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  • 95
    Bio X Cell rat igg2b isotype control
    Rat Igg2b Isotype Control, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 95/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher igg2b
    Lack of C1q affects the level of immunoglobulins IgA (A) , IgG1 (B) , IgG2a (C) , <t>IgG2b</t> (D) , IgG3 (E) , and IgM (F) in infected mice. Whole blood was collected from mice infected with 10 4 Borrelia burgdorferi ) at 7, 10, 21, and 28 days post-infection. Serum level of immunoglobulins was determined by the Bio-Plex system employing the Luminex multianalyte profiling technology. White circles refer to samples from C57BL/6 mice, whereas red squares represent data from C1qα −/− mice ( n = 5; * P
    Igg2b, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson igg2b
    GaMD immune serum from WT mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1 - mice by 5d after immunization a . BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), starting 4, 5, or 6d after GaMD immunization. Day 7 urine samples were analyzed. ND = none detected. b. BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), 5, 6 and 7 d after GaMD immunization. Sera were assayed for total <t>IgG1,</t> <t>IgG2a,</t> IgM and <t>IgG3</t> on d0 (unimmunized) and 8d after GaMD immunization. ND = none detected.For both a and b , * indicates p
    Igg2b, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore igg2a
    GaMD immune serum from WT mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1 - mice by 5d after immunization a . BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), starting 4, 5, or 6d after GaMD immunization. Day 7 urine samples were analyzed. ND = none detected. b. BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), 5, 6 and 7 d after GaMD immunization. Sera were assayed for total <t>IgG1,</t> <t>IgG2a,</t> IgM and <t>IgG3</t> on d0 (unimmunized) and 8d after GaMD immunization. ND = none detected.For both a and b , * indicates p
    Igg2a, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1392 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad anti mouse igg2b
    GaMD immune serum from WT mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1 - mice by 5d after immunization a . BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), starting 4, 5, or 6d after GaMD immunization. Day 7 urine samples were analyzed. ND = none detected. b. BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), 5, 6 and 7 d after GaMD immunization. Sera were assayed for total <t>IgG1,</t> <t>IgG2a,</t> IgM and <t>IgG3</t> on d0 (unimmunized) and 8d after GaMD immunization. ND = none detected.For both a and b , * indicates p
    Anti Mouse Igg2b, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson rat igg2b
    GaMD immune serum from WT mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1 - mice by 5d after immunization a . BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), starting 4, 5, or 6d after GaMD immunization. Day 7 urine samples were analyzed. ND = none detected. b. BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), 5, 6 and 7 d after GaMD immunization. Sera were assayed for total <t>IgG1,</t> <t>IgG2a,</t> IgM and <t>IgG3</t> on d0 (unimmunized) and 8d after GaMD immunization. ND = none detected.For both a and b , * indicates p
    Rat Igg2b, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 95/100, based on 646 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg2b cross adsorbed secondary antibody
    GaMD immune serum from WT mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1 - mice by 5d after immunization a . BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), starting 4, 5, or 6d after GaMD immunization. Day 7 urine samples were analyzed. ND = none detected. b. BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), 5, 6 and 7 d after GaMD immunization. Sera were assayed for total <t>IgG1,</t> <t>IgG2a,</t> IgM and <t>IgG3</t> on d0 (unimmunized) and 8d after GaMD immunization. ND = none detected.For both a and b , * indicates p
    Goat Anti Mouse Igg2b Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Pharmingen igg2b
    Depletion of NK cells promotes long-term kidney allograft survival in B6.CD8 −/− CCR5 −/− recipients Groups of B6.CD8 −/− CCR5 −/− mice (n = 5 per group) received renal allografts from A/J donors or isografts and nephrectomy of the remaining native kidney was performed on day 5 post-transplant. The indicated groups of allograft recipients were treated with 200 µg of control rat <t>IgG</t> (-●-) or with anti-NK1.1 mAb either on days 3, 8 and 13 post-transplant (-■-) or on days 3, 8 and 13 post-transplant and then weekly to day 41 post-transplant (-▲-). (a) Allograft survival was followed by daily examination of overall animal health. * P
    Igg2b, supplied by Pharmingen, used in various techniques. Bioz Stars score: 94/100, based on 294 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad igg2b
    A dominant role of host-derived IL-10 in controlling the pathogenic Th1 type of nephrophilic autoantibody responses . To further assess the roles of host vs DC-derived IL-10 on renal disease, recipient mice of the two mouse strains were injected with donor cells (DC/nec F/T ) from either W/T or IL-10 −/− mice respectively and reciprocally. The animals were sacrificed 4 months after the first injection and kidney sections were stained for mouse IgG ( A ), IgG1 ( B ), IgG2a b ( C ) and <t>IgG2b</t> ( D ), and similarly quantified as described in Figs 2 and 3 . The results shown are quantitative data (dot plots) pooled from three repeated experiments (for details see supplementary Table 6 , available as supplementary data at Rheumatology Online). Statistical differences between groups were determined by the non-parametric Mann–Whitney test, and the P -values of groups having statistical significance are indicated in the graphs.
    Igg2b, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 95/100, based on 245 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rat igg2b
    Anti-aquaporin-4 (AQP4) Ab-positive serum from neuromyelitis optica spectrum disorder (NMOSD) patients activates membrane attack complex deposition on the surface of human astrocytes. (A) Anti-AQP4 Ab-positive serum from NMOSD patients opsonizes human primary astrocytes. Confocal z-stack microscopy analysis confirms formation of <t>AQP4–IgG1</t> immunological complexes on the cell surface in astrocytes incubated with NMOSD serum. White arrows mark colocalization of AQP4 and IgG1 signals (yellow color) (B) . Binding of AQP4 receptors by autoantibodies activates classical complement cascade which can destabilize astrocytes by formation of membrane attack complex (C5b-9) [ (B) , lower panel]. Negative signal in NMOSD serum which has been previously heated causing complement inactivation (56°C, 30 min) excluded false positivity in C5b-9 ICC analysis [ (B) , upper panel].
    Rat Igg2b, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 475 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse igg2b
    Anti-aquaporin-4 (AQP4) Ab-positive serum from neuromyelitis optica spectrum disorder (NMOSD) patients activates membrane attack complex deposition on the surface of human astrocytes. (A) Anti-AQP4 Ab-positive serum from NMOSD patients opsonizes human primary astrocytes. Confocal z-stack microscopy analysis confirms formation of <t>AQP4–IgG1</t> immunological complexes on the cell surface in astrocytes incubated with NMOSD serum. White arrows mark colocalization of AQP4 and IgG1 signals (yellow color) (B) . Binding of AQP4 receptors by autoantibodies activates classical complement cascade which can destabilize astrocytes by formation of membrane attack complex (C5b-9) [ (B) , lower panel]. Negative signal in NMOSD serum which has been previously heated causing complement inactivation (56°C, 30 min) excluded false positivity in C5b-9 ICC analysis [ (B) , upper panel].
    Mouse Igg2b, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    igg2b  (Abcam)
    97
    Abcam igg2b
    GpepIP-specific CD4+ T cells exhibit high potency on helping humoral immune responses to HIV-1 trimer. a Immunization scheme. BALB/c mice were primed twice by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Three weeks later, all groups were immunized with the clade A BG505 gp140 NFL trimer emulsified in incomplete Freund’s adjuvant. Sera were collected at the indicated time points. Mice were euthanized 10 days after trimer immunization. b Splenic and lymph node cells were isolated and stimulated with GpepIP or pepIP in vitro for 5 days. T cell proliferation by CFSE dilution was measured by flow cytometry. c BG505-specific <t>IgG</t> production was examined in all three groups across the indicated time points by ELISA using a serum dilution 1:100. d GC response, defined by the percentage of GL7 + Fas + B cells among B220 + B cells, was evaluated in all three groups on day 10 after trimer immunization by flow cytometry. e – g Expression levels of activation marker CD69 e , CD80 f , and MHCII g were detected on splenic B cells after in vitro stimulation with the BG505 trimer for 3 days. MFI mean fluorescence intensity. Representative results are shown from one of three independent experiments performed. (mean ± s.d.). b medium n = 2; GpepIP or pepIP n = 3 independent experiments. d n = 4 independent experiments. e – g medium n = 2; BG505 n = 3 independent experiments. P -values were determined using Student’s two-sided t -tests. Source data are provided as a Source Data file.
    Igg2b, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems mouse igg2b isotype control
    IL-6 secreted by tumor associated fibroblasts (TAFs) induces hepcidin in breast cancer spheroids (A) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) in patient 113 lobular epithelial cell samples. Samples were 100% 2D tumor epithelial cells (2D TEC), 100% tumor epithelial cell spheroids (3D TEC), spheroids composed of a mixture of 80% TEC and 20% irradiated TAFs (isolated from patient 113), (80% TEC +20%TAF) and 100% irradiated TAF spheroids after 3 days of culture. (B) Western blot analysis of pro-hepcidin and Cyclophilin B for different TEC/TAF (%) combinations after 3 days of spheroid culture. (C) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) and (D) western blot analysis of phosphorylated and total STAT3 of primary TEC spheroids alone, spheroids composed of a mixture of 80% TECs and 20%TAFs, or TEC spheroids exposed to conditioned media (CM) from TAFs for 4 days. Patient 113 tumor ductal (113 Tu Duc), patient 113 tumor lobular (113 Tu Lob) and patient 107 tumor ductal (107 Tu Duc) were used for (C) and patient 113 tumor lobular for (D). For statistical analysis in (C) samples with 20% TAF or TAF CM were compared to their respective 3D TEC sample. (E) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) of patient 113 lobular TEC spheroids after the addition of TAF CM and IL-6 neutralizing antibody (1=1μg/mL and 5=5μg/mL) for 4 days. Neutralizing antibody against <t>IgG</t> (1 and 5 μg/mL) was used as a control. For statistical analysis, samples were compared to TAF CM sample. (F) Western blot analysis of pro-hepcidin and β-actin of patient 113 lobular TEC spheroids after the addition of TAF CM with or without 1μg/mL neutralizing antibodies against IL-6 or IgG for 4 days. All western blot quantifications (B, D and F) were compared to TEC alone.
    Mouse Igg2b Isotype Control, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore murine igg
    IL-6 secreted by tumor associated fibroblasts (TAFs) induces hepcidin in breast cancer spheroids (A) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) in patient 113 lobular epithelial cell samples. Samples were 100% 2D tumor epithelial cells (2D TEC), 100% tumor epithelial cell spheroids (3D TEC), spheroids composed of a mixture of 80% TEC and 20% irradiated TAFs (isolated from patient 113), (80% TEC +20%TAF) and 100% irradiated TAF spheroids after 3 days of culture. (B) Western blot analysis of pro-hepcidin and Cyclophilin B for different TEC/TAF (%) combinations after 3 days of spheroid culture. (C) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) and (D) western blot analysis of phosphorylated and total STAT3 of primary TEC spheroids alone, spheroids composed of a mixture of 80% TECs and 20%TAFs, or TEC spheroids exposed to conditioned media (CM) from TAFs for 4 days. Patient 113 tumor ductal (113 Tu Duc), patient 113 tumor lobular (113 Tu Lob) and patient 107 tumor ductal (107 Tu Duc) were used for (C) and patient 113 tumor lobular for (D). For statistical analysis in (C) samples with 20% TAF or TAF CM were compared to their respective 3D TEC sample. (E) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) of patient 113 lobular TEC spheroids after the addition of TAF CM and IL-6 neutralizing antibody (1=1μg/mL and 5=5μg/mL) for 4 days. Neutralizing antibody against <t>IgG</t> (1 and 5 μg/mL) was used as a control. For statistical analysis, samples were compared to TAF CM sample. (F) Western blot analysis of pro-hepcidin and β-actin of patient 113 lobular TEC spheroids after the addition of TAF CM with or without 1μg/mL neutralizing antibodies against IL-6 or IgG for 4 days. All western blot quantifications (B, D and F) were compared to TEC alone.
    Murine Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems igg2b
    IL-6 secreted by tumor associated fibroblasts (TAFs) induces hepcidin in breast cancer spheroids (A) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) in patient 113 lobular epithelial cell samples. Samples were 100% 2D tumor epithelial cells (2D TEC), 100% tumor epithelial cell spheroids (3D TEC), spheroids composed of a mixture of 80% TEC and 20% irradiated TAFs (isolated from patient 113), (80% TEC +20%TAF) and 100% irradiated TAF spheroids after 3 days of culture. (B) Western blot analysis of pro-hepcidin and Cyclophilin B for different TEC/TAF (%) combinations after 3 days of spheroid culture. (C) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) and (D) western blot analysis of phosphorylated and total STAT3 of primary TEC spheroids alone, spheroids composed of a mixture of 80% TECs and 20%TAFs, or TEC spheroids exposed to conditioned media (CM) from TAFs for 4 days. Patient 113 tumor ductal (113 Tu Duc), patient 113 tumor lobular (113 Tu Lob) and patient 107 tumor ductal (107 Tu Duc) were used for (C) and patient 113 tumor lobular for (D). For statistical analysis in (C) samples with 20% TAF or TAF CM were compared to their respective 3D TEC sample. (E) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) of patient 113 lobular TEC spheroids after the addition of TAF CM and IL-6 neutralizing antibody (1=1μg/mL and 5=5μg/mL) for 4 days. Neutralizing antibody against <t>IgG</t> (1 and 5 μg/mL) was used as a control. For statistical analysis, samples were compared to TAF CM sample. (F) Western blot analysis of pro-hepcidin and β-actin of patient 113 lobular TEC spheroids after the addition of TAF CM with or without 1μg/mL neutralizing antibodies against IL-6 or IgG for 4 days. All western blot quantifications (B, D and F) were compared to TEC alone.
    Igg2b, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher rat igg2b kappa isotype control
    IL-6 secreted by tumor associated fibroblasts (TAFs) induces hepcidin in breast cancer spheroids (A) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) in patient 113 lobular epithelial cell samples. Samples were 100% 2D tumor epithelial cells (2D TEC), 100% tumor epithelial cell spheroids (3D TEC), spheroids composed of a mixture of 80% TEC and 20% irradiated TAFs (isolated from patient 113), (80% TEC +20%TAF) and 100% irradiated TAF spheroids after 3 days of culture. (B) Western blot analysis of pro-hepcidin and Cyclophilin B for different TEC/TAF (%) combinations after 3 days of spheroid culture. (C) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) and (D) western blot analysis of phosphorylated and total STAT3 of primary TEC spheroids alone, spheroids composed of a mixture of 80% TECs and 20%TAFs, or TEC spheroids exposed to conditioned media (CM) from TAFs for 4 days. Patient 113 tumor ductal (113 Tu Duc), patient 113 tumor lobular (113 Tu Lob) and patient 107 tumor ductal (107 Tu Duc) were used for (C) and patient 113 tumor lobular for (D). For statistical analysis in (C) samples with 20% TAF or TAF CM were compared to their respective 3D TEC sample. (E) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) of patient 113 lobular TEC spheroids after the addition of TAF CM and IL-6 neutralizing antibody (1=1μg/mL and 5=5μg/mL) for 4 days. Neutralizing antibody against <t>IgG</t> (1 and 5 μg/mL) was used as a control. For statistical analysis, samples were compared to TAF CM sample. (F) Western blot analysis of pro-hepcidin and β-actin of patient 113 lobular TEC spheroids after the addition of TAF CM with or without 1μg/mL neutralizing antibodies against IL-6 or IgG for 4 days. All western blot quantifications (B, D and F) were compared to TEC alone.
    Rat Igg2b Kappa Isotype Control, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse igg2b
    Human serum <t>IgG</t> response against SGS. (A) ELISA was performed against L. intermedia and L. longipalpis SGS using human sera were from individuals from a CL endemic area (n = 100) or from control individuals (n = 10). (B) ELISA was performed against L. intermedia SGS with human sera from CL individuals (n = 16) or from healthy individuals with either a negative (n = 42) or a positive (n = 32) anti- Leishmania DTH skin test. The ordinate represents the absorbance of the ELISA reaction of these sera against SGS. The symbols indicate results obtained with each serum tested and lines represent their median values (***p
    Mouse Igg2b, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems mouse igg2b
    Human serum <t>IgG</t> response against SGS. (A) ELISA was performed against L. intermedia and L. longipalpis SGS using human sera were from individuals from a CL endemic area (n = 100) or from control individuals (n = 10). (B) ELISA was performed against L. intermedia SGS with human sera from CL individuals (n = 16) or from healthy individuals with either a negative (n = 42) or a positive (n = 32) anti- Leishmania DTH skin test. The ordinate represents the absorbance of the ELISA reaction of these sera against SGS. The symbols indicate results obtained with each serum tested and lines represent their median values (***p
    Mouse Igg2b, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems rat igg isotype control
    Human serum <t>IgG</t> response against SGS. (A) ELISA was performed against L. intermedia and L. longipalpis SGS using human sera were from individuals from a CL endemic area (n = 100) or from control individuals (n = 10). (B) ELISA was performed against L. intermedia SGS with human sera from CL individuals (n = 16) or from healthy individuals with either a negative (n = 42) or a positive (n = 32) anti- Leishmania DTH skin test. The ordinate represents the absorbance of the ELISA reaction of these sera against SGS. The symbols indicate results obtained with each serum tested and lines represent their median values (***p
    Rat Igg Isotype Control, supplied by R&D Systems, used in various techniques. Bioz Stars score: 97/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology igg2b
    Specific humoral responses elicited by immunization of mice with microneme proteins. The reactivity of immunoglobulins anti-STAg was determined by ELISA in serum samples collected from both immunized (TgMICs) and control (PBS) mice 15 days after the last antigen injection. Each point/bar represents the average absorbance ± SD of the serum samples from 4 animals. (A) Absorbance provided by the reaction of serum <t>IgG</t> with STAg. (B) Absorbance provided by the reaction of serum <t>IgG1</t> and <t>IgG2a</t> (diluted 1:25) with STAg. The average absorbance ± SD generated by the reaction of serum IgG1 or IgG2a from each group of immunized mice was significantly higher than the corresponding values provided by control mice, with the exception of the TgMIC6-immunized group, whose results were not significantly different of those of the control group. Three independent experiments were performed, and data from one representative experiment is shown. Asterisks represent statistical significant differences (*p
    Igg2b, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rat igg2b isotype control
    Specific humoral responses elicited by immunization of mice with microneme proteins. The reactivity of immunoglobulins anti-STAg was determined by ELISA in serum samples collected from both immunized (TgMICs) and control (PBS) mice 15 days after the last antigen injection. Each point/bar represents the average absorbance ± SD of the serum samples from 4 animals. (A) Absorbance provided by the reaction of serum <t>IgG</t> with STAg. (B) Absorbance provided by the reaction of serum <t>IgG1</t> and <t>IgG2a</t> (diluted 1:25) with STAg. The average absorbance ± SD generated by the reaction of serum IgG1 or IgG2a from each group of immunized mice was significantly higher than the corresponding values provided by control mice, with the exception of the TgMIC6-immunized group, whose results were not significantly different of those of the control group. Three independent experiments were performed, and data from one representative experiment is shown. Asterisks represent statistical significant differences (*p
    Rat Igg2b Isotype Control, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rat igg2b isotype control/product/Thermo Fisher
    Average 99 stars, based on 212 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Lack of C1q affects the level of immunoglobulins IgA (A) , IgG1 (B) , IgG2a (C) , IgG2b (D) , IgG3 (E) , and IgM (F) in infected mice. Whole blood was collected from mice infected with 10 4 Borrelia burgdorferi ) at 7, 10, 21, and 28 days post-infection. Serum level of immunoglobulins was determined by the Bio-Plex system employing the Luminex multianalyte profiling technology. White circles refer to samples from C57BL/6 mice, whereas red squares represent data from C1qα −/− mice ( n = 5; * P

