC1 activation in natural killer cells

    doi: 10.1074/jbc.RA118.005892

    Figure Lengend Snippet: NK cells show increased glycolysis (ECAR) upon IL-18 stimulation. A , NK cells isolated by flow sorting were stimulated with IL-2 and either IL-12 or IL-18 for 24 h. The graph depicts the transcript levels of several nutrient receptors as quantified by quantitative real-time PCR. B , the ECAR levels of NK cells stimulated with IL-2 or IL-2/18 are shown. C , representative histograms depict the MFI of pS6 in NK cells upon stimulation with the indicated cytokines for 24 h. D , the ECAR levels of stimulated NK cells upon rapamycin treatment are shown. E , NK cell functions are reduced upon inhibition of the amino acid transporter LAT1. NK cells from the spleen of naïve C57BL/6 mice were stimulated with IL-2 and IL-18 cytokines for 18 h in the presence or absence of BCH. Representative histograms depict the proportion of NK cells expressing IFN-γ, granzyme B ( GzymB ), and pS6. Data are from one experiment representative of three independent experiments, with three to five replicates per group. Data represent mean ± S.D. ( error bars ). NS , not significant; *, p

    Article Snippet: WT C57BL/6 mice from Charles River were housed in a specific pathogen-free environment.

    Techniques: Isolation, Real-time Polymerase Chain Reaction, Inhibition, Mouse Assay, Expressing

    IL-18 and IL-1 receptor expression on NK cells. A , surface protein expression of the IL-18 receptor α subunit on various splenic leukocytes from C57BL/6 mice. B , NK cells were isolated by flow sorting from the spleen of C57BL/6 mice and compared with non-NK cells (total splenic leukocytes except NK cells) and total splenic leukocytes. mRNA was extracted from these three populations, and the transcript levels of IL-18 and IL-1 receptors were quantified by quantitative real-time PCR. C , representative plots depict the MFI of CD98 and CD71 expression on enriched NK cells from the spleen of naïve C57BL/6 mice upon stimulation with different cytokines for 24 h. Data are from one experiment representative of three independent experiments, with three replicates per group. Data represent mean ± S.D. ( error bars ). NS , nonsignificant; **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Interleukin-18 up-regulates amino acid transporters and facilitates amino acid–induced mTORC1 activation in natural killer cells

    doi: 10.1074/jbc.RA118.005892

    Figure Lengend Snippet: IL-18 and IL-1 receptor expression on NK cells. A , surface protein expression of the IL-18 receptor α subunit on various splenic leukocytes from C57BL/6 mice. B , NK cells were isolated by flow sorting from the spleen of C57BL/6 mice and compared with non-NK cells (total splenic leukocytes except NK cells) and total splenic leukocytes. mRNA was extracted from these three populations, and the transcript levels of IL-18 and IL-1 receptors were quantified by quantitative real-time PCR. C , representative plots depict the MFI of CD98 and CD71 expression on enriched NK cells from the spleen of naïve C57BL/6 mice upon stimulation with different cytokines for 24 h. Data are from one experiment representative of three independent experiments, with three replicates per group. Data represent mean ± S.D. ( error bars ). NS , nonsignificant; **, p

    Article Snippet: WT C57BL/6 mice from Charles River were housed in a specific pathogen-free environment.

    Techniques: Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction

    IL-18 can induce the up-regulation of nutrient transporters on NK cells in vitro . NK cells were enriched from the spleens of naïve C57BL/6 mice and stimulated with the indicated cytokines in vitro for 24 h. 100 units/ml rhIL-2 was added to maintain NK cell survival. A , representative plots depict the MFI of CD98 and CD71 expression on cytokine-stimulated NK cells. Statistics are comparing samples with IL-2–stimulated NK cells. B , the expression of CD98 and CD71 on NK cells was measured upon stimulation with different concentrations of IL-12 and IL-18. For the statistical analyses of the data, the p values were obtained by comparing the stimulated NK cells with the unstimulated NK cells (without IL-12 or IL-18 treatment). C , representative histograms of the glucose uptake by cytokine-stimulated NK cells as measured by the MFI of 2-NBDG. Statistics are comparing samples with IL-2–stimulated NK cells. Data are from one experiment representative of four independent experiments, with two replicates per group. Data represent mean ± S.D. ( error bars ). NS , not significant; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Interleukin-18 up-regulates amino acid transporters and facilitates amino acid–induced mTORC1 activation in natural killer cells

    doi: 10.1074/jbc.RA118.005892

    Figure Lengend Snippet: IL-18 can induce the up-regulation of nutrient transporters on NK cells in vitro . NK cells were enriched from the spleens of naïve C57BL/6 mice and stimulated with the indicated cytokines in vitro for 24 h. 100 units/ml rhIL-2 was added to maintain NK cell survival. A , representative plots depict the MFI of CD98 and CD71 expression on cytokine-stimulated NK cells. Statistics are comparing samples with IL-2–stimulated NK cells. B , the expression of CD98 and CD71 on NK cells was measured upon stimulation with different concentrations of IL-12 and IL-18. For the statistical analyses of the data, the p values were obtained by comparing the stimulated NK cells with the unstimulated NK cells (without IL-12 or IL-18 treatment). C , representative histograms of the glucose uptake by cytokine-stimulated NK cells as measured by the MFI of 2-NBDG. Statistics are comparing samples with IL-2–stimulated NK cells. Data are from one experiment representative of four independent experiments, with two replicates per group. Data represent mean ± S.D. ( error bars ). NS , not significant; *, p

    Article Snippet: WT C57BL/6 mice from Charles River were housed in a specific pathogen-free environment.

    Techniques: In Vitro, Mouse Assay, Expressing