    Journal: Frontiers in Immunology

    Article Title: The Classical Complement Pathway Is Required to Control Borrelia burgdorferi Levels During Experimental Infection

    doi: 10.3389/fimmu.2018.00959

    Figure Lengend Snippet: Lack of C1q affects the level of immunoglobulins IgA (A) , IgG1 (B) , IgG2a (C) , IgG2b (D) , IgG3 (E) , and IgM (F) in infected mice. Whole blood was collected from mice infected with 10 4 Borrelia burgdorferi ) at 7, 10, 21, and 28 days post-infection. Serum level of immunoglobulins was determined by the Bio-Plex system employing the Luminex multianalyte profiling technology. White circles refer to samples from C57BL/6 mice, whereas red squares represent data from C1qα −/− mice ( n = 5; * P

    Article Snippet: To conduct this assay, 96-well microtiter plates were coated with sonicated B. burgdorferi strain B31 at 5 µg/ml in carbonate buffer (pH 9.3), and one empty column (with no antigen) on each plate was reserved for serial dilution of purified mouse IgG1, IgG2a, IgG2b, IgG3, or IgM (eBioscience) to generate a standard curve.

    Techniques: Infection, Mouse Assay, Luminex

    Lack of C1q alters the level of Borrelia -specific antibody subtypes IgM (A) , IgG1 (B) , IgG2a (C) , IgG2b (D) , and IgG3 (E) in infected mice. Whole blood was collected from mice infected with 10 4 Borrelia burgdorferi ) at 7, 10, 21, and 28 days post-infection. Serum level of Borrelia -specific immunoglobulin subtypes was determined by ELISA using total protein lysate derived from B. burgdorferi strain MSK5. White columns refer to C57BL/6 mice, whereas red columns represent data from C1qα −/− mice ( n = 5; * P

    Journal: Frontiers in Immunology

    Article Title: The Classical Complement Pathway Is Required to Control Borrelia burgdorferi Levels During Experimental Infection

    doi: 10.3389/fimmu.2018.00959

    Figure Lengend Snippet: Lack of C1q alters the level of Borrelia -specific antibody subtypes IgM (A) , IgG1 (B) , IgG2a (C) , IgG2b (D) , and IgG3 (E) in infected mice. Whole blood was collected from mice infected with 10 4 Borrelia burgdorferi ) at 7, 10, 21, and 28 days post-infection. Serum level of Borrelia -specific immunoglobulin subtypes was determined by ELISA using total protein lysate derived from B. burgdorferi strain MSK5. White columns refer to C57BL/6 mice, whereas red columns represent data from C1qα −/− mice ( n = 5; * P

    Article Snippet: To conduct this assay, 96-well microtiter plates were coated with sonicated B. burgdorferi strain B31 at 5 µg/ml in carbonate buffer (pH 9.3), and one empty column (with no antigen) on each plate was reserved for serial dilution of purified mouse IgG1, IgG2a, IgG2b, IgG3, or IgM (eBioscience) to generate a standard curve.

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

    ZNF148 binds the rs2239630 risk allele. a EMSA allele-specific probes for rs2239630 incubated with REH nuclear protein. b Position weighted matrices for ZNF589 and ZNF148 with corresponding genomic sequence below. Yellow boxes highlight base mismatches. c EMSA in REH showing differential allelic binding is caused by ZNF148. Supershift assay performed by addition of anti-ZNF148 antibody or IgG isotype control

    Journal: Leukemia

    Article Title: Genetic predisposition to B-cell acute lymphoblastic leukemia at 14q11.2 is mediated by a CEBPE promoter polymorphism

    doi: 10.1038/s41375-018-0184-z

    Figure Lengend Snippet: ZNF148 binds the rs2239630 risk allele. a EMSA allele-specific probes for rs2239630 incubated with REH nuclear protein. b Position weighted matrices for ZNF589 and ZNF148 with corresponding genomic sequence below. Yellow boxes highlight base mismatches. c EMSA in REH showing differential allelic binding is caused by ZNF148. Supershift assay performed by addition of anti-ZNF148 antibody or IgG isotype control

    Article Snippet: A total of 25 µg of chromatin was incubated with 2 µg of either anti-YY1 (Abcam ab12132), ELF1 (Santa Cruz sc-133096), MAX (Abcam ab53570), E2A (Santa Cruz sc133075), ZNF148 (Atlas Antibodies HPA001656), 0.5 µg of SPI1 (Life Technologies A13971) or an equal amount of control antibody, mouse IgG2b (Thermo Fisher Scientific MA5-14447) or rabbit IgG (AbCam ab37415) and incubated at 4 °C O/N with rotation.

    Techniques: Incubation, Sequencing, Binding Assay

    ZNF148 binds the rs2239630 risk allele and represses CEBPE . a ZNF148 ChIP q-PCR in REH cells. Left x -axis raw signal normalised to input DNA, right y -axis, fold enrichment (hashed bars) vs IgG control. b Allele-specific ChIP q-PCR for ZNF148 in REH. c q-RT PCR for ZNF148 and CEBPE mRNA expression in ZNF148 -overexpressing REH. T-test P -values. Target gene expression normalised to geometric mean of PPIA and TUBB, shown relative to empty vector control. Data points, mean of biological replicates ± SEM. ( d ) H3K27ac ChIP-seq in REH. Reads mapping to each allele lead SNPs in the 14q11.2 risk loci are enumerated on the y -axis, hashed bars show the enrichment for reads mapping to risk alleles. * binomial P -values

    Journal: Leukemia

    Article Title: Genetic predisposition to B-cell acute lymphoblastic leukemia at 14q11.2 is mediated by a CEBPE promoter polymorphism

    doi: 10.1038/s41375-018-0184-z

    Figure Lengend Snippet: ZNF148 binds the rs2239630 risk allele and represses CEBPE . a ZNF148 ChIP q-PCR in REH cells. Left x -axis raw signal normalised to input DNA, right y -axis, fold enrichment (hashed bars) vs IgG control. b Allele-specific ChIP q-PCR for ZNF148 in REH. c q-RT PCR for ZNF148 and CEBPE mRNA expression in ZNF148 -overexpressing REH. T-test P -values. Target gene expression normalised to geometric mean of PPIA and TUBB, shown relative to empty vector control. Data points, mean of biological replicates ± SEM. ( d ) H3K27ac ChIP-seq in REH. Reads mapping to each allele lead SNPs in the 14q11.2 risk loci are enumerated on the y -axis, hashed bars show the enrichment for reads mapping to risk alleles. * binomial P -values

    Article Snippet: A total of 25 µg of chromatin was incubated with 2 µg of either anti-YY1 (Abcam ab12132), ELF1 (Santa Cruz sc-133096), MAX (Abcam ab53570), E2A (Santa Cruz sc133075), ZNF148 (Atlas Antibodies HPA001656), 0.5 µg of SPI1 (Life Technologies A13971) or an equal amount of control antibody, mouse IgG2b (Thermo Fisher Scientific MA5-14447) or rabbit IgG (AbCam ab37415) and incubated at 4 °C O/N with rotation.

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation

    rs2239630 alleles differentially bind SPI1, MAX. a ChIP q-PCR of MAX and SPI1 enriched chromatin in REH cells. x -axis lists genomic loci of PCR primers used to amplify antibody specific (MAX or SPI1, black bars) or negative control (IgG, grey bars) ChIP samples. Raw signal normalised to input DNA (left y -axis). Right y -axis shows fold enriched (hashed bars) of antibody specific % input vs IgG control. b Allele-specific ChIP q-PCR for MAX and SPI1 in REH

    Journal: Leukemia

    Article Title: Genetic predisposition to B-cell acute lymphoblastic leukemia at 14q11.2 is mediated by a CEBPE promoter polymorphism

    doi: 10.1038/s41375-018-0184-z

    Figure Lengend Snippet: rs2239630 alleles differentially bind SPI1, MAX. a ChIP q-PCR of MAX and SPI1 enriched chromatin in REH cells. x -axis lists genomic loci of PCR primers used to amplify antibody specific (MAX or SPI1, black bars) or negative control (IgG, grey bars) ChIP samples. Raw signal normalised to input DNA (left y -axis). Right y -axis shows fold enriched (hashed bars) of antibody specific % input vs IgG control. b Allele-specific ChIP q-PCR for MAX and SPI1 in REH

    Article Snippet: A total of 25 µg of chromatin was incubated with 2 µg of either anti-YY1 (Abcam ab12132), ELF1 (Santa Cruz sc-133096), MAX (Abcam ab53570), E2A (Santa Cruz sc133075), ZNF148 (Atlas Antibodies HPA001656), 0.5 µg of SPI1 (Life Technologies A13971) or an equal amount of control antibody, mouse IgG2b (Thermo Fisher Scientific MA5-14447) or rabbit IgG (AbCam ab37415) and incubated at 4 °C O/N with rotation.

    Techniques: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control

    GaMD immune serum from WT mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1 - mice by 5d after immunization a . BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), starting 4, 5, or 6d after GaMD immunization. Day 7 urine samples were analyzed. ND = none detected. b. BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), 5, 6 and 7 d after GaMD immunization. Sera were assayed for total IgG1, IgG2a, IgM and IgG3 on d0 (unimmunized) and 8d after GaMD immunization. ND = none detected.For both a and b , * indicates p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: GaMD immune serum from WT mice inhibits GaMD-induced renal disease without decreasing other isotypes if injected into GaMD-immunized γ1 - mice by 5d after immunization a . BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), starting 4, 5, or 6d after GaMD immunization. Day 7 urine samples were analyzed. ND = none detected. b. BALB/c γ1 - mice (4 or 8/gp) were injected s.c. with GaMD on day 0 and i.p. with 0.5 ml of pooled serum from GaMD-immunized WT mice (GaMD immune WT serum) or unimmunized WT mice (non-immune serum), 5, 6 and 7 d after GaMD immunization. Sera were assayed for total IgG1, IgG2a, IgM and IgG3 on d0 (unimmunized) and 8d after GaMD immunization. ND = none detected.For both a and b , * indicates p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Injection

    The development of kidney disease in GaMD-immunized γ1 - mice is independent of IFN-γ, IgG2a, C3 and FcRγ BALB/c WT and γ1 - mice (5/gp) were immunized with GaMD on d0 and injected with 1 mg of either anti-IFN-γ or control mAb on days 0 and 5. a , Total levels of all Ig isotypes were determined in 24 hr culture supernatants of spleen cells harvested on days shown. b , GaMD-immunized γ1 - , γ1 - /FcRγ - , γ1 - /C3 - , C3 - / FcRγ - and γ1 - /C3 - /FcγR - mice (5/gp) had their urine tested for LE and blood on days shown. c , d , BALB/c WT and γ1 - mice (5/gp) were immunized with GaMD on d0 and injected with 1 mg of either anti-IFN-γ or control mAb on days 0 and 5. c , Urine obtained on days indicated was assayed for protein, LE and blood. d , BUN levels were determined prior to and 10 days after GaMD immunization. * p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: The development of kidney disease in GaMD-immunized γ1 - mice is independent of IFN-γ, IgG2a, C3 and FcRγ BALB/c WT and γ1 - mice (5/gp) were immunized with GaMD on d0 and injected with 1 mg of either anti-IFN-γ or control mAb on days 0 and 5. a , Total levels of all Ig isotypes were determined in 24 hr culture supernatants of spleen cells harvested on days shown. b , GaMD-immunized γ1 - , γ1 - /FcRγ - , γ1 - /C3 - , C3 - / FcRγ - and γ1 - /C3 - /FcγR - mice (5/gp) had their urine tested for LE and blood on days shown. c , d , BALB/c WT and γ1 - mice (5/gp) were immunized with GaMD on d0 and injected with 1 mg of either anti-IFN-γ or control mAb on days 0 and 5. c , Urine obtained on days indicated was assayed for protein, LE and blood. d , BUN levels were determined prior to and 10 days after GaMD immunization. * p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Injection

    IgG1 inhibits IgG3-induced cryoglobulin kidney disease independent of complement and FcγRIIB and better than IgG2a and IgG2b a , WT mice (4/gp) were injected i.v. with 4 mg of mouse IgG1, IgG2a, IgG2b, or IgG3 anti-TNP mAb and s.c. with 100 μl of TNP-goat serum on days 0 and 1. Urine LE and blood measured prior to injections and on d1 and d2. b , Urine LE and blood for BALB/c WT and C3 - mice (4/gp) injected i.v. with 4 mg of IgG3 anti-TNP mAb and s.c. with 400 μl of TNP-goat serum on d0 and 1. c , WT and FcγRIIB-deficient (FcγRIIB - ) mice (4/gp) were injected s.c. with 100 μl of TNP-goat serum and i.v. with 4 mg of IgG3 anti-TNP ± 5 mg of IgG1 anti-TNP on d0 and 1. Urinalysis on d0, 1 and 2. d , BALB/c mice were injected i.v. with 4 mg of IgG3 anti-TNP and s.c. with 1.4 mg of TNP-BSA on days 0 and 1. Some mice were also injected with 0.625, 1.25, 2.5, or 5 mg of switch variants of IgG1, IgG2a or IgG2b anti-TNP mAbs on d0 and 1. Urine protein was determined on d0 (not shown), d1 (upper panel) and d2 (lower panel). Results are pooled from a total of 7 experiments. Group size: IgG3 alone: 19 mice; 0.625 mg of IgG1, IgG2a, or IgG2b: 4 mice; 1.25 mg of IgG1, IgG2a or IgG2b: 8 mice; 2.5 mg of IgG1, IgG2a or IgG2b: 6 mice; 5 mg of IgG1, IgG2a or IgG2b: 8 or 9 mice. The significance of differences between treatment groups was determined as described in the legend to Fig. 4f . # p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: IgG1 inhibits IgG3-induced cryoglobulin kidney disease independent of complement and FcγRIIB and better than IgG2a and IgG2b a , WT mice (4/gp) were injected i.v. with 4 mg of mouse IgG1, IgG2a, IgG2b, or IgG3 anti-TNP mAb and s.c. with 100 μl of TNP-goat serum on days 0 and 1. Urine LE and blood measured prior to injections and on d1 and d2. b , Urine LE and blood for BALB/c WT and C3 - mice (4/gp) injected i.v. with 4 mg of IgG3 anti-TNP mAb and s.c. with 400 μl of TNP-goat serum on d0 and 1. c , WT and FcγRIIB-deficient (FcγRIIB - ) mice (4/gp) were injected s.c. with 100 μl of TNP-goat serum and i.v. with 4 mg of IgG3 anti-TNP ± 5 mg of IgG1 anti-TNP on d0 and 1. Urinalysis on d0, 1 and 2. d , BALB/c mice were injected i.v. with 4 mg of IgG3 anti-TNP and s.c. with 1.4 mg of TNP-BSA on days 0 and 1. Some mice were also injected with 0.625, 1.25, 2.5, or 5 mg of switch variants of IgG1, IgG2a or IgG2b anti-TNP mAbs on d0 and 1. Urine protein was determined on d0 (not shown), d1 (upper panel) and d2 (lower panel). Results are pooled from a total of 7 experiments. Group size: IgG3 alone: 19 mice; 0.625 mg of IgG1, IgG2a, or IgG2b: 4 mice; 1.25 mg of IgG1, IgG2a or IgG2b: 8 mice; 2.5 mg of IgG1, IgG2a or IgG2b: 6 mice; 5 mg of IgG1, IgG2a or IgG2b: 8 or 9 mice. The significance of differences between treatment groups was determined as described in the legend to Fig. 4f . # p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Injection

    Concurrent injection of WT mice with IgG3 anti-TNP mAb and TNP-goat serum induces glomerulopathy a , WT mice (4/gp) were injected i.v.with 4 mg of mouse IgG1, IgG2a, IgG2b, or IgG3 anti-TNP mAb and s.c. with 100 μl of TNP-goat serum on days 0 and 1. Urine protein measured prior to injections and on d1 and d2. b , c , WT mice (4/gp) were injected with mouse IgG3 anti-TNP mAb +/- TNP-goat serum as in “a.” Day 2 mouse sera were analyzed for BUN (b). Day 2 kidneys were stained with PAS (c, panel 1: glomerulus from mouse that received only IgG3; panels 2: glomerulus from mouse that received IgG3 + TNP-goat serum). Representative of 3 mice/group. d , Urine protein for BALB/c WT and C3 - mice (4/gp) injected i.v. with 4 mg of IgG3 anti-TNP mAb and s.c. with 400 μl of TNP-goat serum on d0 and 1. * p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: Concurrent injection of WT mice with IgG3 anti-TNP mAb and TNP-goat serum induces glomerulopathy a , WT mice (4/gp) were injected i.v.with 4 mg of mouse IgG1, IgG2a, IgG2b, or IgG3 anti-TNP mAb and s.c. with 100 μl of TNP-goat serum on days 0 and 1. Urine protein measured prior to injections and on d1 and d2. b , c , WT mice (4/gp) were injected with mouse IgG3 anti-TNP mAb +/- TNP-goat serum as in “a.” Day 2 mouse sera were analyzed for BUN (b). Day 2 kidneys were stained with PAS (c, panel 1: glomerulus from mouse that received only IgG3; panels 2: glomerulus from mouse that received IgG3 + TNP-goat serum). Representative of 3 mice/group. d , Urine protein for BALB/c WT and C3 - mice (4/gp) injected i.v. with 4 mg of IgG3 anti-TNP mAb and s.c. with 400 μl of TNP-goat serum on d0 and 1. * p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Injection, Mouse Assay, Staining

    Glomerulopathy in GaMD-immunized γ1 - mice is complement- and FcRγ -independent and associated with IgG3 cryoglobulinemia a . Serum anti-goat IgG titers in WT and γ1 - mice (4/gp) 8d after GaMD immunization. b , c . Urine protein (b) and BUN (c) of GaMD-immunized γ1 - , γ1 - /FcRγ - , γ1 - /C3 - , C3 - / FcRγ - and γ1 - /C3 - /FcγR - mice (5/gp). d , e . Serum cryoprecipitate protein and Ig isotype concentrations 6-7 d after GaMD immunization of WT and γ1 - mice (7 or 8/gp). Only cryoprecipitates from γ1 - mice contained detectable Ig. f. IgG3 (brown color) in glomerular capillaries (arrows) of γ1 - mice 8d after GaMD (panels 1, low magnification; panel 2, high magnification). * p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: Glomerulopathy in GaMD-immunized γ1 - mice is complement- and FcRγ -independent and associated with IgG3 cryoglobulinemia a . Serum anti-goat IgG titers in WT and γ1 - mice (4/gp) 8d after GaMD immunization. b , c . Urine protein (b) and BUN (c) of GaMD-immunized γ1 - , γ1 - /FcRγ - , γ1 - /C3 - , C3 - / FcRγ - and γ1 - /C3 - /FcγR - mice (5/gp). d , e . Serum cryoprecipitate protein and Ig isotype concentrations 6-7 d after GaMD immunization of WT and γ1 - mice (7 or 8/gp). Only cryoprecipitates from γ1 - mice contained detectable Ig. f. IgG3 (brown color) in glomerular capillaries (arrows) of γ1 - mice 8d after GaMD (panels 1, low magnification; panel 2, high magnification). * p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay

    Delayed antigen elimination does not account for renal disease in GaMD-immunized γ1 - mice a . BALB/c WT and γ1 - mice (10/gp) were immunized s.c. withGaMD. Sera obtained 5, 6, 7 and 9 days later were evaluated by gel double diffusion for the presence of goat IgG. b-e. BALB/c WT mice (4 or 5/gp) were injected s.c. with a total of 0.2 ml of different mixtures of GaMD and goat anti-KLH antisera. b, c , Mouse sera collected 9d later were assayed for BUN ( b ) and IgG1 anti-goat IgG Ab ( c ). d , Sera obtained 6-13d post-immunization were evaluated by gel double diffusion for the presence of goat IgG. e , Urine samples collected 4-12 days post-immunization were analyzed for protein. * p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: Delayed antigen elimination does not account for renal disease in GaMD-immunized γ1 - mice a . BALB/c WT and γ1 - mice (10/gp) were immunized s.c. withGaMD. Sera obtained 5, 6, 7 and 9 days later were evaluated by gel double diffusion for the presence of goat IgG. b-e. BALB/c WT mice (4 or 5/gp) were injected s.c. with a total of 0.2 ml of different mixtures of GaMD and goat anti-KLH antisera. b, c , Mouse sera collected 9d later were assayed for BUN ( b ) and IgG1 anti-goat IgG Ab ( c ). d , Sera obtained 6-13d post-immunization were evaluated by gel double diffusion for the presence of goat IgG. e , Urine samples collected 4-12 days post-immunization were analyzed for protein. * p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Diffusion-based Assay, Injection

    Ag-specific IgG1 prevents IgG3-mediated glomerulopathy BALB/c γ1 - mice (5/gp) were injected with GaMD on day 0 and/or GaMD-immune or rabbit anti-mouse IgD (RaMD) immune WT serum daily on d4-7. a , b , Urine protein ( a ) and d12 serum albumin and BUN levels ( b ). * p

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: Ag-specific IgG1 prevents IgG3-mediated glomerulopathy BALB/c γ1 - mice (5/gp) were injected with GaMD on day 0 and/or GaMD-immune or rabbit anti-mouse IgD (RaMD) immune WT serum daily on d4-7. a , b , Urine protein ( a ) and d12 serum albumin and BUN levels ( b ). * p

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Injection

    GaMD immunization of γ1 - mice induces renal dysfunction and glomerular deposition of PAS + material that includes IgG and complement a , WT and γ1 - mice (4/gp) were immunized with GaMD. Urine LE and blood were obtained. b , Representative photomicrographs of glomeruli stained for C3 (top panels) or total mouse IgG (bottom panels) from WT (right panels) and γ1 -/- mice (left panels) 12 d post-GaMD immunization 3 mice/group. c , Deposition of amorphous PAS + material in glomeruli of γ1 - , but not WT begins ∼7 days post GaMD-immunization and leads to glomerular destruction by day 9. Note the scarcity of inflammatory cells in glomeruli. Representative data of 6 mice/group.

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: GaMD immunization of γ1 - mice induces renal dysfunction and glomerular deposition of PAS + material that includes IgG and complement a , WT and γ1 - mice (4/gp) were immunized with GaMD. Urine LE and blood were obtained. b , Representative photomicrographs of glomeruli stained for C3 (top panels) or total mouse IgG (bottom panels) from WT (right panels) and γ1 -/- mice (left panels) 12 d post-GaMD immunization 3 mice/group. c , Deposition of amorphous PAS + material in glomeruli of γ1 - , but not WT begins ∼7 days post GaMD-immunization and leads to glomerular destruction by day 9. Note the scarcity of inflammatory cells in glomeruli. Representative data of 6 mice/group.

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Staining

    Antigen-specific IgG1 can prevent IgG3 immune complex glomerular deposition BALB/c WT mice were injected i.v.with mouse IgG1 and/or IgG3 anti-TNP mAb with or without s.c.injection of TNP-BSA on days 0 and 1. a. Kidneys were stained with PAS on day 2. Representative micrographs from 3 mice/group are shown. b. Kidney serial sections were stained with PAS or for IgG3 or IgG1 (brown pigment). Representative micrographs from 4 mice/group are shown.

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: Antigen-specific IgG1 can prevent IgG3 immune complex glomerular deposition BALB/c WT mice were injected i.v.with mouse IgG1 and/or IgG3 anti-TNP mAb with or without s.c.injection of TNP-BSA on days 0 and 1. a. Kidneys were stained with PAS on day 2. Representative micrographs from 3 mice/group are shown. b. Kidney serial sections were stained with PAS or for IgG3 or IgG1 (brown pigment). Representative micrographs from 4 mice/group are shown.

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Injection, Staining

    IgG3 IC persist and accumulate in the glomeruli of GaMD-immunized γ1 - BALB/c WT and γ1 - mice were left untreated or were immunized with GaMD. Kidney sections were stained for mouse IgG1, IgG2a, IgG2b, IgG3 and IgM 8 and 12d later. Representative photomicrographs from 3 GaMD-immunized mice are shown. Insets show magnified views. No staining was observed with sections from unimmunized mice (not shown).

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: IgG3 IC persist and accumulate in the glomeruli of GaMD-immunized γ1 - BALB/c WT and γ1 - mice were left untreated or were immunized with GaMD. Kidney sections were stained for mouse IgG1, IgG2a, IgG2b, IgG3 and IgM 8 and 12d later. Representative photomicrographs from 3 GaMD-immunized mice are shown. Insets show magnified views. No staining was observed with sections from unimmunized mice (not shown).

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Staining

    GaMD-immunized γ1 +/- mice generate large IgG3 responses but develop mild renal disease BALB/c mice homozygous (γ1 +/+ ), heterozygous (γ1 +/- ) and null (γ1 -/- ) for a functional γ1 allele (6/gp) were injected s.c. with GaMD. a , Sera were titered for goat IgG-specific IgG1, IgG2a and IgG3 0, 8 and 12 days later. Day 0 titers were zero for all Ig isotypes (data not shown). b , Urine samples from the same mice were assayed for protein and leukocyte esterase. ND = none detected.

    Journal: Nature

    Article Title: IgG1 protects against renal disease in a mouse model of cryoglobulinemia

    doi: 10.1038/nature13868

    Figure Lengend Snippet: GaMD-immunized γ1 +/- mice generate large IgG3 responses but develop mild renal disease BALB/c mice homozygous (γ1 +/+ ), heterozygous (γ1 +/- ) and null (γ1 -/- ) for a functional γ1 allele (6/gp) were injected s.c. with GaMD. a , Sera were titered for goat IgG-specific IgG1, IgG2a and IgG3 0, 8 and 12 days later. Day 0 titers were zero for all Ig isotypes (data not shown). b , Urine samples from the same mice were assayed for protein and leukocyte esterase. ND = none detected.

    Article Snippet: Serum, splenic supernatants and re-suspended cryoglobulin pellets were analyzed for total and GIgG-specific IgG1, IgG2a/c, IgG2b, IgG3, IgE, IgA and IgM content, using standard sandwich ELISA with paired anti-Ig isotype mAbs for each Ig isotype (BD-Pharmingen and eBioscience).

    Techniques: Mouse Assay, Functional Assay, Injection

    Depletion of NK cells promotes long-term kidney allograft survival in B6.CD8 −/− CCR5 −/− recipients Groups of B6.CD8 −/− CCR5 −/− mice (n = 5 per group) received renal allografts from A/J donors or isografts and nephrectomy of the remaining native kidney was performed on day 5 post-transplant. The indicated groups of allograft recipients were treated with 200 µg of control rat IgG (-●-) or with anti-NK1.1 mAb either on days 3, 8 and 13 post-transplant (-■-) or on days 3, 8 and 13 post-transplant and then weekly to day 41 post-transplant (-▲-). (a) Allograft survival was followed by daily examination of overall animal health. * P

    Journal: Kidney international

    Article Title: Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts

    doi: 10.1016/j.kint.2016.02.030

    Figure Lengend Snippet: Depletion of NK cells promotes long-term kidney allograft survival in B6.CD8 −/− CCR5 −/− recipients Groups of B6.CD8 −/− CCR5 −/− mice (n = 5 per group) received renal allografts from A/J donors or isografts and nephrectomy of the remaining native kidney was performed on day 5 post-transplant. The indicated groups of allograft recipients were treated with 200 µg of control rat IgG (-●-) or with anti-NK1.1 mAb either on days 3, 8 and 13 post-transplant (-■-) or on days 3, 8 and 13 post-transplant and then weekly to day 41 post-transplant (-▲-). (a) Allograft survival was followed by daily examination of overall animal health. * P

    Article Snippet: After washing, the cells were resuspended in staining buffer (Dulbecco’s PBS with 2% FCS/0.02% NaN3) containing FITC-conjugated rat anti-mouse IgG1, IgG2a, IgG2b or IgG3 mAb (Pharmingen, San Diego, CA) for 30 min on ice.

    Techniques: Mouse Assay

    Production of donor-reactive IgG antibody in wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− kidney allograft recipients Serum was collected from individual wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− recipients of A/J kidney allografts on days 3, 7, and 14 post-transplant and at the time of rejection. The sera were tested for reactivity to A/J thymocytes using a flow cytometry-based approach to determine the titer of donor-reactive antibody for each of the IgG subclasses. Data indicate mean titer for each recipient group ± SEM.

    Journal: Kidney international

    Article Title: Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts

    doi: 10.1016/j.kint.2016.02.030

    Figure Lengend Snippet: Production of donor-reactive IgG antibody in wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− kidney allograft recipients Serum was collected from individual wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− recipients of A/J kidney allografts on days 3, 7, and 14 post-transplant and at the time of rejection. The sera were tested for reactivity to A/J thymocytes using a flow cytometry-based approach to determine the titer of donor-reactive antibody for each of the IgG subclasses. Data indicate mean titer for each recipient group ± SEM.

    Article Snippet: After washing, the cells were resuspended in staining buffer (Dulbecco’s PBS with 2% FCS/0.02% NaN3) containing FITC-conjugated rat anti-mouse IgG1, IgG2a, IgG2b or IgG3 mAb (Pharmingen, San Diego, CA) for 30 min on ice.

    Techniques: Flow Cytometry, Cytometry

    Titers of donor-reactive IgG antibody at the time of kidney allograft rejection in wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− recipients (a) Titers of donor-reactive IgG antibody were determined in serum collected from allograft recipients at the time of graft rejection or from C57BL/7 recipients with long-term surviving allografts collected on day 40 post-transplant. Data indicate individual titers within each recipient group and the mean titer for each recipient group ± SEM. (b–d) Histological evaluation of a rejecting kidney allograft from a wild C57BL/6 recipient on day 36 post-transplant stained with: (b) hematoxylin and eosin; (c) anti-C4d antibody; and (d) anti-Mac2 antibody. Arrows indicate marginating leukocytes. Images shown are representative of 2 individual allografts in the group.

    Journal: Kidney international

    Article Title: Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts

    doi: 10.1016/j.kint.2016.02.030

    Figure Lengend Snippet: Titers of donor-reactive IgG antibody at the time of kidney allograft rejection in wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− recipients (a) Titers of donor-reactive IgG antibody were determined in serum collected from allograft recipients at the time of graft rejection or from C57BL/7 recipients with long-term surviving allografts collected on day 40 post-transplant. Data indicate individual titers within each recipient group and the mean titer for each recipient group ± SEM. (b–d) Histological evaluation of a rejecting kidney allograft from a wild C57BL/6 recipient on day 36 post-transplant stained with: (b) hematoxylin and eosin; (c) anti-C4d antibody; and (d) anti-Mac2 antibody. Arrows indicate marginating leukocytes. Images shown are representative of 2 individual allografts in the group.

    Article Snippet: After washing, the cells were resuspended in staining buffer (Dulbecco’s PBS with 2% FCS/0.02% NaN3) containing FITC-conjugated rat anti-mouse IgG1, IgG2a, IgG2b or IgG3 mAb (Pharmingen, San Diego, CA) for 30 min on ice.

    Techniques: Staining

    Expression of inflammatory mediator mRNA in kidney allografts in B6.CD8 −/− CCR5 −/− recipients treated with mAb depleting NK or Gr-1 + cells Wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− received renal allografts from A/J donors and a group of wild type C57BL/6 mice received isografts. Groups of 3–5 recipients were treated with 250 µg anti-Gr-1 (RB6.8C5) or anti-NK 1.1 (PK136) mAb to deplete Gr-1 + or NK cells or rat IgG as a control on days 5 and 6 post-transplant. Grafts were harvested on day 7 post-transplant and whole cell RNA was prepared and analyzed by qRT/PCR for expression of the indicated cytokine, chemokine and inflammatory mediator genes. Data indicate the mean expression levels of each test gene in allografts for each group. * P

    Journal: Kidney international

    Article Title: Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts

    doi: 10.1016/j.kint.2016.02.030

    Figure Lengend Snippet: Expression of inflammatory mediator mRNA in kidney allografts in B6.CD8 −/− CCR5 −/− recipients treated with mAb depleting NK or Gr-1 + cells Wild type C57BL/6, B6.CCR5 −/− and B6.CD8 −/− CCR5 −/− received renal allografts from A/J donors and a group of wild type C57BL/6 mice received isografts. Groups of 3–5 recipients were treated with 250 µg anti-Gr-1 (RB6.8C5) or anti-NK 1.1 (PK136) mAb to deplete Gr-1 + or NK cells or rat IgG as a control on days 5 and 6 post-transplant. Grafts were harvested on day 7 post-transplant and whole cell RNA was prepared and analyzed by qRT/PCR for expression of the indicated cytokine, chemokine and inflammatory mediator genes. Data indicate the mean expression levels of each test gene in allografts for each group. * P

    Article Snippet: After washing, the cells were resuspended in staining buffer (Dulbecco’s PBS with 2% FCS/0.02% NaN3) containing FITC-conjugated rat anti-mouse IgG1, IgG2a, IgG2b or IgG3 mAb (Pharmingen, San Diego, CA) for 30 min on ice.

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR

    A dominant role of host-derived IL-10 in controlling the pathogenic Th1 type of nephrophilic autoantibody responses . To further assess the roles of host vs DC-derived IL-10 on renal disease, recipient mice of the two mouse strains were injected with donor cells (DC/nec F/T ) from either W/T or IL-10 −/− mice respectively and reciprocally. The animals were sacrificed 4 months after the first injection and kidney sections were stained for mouse IgG ( A ), IgG1 ( B ), IgG2a b ( C ) and IgG2b ( D ), and similarly quantified as described in Figs 2 and 3 . The results shown are quantitative data (dot plots) pooled from three repeated experiments (for details see supplementary Table 6 , available as supplementary data at Rheumatology Online). Statistical differences between groups were determined by the non-parametric Mann–Whitney test, and the P -values of groups having statistical significance are indicated in the graphs.

    Journal: Rheumatology (Oxford, England)

    Article Title: An essential protective role of IL-10 in the immunological mechanism underlying resistance vs susceptibility to lupus induction by dendritic cells and dying cells

    doi: 10.1093/rheumatology/ker198

    Figure Lengend Snippet: A dominant role of host-derived IL-10 in controlling the pathogenic Th1 type of nephrophilic autoantibody responses . To further assess the roles of host vs DC-derived IL-10 on renal disease, recipient mice of the two mouse strains were injected with donor cells (DC/nec F/T ) from either W/T or IL-10 −/− mice respectively and reciprocally. The animals were sacrificed 4 months after the first injection and kidney sections were stained for mouse IgG ( A ), IgG1 ( B ), IgG2a b ( C ) and IgG2b ( D ), and similarly quantified as described in Figs 2 and 3 . The results shown are quantitative data (dot plots) pooled from three repeated experiments (for details see supplementary Table 6 , available as supplementary data at Rheumatology Online). Statistical differences between groups were determined by the non-parametric Mann–Whitney test, and the P -values of groups having statistical significance are indicated in the graphs.

    Article Snippet: For detection of renal IC deposition, FITC-conjugated goat polyclonal antibodies against mouse IgG (1/200 dilution; Sigma-Aldrich), IgG1 (1/100 dilution; Serotec, STAR81F), IgG2b (1/50 dilution; Serotec, STAR83F) and C3 (1/100 dilution; Cappel, #55500), and a rat anti-mouse CD68 antibody (1/200 dilution), were used on the frozen tissue sections.

    Techniques: Derivative Assay, Mouse Assay, Injection, Staining, MANN-WHITNEY

    Characterization of renal reactive autoantibodies—isotypic analysis of the renal ICs. The antibody isotypes of renal Ig deposits in the DC/nec F/T - and DC/nec H/S -treated IL-10 −/− mice were assessed by immunostaining using FITC-conjugated antibodies specific for mouse IgG1, IgG2a b (C57/BL6 allotype specific Abs) and IgG2b, respectively. Photomicrographs representative of each of the two treatment groups ( DC/nec F/T , DC/nec H/S ) are shown. The intensity of the staining was quantified (AFU, dot plots). Each symbol represents a single mouse, the horizontal bar is the mean value for each group, and the data shown were results pooled from two repeated experiments (for details see supplementary table 5 , available as supplementary data at Rheumatology Online). Control group: PBS-treated mice. Original magnification: ×20; statistical differences between groups were determined by the non-parametric Mann–Whitney test, and the P -values indicated in the graphs.

    Journal: Rheumatology (Oxford, England)

    Article Title: An essential protective role of IL-10 in the immunological mechanism underlying resistance vs susceptibility to lupus induction by dendritic cells and dying cells

    doi: 10.1093/rheumatology/ker198

    Figure Lengend Snippet: Characterization of renal reactive autoantibodies—isotypic analysis of the renal ICs. The antibody isotypes of renal Ig deposits in the DC/nec F/T - and DC/nec H/S -treated IL-10 −/− mice were assessed by immunostaining using FITC-conjugated antibodies specific for mouse IgG1, IgG2a b (C57/BL6 allotype specific Abs) and IgG2b, respectively. Photomicrographs representative of each of the two treatment groups ( DC/nec F/T , DC/nec H/S ) are shown. The intensity of the staining was quantified (AFU, dot plots). Each symbol represents a single mouse, the horizontal bar is the mean value for each group, and the data shown were results pooled from two repeated experiments (for details see supplementary table 5 , available as supplementary data at Rheumatology Online). Control group: PBS-treated mice. Original magnification: ×20; statistical differences between groups were determined by the non-parametric Mann–Whitney test, and the P -values indicated in the graphs.

    Article Snippet: For detection of renal IC deposition, FITC-conjugated goat polyclonal antibodies against mouse IgG (1/200 dilution; Sigma-Aldrich), IgG1 (1/100 dilution; Serotec, STAR81F), IgG2b (1/50 dilution; Serotec, STAR83F) and C3 (1/100 dilution; Cappel, #55500), and a rat anti-mouse CD68 antibody (1/200 dilution), were used on the frozen tissue sections.

    Techniques: Mouse Assay, Immunostaining, Staining, MANN-WHITNEY

    Renal IC deposition: kidney tissue samples were from mice of the different treatment groups described in Fig. 1 . Immunostaining for mouse IgG ( A ) and C3 ( B ) on frozen kidney sections revealed severe glomerular deposits but only in DC/nec F/T -treated IL-10 −/− mice. The scattered dot plots compare the relative levels of renal IgG and C3 deposits, as quantified by the IF method (see Materials and methods section). Each symbol represents a single mouse and the horizontal bars are the mean values for each of the four treatment groups of each mouse strain. In these experiments, the donor cells and recipient mice were of the same strain, i.e. W/T and IL-10 −/− mice, respectively. Original magnification ×20 ( A ). The quantitative data (dot plots) shown in ( A ) and in ( B ) were results pooled from three or two repeated experiments, respectively (for details see supplementary tables 3 and 4 , available as supplementary data at Rheumatology Online). Statistical differences between groups are determined by the non-parametric Mann–Whitney test, and the P -values indicated in the graphs. The results were also reanalysed and reconfirmed by the non-parametric one-way ANOVA test to be highly significant among all of the experimental groups ( P

    Journal: Rheumatology (Oxford, England)

    Article Title: An essential protective role of IL-10 in the immunological mechanism underlying resistance vs susceptibility to lupus induction by dendritic cells and dying cells

    doi: 10.1093/rheumatology/ker198

    Figure Lengend Snippet: Renal IC deposition: kidney tissue samples were from mice of the different treatment groups described in Fig. 1 . Immunostaining for mouse IgG ( A ) and C3 ( B ) on frozen kidney sections revealed severe glomerular deposits but only in DC/nec F/T -treated IL-10 −/− mice. The scattered dot plots compare the relative levels of renal IgG and C3 deposits, as quantified by the IF method (see Materials and methods section). Each symbol represents a single mouse and the horizontal bars are the mean values for each of the four treatment groups of each mouse strain. In these experiments, the donor cells and recipient mice were of the same strain, i.e. W/T and IL-10 −/− mice, respectively. Original magnification ×20 ( A ). The quantitative data (dot plots) shown in ( A ) and in ( B ) were results pooled from three or two repeated experiments, respectively (for details see supplementary tables 3 and 4 , available as supplementary data at Rheumatology Online). Statistical differences between groups are determined by the non-parametric Mann–Whitney test, and the P -values indicated in the graphs. The results were also reanalysed and reconfirmed by the non-parametric one-way ANOVA test to be highly significant among all of the experimental groups ( P

    Article Snippet: For detection of renal IC deposition, FITC-conjugated goat polyclonal antibodies against mouse IgG (1/200 dilution; Sigma-Aldrich), IgG1 (1/100 dilution; Serotec, STAR81F), IgG2b (1/50 dilution; Serotec, STAR83F) and C3 (1/100 dilution; Cappel, #55500), and a rat anti-mouse CD68 antibody (1/200 dilution), were used on the frozen tissue sections.

    Techniques: Mouse Assay, Immunostaining, MANN-WHITNEY

    Anti-aquaporin-4 (AQP4) Ab-positive serum from neuromyelitis optica spectrum disorder (NMOSD) patients activates membrane attack complex deposition on the surface of human astrocytes. (A) Anti-AQP4 Ab-positive serum from NMOSD patients opsonizes human primary astrocytes. Confocal z-stack microscopy analysis confirms formation of AQP4–IgG1 immunological complexes on the cell surface in astrocytes incubated with NMOSD serum. White arrows mark colocalization of AQP4 and IgG1 signals (yellow color) (B) . Binding of AQP4 receptors by autoantibodies activates classical complement cascade which can destabilize astrocytes by formation of membrane attack complex (C5b-9) [ (B) , lower panel]. Negative signal in NMOSD serum which has been previously heated causing complement inactivation (56°C, 30 min) excluded false positivity in C5b-9 ICC analysis [ (B) , upper panel].

    Journal: Frontiers in Immunology

    Article Title: C5a-Preactivated Neutrophils Are Critical for Autoimmune-Induced Astrocyte Dysregulation in Neuromyelitis Optica Spectrum Disorder

    doi: 10.3389/fimmu.2018.01694

    Figure Lengend Snippet: Anti-aquaporin-4 (AQP4) Ab-positive serum from neuromyelitis optica spectrum disorder (NMOSD) patients activates membrane attack complex deposition on the surface of human astrocytes. (A) Anti-AQP4 Ab-positive serum from NMOSD patients opsonizes human primary astrocytes. Confocal z-stack microscopy analysis confirms formation of AQP4–IgG1 immunological complexes on the cell surface in astrocytes incubated with NMOSD serum. White arrows mark colocalization of AQP4 and IgG1 signals (yellow color) (B) . Binding of AQP4 receptors by autoantibodies activates classical complement cascade which can destabilize astrocytes by formation of membrane attack complex (C5b-9) [ (B) , lower panel]. Negative signal in NMOSD serum which has been previously heated causing complement inactivation (56°C, 30 min) excluded false positivity in C5b-9 ICC analysis [ (B) , upper panel].

    Article Snippet: Anti-IgG1/FITC (Dako Cytomation), anti-AQP4 (Santa Cruz Biotechnology), anti-C5b-9 (Dako Cytomation), phalloidin (Invitrogen), anti-GFAP (Santa Cruz Biotechnology), and rat IgG2b (Invitrogen), as negative isotype control were used.

    Techniques: Microscopy, Incubation, Binding Assay, Immunocytochemistry

    Human astrocytes recruit healthy control (HC) neutrophils in the presence of anti-aquaporin-4 (AQP4) Ab-positive neuromyelitis optica spectrum disorder (NMOSD) serum as a result of C5a release. (A) Astrocytes preincubated with NMOSD serum induce chemotaxis of HC neutrophils as the result of C5a release. The example of analysis representative of four independent experiments. R1—region of beads; R2—region of neutrophils. (B) C5a release following AQP4–IgG1 binding to AQP4 during incubation of primary human astrocytes with NMOSD serum. Concentration of C5a was measured in supernatants after 1 h incubation with NMOSD, remitting–relapsing multiple sclerosis, or HC serum. (C) Concentration of C5a in supernatants of astrocytes incubated with NMOSD or control serum. The bars represent the means ± SD from four independent experiments.

    Journal: Frontiers in Immunology

    Article Title: C5a-Preactivated Neutrophils Are Critical for Autoimmune-Induced Astrocyte Dysregulation in Neuromyelitis Optica Spectrum Disorder

    doi: 10.3389/fimmu.2018.01694

    Figure Lengend Snippet: Human astrocytes recruit healthy control (HC) neutrophils in the presence of anti-aquaporin-4 (AQP4) Ab-positive neuromyelitis optica spectrum disorder (NMOSD) serum as a result of C5a release. (A) Astrocytes preincubated with NMOSD serum induce chemotaxis of HC neutrophils as the result of C5a release. The example of analysis representative of four independent experiments. R1—region of beads; R2—region of neutrophils. (B) C5a release following AQP4–IgG1 binding to AQP4 during incubation of primary human astrocytes with NMOSD serum. Concentration of C5a was measured in supernatants after 1 h incubation with NMOSD, remitting–relapsing multiple sclerosis, or HC serum. (C) Concentration of C5a in supernatants of astrocytes incubated with NMOSD or control serum. The bars represent the means ± SD from four independent experiments.

    Article Snippet: Anti-IgG1/FITC (Dako Cytomation), anti-AQP4 (Santa Cruz Biotechnology), anti-C5b-9 (Dako Cytomation), phalloidin (Invitrogen), anti-GFAP (Santa Cruz Biotechnology), and rat IgG2b (Invitrogen), as negative isotype control were used.

    Techniques: Chemotaxis Assay, Binding Assay, Incubation, Concentration Assay

    Increased mortality in streptomycin-treated mice is IFNγ dependent. (A) Mice were given streptomycin for 2 weeks before infection with Sendai virus (SeV). At day 5 and 9 PI SeV, 100 µg of an IFNγ blocking or control IgG monoclonal antibody was given SQ and mortality measured ( n = 5/group). (B) Mice were treated as in panel (A) but with the indicated doses of anti-IFNγ antibodies being administered ( n = 5/group). p

    Journal: Frontiers in Immunology

    Article Title: Intestinal Microbiota Disruption Reduces Regulatory T Cells and Increases Respiratory Viral Infection Mortality Through Increased IFNγ Production

    doi: 10.3389/fimmu.2018.01587

    Figure Lengend Snippet: Increased mortality in streptomycin-treated mice is IFNγ dependent. (A) Mice were given streptomycin for 2 weeks before infection with Sendai virus (SeV). At day 5 and 9 PI SeV, 100 µg of an IFNγ blocking or control IgG monoclonal antibody was given SQ and mortality measured ( n = 5/group). (B) Mice were treated as in panel (A) but with the indicated doses of anti-IFNγ antibodies being administered ( n = 5/group). p

    Article Snippet: For NK1.1 depletion, anti-mouse NK1.1 antibody (clone PK136; BioXCell) or control mouse IgG2a (clone C1.18.4; BioXCell) was used, and for CD4 depletion anti-mouse CD4 (clone RM4-5; eBioscience) or control rat IgG2b (eBR2a; eBioscience) were used.

    Techniques: Mouse Assay, Infection, Blocking Assay

    GpepIP-specific CD4+ T cells exhibit high potency on helping humoral immune responses to HIV-1 trimer. a Immunization scheme. BALB/c mice were primed twice by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Three weeks later, all groups were immunized with the clade A BG505 gp140 NFL trimer emulsified in incomplete Freund’s adjuvant. Sera were collected at the indicated time points. Mice were euthanized 10 days after trimer immunization. b Splenic and lymph node cells were isolated and stimulated with GpepIP or pepIP in vitro for 5 days. T cell proliferation by CFSE dilution was measured by flow cytometry. c BG505-specific IgG production was examined in all three groups across the indicated time points by ELISA using a serum dilution 1:100. d GC response, defined by the percentage of GL7 + Fas + B cells among B220 + B cells, was evaluated in all three groups on day 10 after trimer immunization by flow cytometry. e – g Expression levels of activation marker CD69 e , CD80 f , and MHCII g were detected on splenic B cells after in vitro stimulation with the BG505 trimer for 3 days. MFI mean fluorescence intensity. Representative results are shown from one of three independent experiments performed. (mean ± s.d.). b medium n = 2; GpepIP or pepIP n = 3 independent experiments. d n = 4 independent experiments. e – g medium n = 2; BG505 n = 3 independent experiments. P -values were determined using Student’s two-sided t -tests. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Glycopeptide epitope facilitates HIV-1 envelope specific humoral immune responses by eliciting T cell help

    doi: 10.1038/s41467-020-16319-0

    Figure Lengend Snippet: GpepIP-specific CD4+ T cells exhibit high potency on helping humoral immune responses to HIV-1 trimer. a Immunization scheme. BALB/c mice were primed twice by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Three weeks later, all groups were immunized with the clade A BG505 gp140 NFL trimer emulsified in incomplete Freund’s adjuvant. Sera were collected at the indicated time points. Mice were euthanized 10 days after trimer immunization. b Splenic and lymph node cells were isolated and stimulated with GpepIP or pepIP in vitro for 5 days. T cell proliferation by CFSE dilution was measured by flow cytometry. c BG505-specific IgG production was examined in all three groups across the indicated time points by ELISA using a serum dilution 1:100. d GC response, defined by the percentage of GL7 + Fas + B cells among B220 + B cells, was evaluated in all three groups on day 10 after trimer immunization by flow cytometry. e – g Expression levels of activation marker CD69 e , CD80 f , and MHCII g were detected on splenic B cells after in vitro stimulation with the BG505 trimer for 3 days. MFI mean fluorescence intensity. Representative results are shown from one of three independent experiments performed. (mean ± s.d.). b medium n = 2; GpepIP or pepIP n = 3 independent experiments. d n = 4 independent experiments. e – g medium n = 2; BG505 n = 3 independent experiments. P -values were determined using Student’s two-sided t -tests. Source data are provided as a Source Data file.

    Article Snippet: IgG subclasses were detected by HRP-conjugated anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (dilution 1:10,000, Abcam) and TMB substrate as described above.

    Techniques: Mouse Assay, Injection, Isolation, In Vitro, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Activation Assay, Marker, Fluorescence

    GpepIP primary immunization prior to trimer immunization elicits functional antibody production. a Immunization scheme. BALB/c mice were primed three times (with a 3-week interval) by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Subsequently, all groups were immunized with the clade A BG505 gp140 NFL trimer adjuvanted with Alum for three times (with a 3-week interval). Sera were collected 7 days after each trimer immunization (post 1–3). b BG505-specific IgG production was examined in all three groups post each trimer immunization by ELISA using a serum dilution 1:400. c Antisera from all three groups after the third trimer immunizations were analyzed for IgG subclass switching by ELISA using a serum dilution 1:1600. d The neutralizing activity (neutralization 50% inhibitory dilution (ID 50 )) of antisera from adjuvant, GpepIP, and pepIP primary followed by three BG505 booster immunizations were tested against tier 1 and tier 2 HIV-1 viruses via a TZM-bl cell-based neutralization assays. MLV-pseudotyped virus was used as negative control for non-HIV-1-specific inhibitory activity in the assay. Antibody CH01-31 was used as positive control (shown as antibody concentration). e BG505 was chemically labeled with fluorescein isothiocyanate (FITC) and incubated with BMDCs at 37 °C for 2 h. Cells were then collected and antigen uptake was measured by flow cytometry. To evaluate antisera for their function, fluorophore-labeled BG505 was pre-incubated with antisera used in e at different dilutions before adding into BMDCs. The uptake rate is defined as FITC-positive cells compared to no BG505 (medium) group. Representative results are shown from one of three independent experiments performed. (mean ± s.d.). b n = 6 for pre-bleed; n = 2 independent experiments for post 1–3. n = 4 and 3 independent experiments for c and e , respectively. P -values were determined using Student’s two-sided t -tests. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Glycopeptide epitope facilitates HIV-1 envelope specific humoral immune responses by eliciting T cell help

    doi: 10.1038/s41467-020-16319-0

    Figure Lengend Snippet: GpepIP primary immunization prior to trimer immunization elicits functional antibody production. a Immunization scheme. BALB/c mice were primed three times (with a 3-week interval) by subcutaneous injection of GpepIP or pepIP emulsified in Freund’s adjuvant or of adjuvant alone. Subsequently, all groups were immunized with the clade A BG505 gp140 NFL trimer adjuvanted with Alum for three times (with a 3-week interval). Sera were collected 7 days after each trimer immunization (post 1–3). b BG505-specific IgG production was examined in all three groups post each trimer immunization by ELISA using a serum dilution 1:400. c Antisera from all three groups after the third trimer immunizations were analyzed for IgG subclass switching by ELISA using a serum dilution 1:1600. d The neutralizing activity (neutralization 50% inhibitory dilution (ID 50 )) of antisera from adjuvant, GpepIP, and pepIP primary followed by three BG505 booster immunizations were tested against tier 1 and tier 2 HIV-1 viruses via a TZM-bl cell-based neutralization assays. MLV-pseudotyped virus was used as negative control for non-HIV-1-specific inhibitory activity in the assay. Antibody CH01-31 was used as positive control (shown as antibody concentration). e BG505 was chemically labeled with fluorescein isothiocyanate (FITC) and incubated with BMDCs at 37 °C for 2 h. Cells were then collected and antigen uptake was measured by flow cytometry. To evaluate antisera for their function, fluorophore-labeled BG505 was pre-incubated with antisera used in e at different dilutions before adding into BMDCs. The uptake rate is defined as FITC-positive cells compared to no BG505 (medium) group. Representative results are shown from one of three independent experiments performed. (mean ± s.d.). b n = 6 for pre-bleed; n = 2 independent experiments for post 1–3. n = 4 and 3 independent experiments for c and e , respectively. P -values were determined using Student’s two-sided t -tests. Source data are provided as a Source Data file.

    Article Snippet: IgG subclasses were detected by HRP-conjugated anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (dilution 1:10,000, Abcam) and TMB substrate as described above.

    Techniques: Functional Assay, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Activity Assay, Neutralization, Negative Control, Positive Control, Concentration Assay, Labeling, Incubation, Flow Cytometry

    gp120 glycopeptide epitopes are recognized by CD4+ T cells. BALB/c mice were immunized with pooled gp120 glycopeptides (prepared by protease digestion of gp120). After booster immunization, CD4 + T cells were isolated and stimulated in vitro with either intact gp120 or PNGase F–treated, deglycosylated gp120 (DG-gp120) in the presence of mitomycin C-treated APCs for 5 days. a , b Flow cytometric analysis of T cell proliferation by CFSE division among CD4 + T cells. c , d Production of IL-4 c and IFN-γ d in the culture supernatant after T cell stimulation was measured by ELISA. e Recognition of coated gp120 by antiserum from mice immunized with gp120 glycopeptides in the presence of the indicated inhibitors was examined by competition ELISA using a serum dilution 1:1600. Serum titers are reported as OD at 405 nm. f – h Serum from mice immunized with pooled gp120 glycopeptides were collected 7 days after booster immunization. Titers of IgG1 f , IgG2a g , and IgG3 h for recognition of glycosylated gp120 or deglycosylated gp120 were measured by ELISA. Representative results are shown from one of three independent experiments performed. (mean ± s.d.). b n = 6 independent experiments. c n = 5 for medium and DG-gp120; n = 4 for gp120. n = 4, 3, 2, 2, 2 independent experiments for d – h , respectively. P -values were determined using Student’s two-sided t -tests. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Glycopeptide epitope facilitates HIV-1 envelope specific humoral immune responses by eliciting T cell help

    doi: 10.1038/s41467-020-16319-0

    Figure Lengend Snippet: gp120 glycopeptide epitopes are recognized by CD4+ T cells. BALB/c mice were immunized with pooled gp120 glycopeptides (prepared by protease digestion of gp120). After booster immunization, CD4 + T cells were isolated and stimulated in vitro with either intact gp120 or PNGase F–treated, deglycosylated gp120 (DG-gp120) in the presence of mitomycin C-treated APCs for 5 days. a , b Flow cytometric analysis of T cell proliferation by CFSE division among CD4 + T cells. c , d Production of IL-4 c and IFN-γ d in the culture supernatant after T cell stimulation was measured by ELISA. e Recognition of coated gp120 by antiserum from mice immunized with gp120 glycopeptides in the presence of the indicated inhibitors was examined by competition ELISA using a serum dilution 1:1600. Serum titers are reported as OD at 405 nm. f – h Serum from mice immunized with pooled gp120 glycopeptides were collected 7 days after booster immunization. Titers of IgG1 f , IgG2a g , and IgG3 h for recognition of glycosylated gp120 or deglycosylated gp120 were measured by ELISA. Representative results are shown from one of three independent experiments performed. (mean ± s.d.). b n = 6 independent experiments. c n = 5 for medium and DG-gp120; n = 4 for gp120. n = 4, 3, 2, 2, 2 independent experiments for d – h , respectively. P -values were determined using Student’s two-sided t -tests. Source data are provided as a Source Data file.

    Article Snippet: IgG subclasses were detected by HRP-conjugated anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (dilution 1:10,000, Abcam) and TMB substrate as described above.

    Techniques: Mouse Assay, Isolation, In Vitro, Cell Stimulation, Enzyme-linked Immunosorbent Assay

    The glycopeptide epitope GpepIP elicits a glycan-dependent cellular and humoral immune response. a , b CD4 + T cells obtained from mice immunized with gp120 were stimulated in vitro in the presence of mitomycin C-treated APCs pulsed with either GpepIP or pepIP, and T cell proliferation was examined by flow cytometry with use of CFSE fluorescence dilution through cell division. c , d CD4 + T cells obtained from mice immunized with GpepIP expressed in GnT1 −/− cells c or with pepIP d were stimulated with GpepIP expressed in GnTI −/− or 293F cells or with pepIP in the presence of mitomycin C-treated APCs, and T cell proliferation was examined by CFSE flow cytometry. e , f Antisera from mice immunized with GpepIP or pepIP were titrated for IgG binding to immobilized GpepIP e or pepIP f by ELISA. g Antisera from GpepIP and pepIP immunization groups recognize gp120 expressed in 293-F and GnTI −/− cells differentially as measured by ELISA using a serum dilution 1:400. h – j Serum from mice immunized with GpepIP expressed in GnT1 −/− cells were collected 7 days after booster immunization. As a control, serum from naïve mice was used as background. Titers of IgG1 h , IgG2a i , and IgG3 j for recognition of glycosylated gp120 or deglycosylated gp120 were measured by ELISA. Data was presented after subtracting background. Representative results are shown from one of three independent experiments performed. (mean ± s.d.). b medium n = 4; GpepIP and pepIP n = 6 independent experiments. c medium and GpepIP (GnTI) n = 4; GpepIP (293-F) and pepIP n = 5 independent experiments. n = 4 for e and f ; n = 8 for g ; n = 2 independent experiments for h , i , and j . P -values were determined using Student’s two-sided t -tests. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Glycopeptide epitope facilitates HIV-1 envelope specific humoral immune responses by eliciting T cell help

    doi: 10.1038/s41467-020-16319-0

    Figure Lengend Snippet: The glycopeptide epitope GpepIP elicits a glycan-dependent cellular and humoral immune response. a , b CD4 + T cells obtained from mice immunized with gp120 were stimulated in vitro in the presence of mitomycin C-treated APCs pulsed with either GpepIP or pepIP, and T cell proliferation was examined by flow cytometry with use of CFSE fluorescence dilution through cell division. c , d CD4 + T cells obtained from mice immunized with GpepIP expressed in GnT1 −/− cells c or with pepIP d were stimulated with GpepIP expressed in GnTI −/− or 293F cells or with pepIP in the presence of mitomycin C-treated APCs, and T cell proliferation was examined by CFSE flow cytometry. e , f Antisera from mice immunized with GpepIP or pepIP were titrated for IgG binding to immobilized GpepIP e or pepIP f by ELISA. g Antisera from GpepIP and pepIP immunization groups recognize gp120 expressed in 293-F and GnTI −/− cells differentially as measured by ELISA using a serum dilution 1:400. h – j Serum from mice immunized with GpepIP expressed in GnT1 −/− cells were collected 7 days after booster immunization. As a control, serum from naïve mice was used as background. Titers of IgG1 h , IgG2a i , and IgG3 j for recognition of glycosylated gp120 or deglycosylated gp120 were measured by ELISA. Data was presented after subtracting background. Representative results are shown from one of three independent experiments performed. (mean ± s.d.). b medium n = 4; GpepIP and pepIP n = 6 independent experiments. c medium and GpepIP (GnTI) n = 4; GpepIP (293-F) and pepIP n = 5 independent experiments. n = 4 for e and f ; n = 8 for g ; n = 2 independent experiments for h , i , and j . P -values were determined using Student’s two-sided t -tests. Source data are provided as a Source Data file.

    Article Snippet: IgG subclasses were detected by HRP-conjugated anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (dilution 1:10,000, Abcam) and TMB substrate as described above.

    Techniques: Mouse Assay, In Vitro, Flow Cytometry, Fluorescence, Binding Assay, Enzyme-linked Immunosorbent Assay

    IL-6 secreted by tumor associated fibroblasts (TAFs) induces hepcidin in breast cancer spheroids (A) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) in patient 113 lobular epithelial cell samples. Samples were 100% 2D tumor epithelial cells (2D TEC), 100% tumor epithelial cell spheroids (3D TEC), spheroids composed of a mixture of 80% TEC and 20% irradiated TAFs (isolated from patient 113), (80% TEC +20%TAF) and 100% irradiated TAF spheroids after 3 days of culture. (B) Western blot analysis of pro-hepcidin and Cyclophilin B for different TEC/TAF (%) combinations after 3 days of spheroid culture. (C) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) and (D) western blot analysis of phosphorylated and total STAT3 of primary TEC spheroids alone, spheroids composed of a mixture of 80% TECs and 20%TAFs, or TEC spheroids exposed to conditioned media (CM) from TAFs for 4 days. Patient 113 tumor ductal (113 Tu Duc), patient 113 tumor lobular (113 Tu Lob) and patient 107 tumor ductal (107 Tu Duc) were used for (C) and patient 113 tumor lobular for (D). For statistical analysis in (C) samples with 20% TAF or TAF CM were compared to their respective 3D TEC sample. (E) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) of patient 113 lobular TEC spheroids after the addition of TAF CM and IL-6 neutralizing antibody (1=1μg/mL and 5=5μg/mL) for 4 days. Neutralizing antibody against IgG (1 and 5 μg/mL) was used as a control. For statistical analysis, samples were compared to TAF CM sample. (F) Western blot analysis of pro-hepcidin and β-actin of patient 113 lobular TEC spheroids after the addition of TAF CM with or without 1μg/mL neutralizing antibodies against IL-6 or IgG for 4 days. All western blot quantifications (B, D and F) were compared to TEC alone.

    Journal: Oncogene

    Article Title: Contribution of three dimensional architecture and tumor-associated fibroblasts to hepcidin regulation in breast cancer

    doi: 10.1038/s41388-018-0243-y

    Figure Lengend Snippet: IL-6 secreted by tumor associated fibroblasts (TAFs) induces hepcidin in breast cancer spheroids (A) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) in patient 113 lobular epithelial cell samples. Samples were 100% 2D tumor epithelial cells (2D TEC), 100% tumor epithelial cell spheroids (3D TEC), spheroids composed of a mixture of 80% TEC and 20% irradiated TAFs (isolated from patient 113), (80% TEC +20%TAF) and 100% irradiated TAF spheroids after 3 days of culture. (B) Western blot analysis of pro-hepcidin and Cyclophilin B for different TEC/TAF (%) combinations after 3 days of spheroid culture. (C) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) and (D) western blot analysis of phosphorylated and total STAT3 of primary TEC spheroids alone, spheroids composed of a mixture of 80% TECs and 20%TAFs, or TEC spheroids exposed to conditioned media (CM) from TAFs for 4 days. Patient 113 tumor ductal (113 Tu Duc), patient 113 tumor lobular (113 Tu Lob) and patient 107 tumor ductal (107 Tu Duc) were used for (C) and patient 113 tumor lobular for (D). For statistical analysis in (C) samples with 20% TAF or TAF CM were compared to their respective 3D TEC sample. (E) RT-qPCR of hepcidin mRNA (normalized to Cyclophilin A) of patient 113 lobular TEC spheroids after the addition of TAF CM and IL-6 neutralizing antibody (1=1μg/mL and 5=5μg/mL) for 4 days. Neutralizing antibody against IgG (1 and 5 μg/mL) was used as a control. For statistical analysis, samples were compared to TAF CM sample. (F) Western blot analysis of pro-hepcidin and β-actin of patient 113 lobular TEC spheroids after the addition of TAF CM with or without 1μg/mL neutralizing antibodies against IL-6 or IgG for 4 days. All western blot quantifications (B, D and F) were compared to TEC alone.

    Article Snippet: For neutralization of IL-6 in TAF conditioned media, TAF conditioned media was collected as described above and pre-treated with 1 or 5 μg/mL anti-IL-6 or isotope-matched anti-IgG neutralizing antibodies (R & D Systems, cat#MAB2061-100, MAB004) for one hour before addition of CM to TECs during spheroid plating.

    Techniques: Quantitative RT-PCR, Irradiation, Isolation, Western Blot

    Hepcidin and GDF-15 are increased and their expression is correlated in breast tumors (A and B) Box plot with Tukey whisker of (A) GDF15 and (B) HAMP mRNA expression (log2 transformed) in normal adjacent tissue (n=61) compared to primary tumor tissue (n=526) in the TCGA breast cancer dataset. (C) GDF15 transcripts in TCGA samples from breast cancer patients divided by HAMP expression (below and above the mean) shown as box and whisker plot. (D) HAMP transcripts in TCGA samples from breast cancer patients divided by GDF15 expression (below and above the mean) shown as box and whisker. (E) Representative images of immunohistochemical staining of tumor tissue from patients with invasive ductal carcinoma (IDC). Proteins stained are Hepcidin, GDF-15, Pan-Cytokeratin and IgG control. (F) Scatter plot displays quantification of staining of epithelial cells from tissues from 56 BRCA patients. A regression analysis was performed to examine correlation of staining intensities (R 2 =0.4434 p

    Journal: Oncogene

    Article Title: Contribution of three dimensional architecture and tumor-associated fibroblasts to hepcidin regulation in breast cancer

    doi: 10.1038/s41388-018-0243-y

    Figure Lengend Snippet: Hepcidin and GDF-15 are increased and their expression is correlated in breast tumors (A and B) Box plot with Tukey whisker of (A) GDF15 and (B) HAMP mRNA expression (log2 transformed) in normal adjacent tissue (n=61) compared to primary tumor tissue (n=526) in the TCGA breast cancer dataset. (C) GDF15 transcripts in TCGA samples from breast cancer patients divided by HAMP expression (below and above the mean) shown as box and whisker plot. (D) HAMP transcripts in TCGA samples from breast cancer patients divided by GDF15 expression (below and above the mean) shown as box and whisker. (E) Representative images of immunohistochemical staining of tumor tissue from patients with invasive ductal carcinoma (IDC). Proteins stained are Hepcidin, GDF-15, Pan-Cytokeratin and IgG control. (F) Scatter plot displays quantification of staining of epithelial cells from tissues from 56 BRCA patients. A regression analysis was performed to examine correlation of staining intensities (R 2 =0.4434 p

    Article Snippet: For neutralization of IL-6 in TAF conditioned media, TAF conditioned media was collected as described above and pre-treated with 1 or 5 μg/mL anti-IL-6 or isotope-matched anti-IgG neutralizing antibodies (R & D Systems, cat#MAB2061-100, MAB004) for one hour before addition of CM to TECs during spheroid plating.

    Techniques: Expressing, Whisker Assay, Transformation Assay, Immunohistochemistry, Staining

    BMPs regulate hepcidin expression in MCF-7 breast cancer cells (A) RT-qPCR of BMP4, BMP6 or BMP7 mRNA (normalized to β-actin) in MCF-7 and MCF-10A monolayer cells. (B) Western blot analysis of pro-hepcidin and β-actin following siRNA knock-down of non-target control (NTC), BMP4, BMP6, and BMP7 for 48 hours in MCF-7 cells. For quantification, samples were compared to NTC. GAPDH siRNA was used as an additional control. (C) Western blot analysis of pro-hepcidin and β-actin after the addition of 1 and 3 μg/mL neutralizing antibodies against BMP4, BMP6, BMP7 or IgG (3μg/ml) isotope control for 48 hrs in MCF-7 cells. For quantification, samples were compared to untreated sample. (D) Western blot analysis of phosporylated-STAT3 (pSTAT3), total STAT3 (tSTAT3), pro-hepcidin, and β-actin following the addition of recombinant IL-6 for 24 hours in MCF-7 cells.

    Journal: Oncogene

    Article Title: Contribution of three dimensional architecture and tumor-associated fibroblasts to hepcidin regulation in breast cancer

    doi: 10.1038/s41388-018-0243-y

    Figure Lengend Snippet: BMPs regulate hepcidin expression in MCF-7 breast cancer cells (A) RT-qPCR of BMP4, BMP6 or BMP7 mRNA (normalized to β-actin) in MCF-7 and MCF-10A monolayer cells. (B) Western blot analysis of pro-hepcidin and β-actin following siRNA knock-down of non-target control (NTC), BMP4, BMP6, and BMP7 for 48 hours in MCF-7 cells. For quantification, samples were compared to NTC. GAPDH siRNA was used as an additional control. (C) Western blot analysis of pro-hepcidin and β-actin after the addition of 1 and 3 μg/mL neutralizing antibodies against BMP4, BMP6, BMP7 or IgG (3μg/ml) isotope control for 48 hrs in MCF-7 cells. For quantification, samples were compared to untreated sample. (D) Western blot analysis of phosporylated-STAT3 (pSTAT3), total STAT3 (tSTAT3), pro-hepcidin, and β-actin following the addition of recombinant IL-6 for 24 hours in MCF-7 cells.

    Article Snippet: For neutralization of IL-6 in TAF conditioned media, TAF conditioned media was collected as described above and pre-treated with 1 or 5 μg/mL anti-IL-6 or isotope-matched anti-IgG neutralizing antibodies (R & D Systems, cat#MAB2061-100, MAB004) for one hour before addition of CM to TECs during spheroid plating.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Recombinant

    Human serum IgG response against SGS. (A) ELISA was performed against L. intermedia and L. longipalpis SGS using human sera were from individuals from a CL endemic area (n = 100) or from control individuals (n = 10). (B) ELISA was performed against L. intermedia SGS with human sera from CL individuals (n = 16) or from healthy individuals with either a negative (n = 42) or a positive (n = 32) anti- Leishmania DTH skin test. The ordinate represents the absorbance of the ELISA reaction of these sera against SGS. The symbols indicate results obtained with each serum tested and lines represent their median values (***p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Enhanced Leishmania braziliensis Infection Following Pre-Exposure to Sandfly Saliva

    doi: 10.1371/journal.pntd.0000084

    Figure Lengend Snippet: Human serum IgG response against SGS. (A) ELISA was performed against L. intermedia and L. longipalpis SGS using human sera were from individuals from a CL endemic area (n = 100) or from control individuals (n = 10). (B) ELISA was performed against L. intermedia SGS with human sera from CL individuals (n = 16) or from healthy individuals with either a negative (n = 42) or a positive (n = 32) anti- Leishmania DTH skin test. The ordinate represents the absorbance of the ELISA reaction of these sera against SGS. The symbols indicate results obtained with each serum tested and lines represent their median values (***p

    Article Snippet: To check that anti-IgG1, anti-IgG2a and anti-IgG2b were working properly, purified mouse IgG1 (clone CD28.2 [2 µg/mL]); mouse IgG2a (clone HIT3a [2 µg/mL]) and mouse IgG2b (clone G265-5 [2 µg/mL]), all from BD Pharmingen) were employed as positive controls.

    Techniques: Enzyme-linked Immunosorbent Assay

    Anti-saliva antibody response following immunization of BALB/c mice with Lutzomyia intermedia SGS. BALB/c mice (3–5 per group) received 3 inoculations of PBS (open bars) or SGS (closed bars). (A) ELISA was performed with sera from mice inoculated with PBS (open bars) or SGS (closed bars). (B) IgG subclasses present in immune sera were determined by ELISA using IgG1, IgG2a and IgG2b conjugates. Purified IgG1, IgG2a and IgG2b (dotted bars) were employed as control IgG subclass (positive controls). Bars represent the means and standard errors of the means from three independent experiments (*p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Enhanced Leishmania braziliensis Infection Following Pre-Exposure to Sandfly Saliva

    doi: 10.1371/journal.pntd.0000084

    Figure Lengend Snippet: Anti-saliva antibody response following immunization of BALB/c mice with Lutzomyia intermedia SGS. BALB/c mice (3–5 per group) received 3 inoculations of PBS (open bars) or SGS (closed bars). (A) ELISA was performed with sera from mice inoculated with PBS (open bars) or SGS (closed bars). (B) IgG subclasses present in immune sera were determined by ELISA using IgG1, IgG2a and IgG2b conjugates. Purified IgG1, IgG2a and IgG2b (dotted bars) were employed as control IgG subclass (positive controls). Bars represent the means and standard errors of the means from three independent experiments (*p

    Article Snippet: To check that anti-IgG1, anti-IgG2a and anti-IgG2b were working properly, purified mouse IgG1 (clone CD28.2 [2 µg/mL]); mouse IgG2a (clone HIT3a [2 µg/mL]) and mouse IgG2b (clone G265-5 [2 µg/mL]), all from BD Pharmingen) were employed as positive controls.

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

    Specific humoral responses elicited by immunization of mice with microneme proteins. The reactivity of immunoglobulins anti-STAg was determined by ELISA in serum samples collected from both immunized (TgMICs) and control (PBS) mice 15 days after the last antigen injection. Each point/bar represents the average absorbance ± SD of the serum samples from 4 animals. (A) Absorbance provided by the reaction of serum IgG with STAg. (B) Absorbance provided by the reaction of serum IgG1 and IgG2a (diluted 1:25) with STAg. The average absorbance ± SD generated by the reaction of serum IgG1 or IgG2a from each group of immunized mice was significantly higher than the corresponding values provided by control mice, with the exception of the TgMIC6-immunized group, whose results were not significantly different of those of the control group. Three independent experiments were performed, and data from one representative experiment is shown. Asterisks represent statistical significant differences (*p

    Journal: PLoS ONE

    Article Title: Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice

    doi: 10.1371/journal.pone.0143087

    Figure Lengend Snippet: Specific humoral responses elicited by immunization of mice with microneme proteins. The reactivity of immunoglobulins anti-STAg was determined by ELISA in serum samples collected from both immunized (TgMICs) and control (PBS) mice 15 days after the last antigen injection. Each point/bar represents the average absorbance ± SD of the serum samples from 4 animals. (A) Absorbance provided by the reaction of serum IgG with STAg. (B) Absorbance provided by the reaction of serum IgG1 and IgG2a (diluted 1:25) with STAg. The average absorbance ± SD generated by the reaction of serum IgG1 or IgG2a from each group of immunized mice was significantly higher than the corresponding values provided by control mice, with the exception of the TgMIC6-immunized group, whose results were not significantly different of those of the control group. Three independent experiments were performed, and data from one representative experiment is shown. Asterisks represent statistical significant differences (*p

    Article Snippet: Plates were then incubated at 37°C for 1 h, washed four times, and incubated with horseradish-peroxidase-conjugated goat anti-mouse IgG, IgG1, or IgG2b (Santa Cruz Biotechnology), at 1:5000 dilution in blocking buffer, for 1 h, at 37°C.

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

    SDS-PAGE and western blot analysis of native and recombinant microneme proteins. SDS-PAGE of recombinant proteins (panels A, B, and C, Coomassie Blue stained) or native (panel D, silver-stained) proteins. Heterologous expression was noted in E . coli (DE3) and recombinant proteins were detected in inclusion bodies. Lane 1: protein expression before induction. Lane 2: protein expression after induction. Lane 3: purified and refolded histidine-tagged recombinant proteins, displayed apparent molecular masses of 70-kDa (TgMIC1, panel A), 80-kDa (TgMIC4, panel B), and 30-kDa (TgMIC6, panel C). Panel E shows the electrophoretical profile of the Lac+ fraction, composed of the native proteins TgMIC1 (53-kDa) and TgMIC4 (68-kDa). Lane M: Molecular mass markers. Reactivity of recombinant and native microneme proteins with anti-Lac+ mouse serum was examined by western blot (Panel E), developed with peroxidase-conjugated goat anti-mouse IgG.

    Journal: PLoS ONE

    Article Title: Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice

    doi: 10.1371/journal.pone.0143087

    Figure Lengend Snippet: SDS-PAGE and western blot analysis of native and recombinant microneme proteins. SDS-PAGE of recombinant proteins (panels A, B, and C, Coomassie Blue stained) or native (panel D, silver-stained) proteins. Heterologous expression was noted in E . coli (DE3) and recombinant proteins were detected in inclusion bodies. Lane 1: protein expression before induction. Lane 2: protein expression after induction. Lane 3: purified and refolded histidine-tagged recombinant proteins, displayed apparent molecular masses of 70-kDa (TgMIC1, panel A), 80-kDa (TgMIC4, panel B), and 30-kDa (TgMIC6, panel C). Panel E shows the electrophoretical profile of the Lac+ fraction, composed of the native proteins TgMIC1 (53-kDa) and TgMIC4 (68-kDa). Lane M: Molecular mass markers. Reactivity of recombinant and native microneme proteins with anti-Lac+ mouse serum was examined by western blot (Panel E), developed with peroxidase-conjugated goat anti-mouse IgG.

    Article Snippet: Plates were then incubated at 37°C for 1 h, washed four times, and incubated with horseradish-peroxidase-conjugated goat anti-mouse IgG, IgG1, or IgG2b (Santa Cruz Biotechnology), at 1:5000 dilution in blocking buffer, for 1 h, at 37°C.

    Techniques: SDS Page, Western Blot, Recombinant, Staining, Expressing, Purification

    Experimental Protocol. (A) In the first experimental procedure mice were subcutaneously (s.c.) vaccinated with microneme proteins emulsified in Freund’s complete adjuvant. Mice were boosted at the same dose and regimen on day 15 and 30 after first injection, now emulsified in Freund’s incomplete adjuvant. Fifteen days after the last injection, blood and spleen samples were collected to assess serum IgG, in vitro T cell proliferation, and cytokine concentrations. (B) One month after the last immunization procedure, the mice were orally infected with 80 cysts of the ME49 strain and the mortality was monitored daily for 1 month. To evaluate the tissue cyst burden, the brain of the mice infected with 40 cysts was removed 1 month after the challenge and the mean number of cysts per brain was determined. Additionally, blood samples from mice challenged with 40 cysts were collected 30 days after T . gondii challenge and the serum cytokine levels were analyzed.

    Journal: PLoS ONE

    Article Title: Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice

    doi: 10.1371/journal.pone.0143087

    Figure Lengend Snippet: Experimental Protocol. (A) In the first experimental procedure mice were subcutaneously (s.c.) vaccinated with microneme proteins emulsified in Freund’s complete adjuvant. Mice were boosted at the same dose and regimen on day 15 and 30 after first injection, now emulsified in Freund’s incomplete adjuvant. Fifteen days after the last injection, blood and spleen samples were collected to assess serum IgG, in vitro T cell proliferation, and cytokine concentrations. (B) One month after the last immunization procedure, the mice were orally infected with 80 cysts of the ME49 strain and the mortality was monitored daily for 1 month. To evaluate the tissue cyst burden, the brain of the mice infected with 40 cysts was removed 1 month after the challenge and the mean number of cysts per brain was determined. Additionally, blood samples from mice challenged with 40 cysts were collected 30 days after T . gondii challenge and the serum cytokine levels were analyzed.

    Article Snippet: Plates were then incubated at 37°C for 1 h, washed four times, and incubated with horseradish-peroxidase-conjugated goat anti-mouse IgG, IgG1, or IgG2b (Santa Cruz Biotechnology), at 1:5000 dilution in blocking buffer, for 1 h, at 37°C.

    Techniques: Mouse Assay, Injection, In Vitro, Infection