Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Comparison of L5-VRT and SNI-induced expression of CX3CL1 in bilateral ACC. Representative protein microarray results ( A ) and the quantification analysis ( B ) showing that SNI triggered upregulations of chemokines CX3CL1 and CCL11 in contralateral ACC.* p < 0.05, ** p < 0.01 versus sham group ( n = 3 rats/group, one-way ANOVA). C , D Representative western blotting of CX3CL1 protein levels in bilateral ACC following SNI ( C ) and L5-VRT ( D ) are shown on the top and the quantification results are shown below. Significant differences are observed on the contralateral side in the SNI group but on the bilateral side in the L5-VRT group. * p < 0.05 versus sham group ( n = 3 rats/group, one-way ANOVA)

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques: Comparison, Expressing, Microarray, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Nerve injury-induced CX3CL1 is expressed in ACC neurons. Double-immunofluorescence staining showing the co-localization of CX3CL1-IR (red) with NeuN (green, top), but not Iba1 (green, medial) and GFAP (green, below) 7d post-SNI. Scale bar = 50 μm. The fluorescence intensity curves for red and green from boxed areas are shown on the right side of each group

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques: Double Immunofluorescence Staining, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Effects of DREADD-hM3Dq and hM4Di on ACC CX3CL1 protein levels and the mechanical paw withdrawal thresholds. A Behavioral test paradigms are shown on the top and representative western blotting for bilateral ACC CX3CL1 in the mice injected with DREADD-Gq or DREADD-Gi are shown below. B The injection site of virus carrying hM3Dq-, hM4Di-mCherry or mCherry in contralateral ACC. C Quantification of bilateral CX3CL1 in the mice injected with DREADD-Gq and DREADD-Gi, respectively. * p < 0.05, *** p < 0.001 versus mCherry control groups ( n = 3 mice/group, two-way ANOVA). D Compared to the mCherry control mice, DREADD-hM3Dq induced significant decrease of threshold in ipsilateral hind paws of normal mice (left); DREADD-hM4Di reversed the SNI-induced decrease of ipsilateral threshold (right). * p < 0.05 versus mCherry control groups ( n = 6 mice/group, multiple t tests)

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques: Western Blot, Injection, Virus, Control

Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Microinjection of anti-CX3CL1 antibody into ACC attenuates SNI-induced mechanical allodynia and inhibits ACC TNF-α and Nav1.6 protein upregulations. A , B Behavioral test paradigms are shown on the first left, and the ipsilateral and contralateral test results are shown on the second and first right, respectively. Statistical significance was determined by Dunn's multiple comparisons test (* p < 0.05; ** p < 0.01 versus PO -1d) or multiple t tests (# p < 0.05 versus IgG control). C The mark left by the cannula implantation showing the drug injection site in Cg1 region. D Representative western blotting of CX3CL1, TNF-α and Nav1.6 in bilateral ACC following contralateral administration of anti-CX3CL1 antibody (1 μg/μl, 3 μl) 7d post-SNI. E The quantification results showing the inhibitory effects of anti-CX3CL1 antibody on SNI-induced CX3CL1, TNF-α and Nav1.6 expressions. * p < 0.05, ** p < 0.01, *** p < 0.001 versus IgG control (one-way ANOVA)

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques: Microinjection, Control, Injection, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Pretreatment with anti-CX3CL1 antibody in contralateral ACC modulates the expression levels of spinal c-Fos, Iba1, TNF-α, IL-6 after SNI surgery. A Pretreatment with anti-CX3CL1 antibody in contralateral ACC (1 μg/μl, 3 μl) 30 min before nerve injury downregulates Iba1-IR in ipsilateral SDH 7d post-SNI. The injection site in ACC and the detection area by grey rectangular box in spinal cord are shown on the left. Representative images obtained from sham and SNI rats pretreated with IgG or anti-CX3CL1 antibody (upper right). Scale bar = 500 μm. Insets from ipsilateral SDH (lower right). Scale bar = 50 μm. B Quantification for mean fluorescence intensity of Iba1 from bilateral SDH (*** p < 0.001, two-way ANOVA). C – G The expression levels of Iba1, CD11b, c-Fos, TNF-α and IL-6 in ipsilateral SDH following anti-CX3CL1 treatment in contralateral ACC and quantification for western blot data. * p < 0.05, ** p < 0.01, *** p < 0.001 (paired t test or one-way ANOVA)

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques: Expressing, Injection, Fluorescence, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury

doi: 10.1186/s12974-022-02525-8

Figure Lengend Snippet: Graphical abstract: anterior cingulate cortex (ACC), a first-order cortical region that responds to painful stimuli, may play important roles in the occurrence of mirror-image pain. In this study, we provide evidence that in ACC, stronger immune response to L5-VRT surgery contributes to mirror pain, and CX3CL1 mediates the descending facilitation and aggravates the spinal neuroinflammation. Strategies targeting chemokine-mediated ACC hyperexcitability may develop novel therapeutics for neuropathic pain

Article Snippet: After a week of recovery from catheterization, anti-rat CX3CL1 antibody (AF-510-NA, R&D systems, Inc.) or normal goat IgG control (AB-108-C, R&D systems, Inc.) were injected into the ACC at a dose of 10 μg/ml (10 μl, R&D) over a 5 min period, 30 min before or 7 d after SNI.

Techniques:

Journal: Nature Communications

Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation

doi: 10.1038/s41467-018-07626-8

Figure Lengend Snippet: B cells inhibit osteoblast differentiation in vitro by secreting CCL3 and TNF. a – c BM B cells from WT or TNF-Tg mice were purified, as in Fig. . B cells were co-cultured with MPCs ± anti-CCL3 neutralizing Ab ± anti-TNF neutralizing Ab in OB-inducing medium for 2 days. a ALP+ area was measured. * p < 0.05 vs. WT, # p < 0.05 vs. TNF-Tg treated with IgG. ( b , c ) B cells were removed and protein lysates from the MPCs were subjected to Western blot analyses. Expression levels of NF-κB ( b ) and ERK signaling ( c ) molecules in cell lysates from MPCs were assessed. Supplementary Fig. shows uncropped gel images. Each experiment was performed 3 to 5 times. Representative images and quantifications shown in the figure come from one independent experiment. All error bars represent s.e.m. One way ANOVA followed by Dunnett’s post-hoc multiple comparisons was performed

Article Snippet: After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA.

Techniques: In Vitro, Purification, Cell Culture, Western Blot, Expressing

Journal: Nature Communications

Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation

doi: 10.1038/s41467-018-07626-8

Figure Lengend Snippet: RA B cells express high levels of CCL3 and TNF and inhibit osteoblast bone formation in vivo. a MPCs were treated with different doses of CCL3 in OB-inducing medium for 2 days. The area of ALP+ cells was measured. * p < 0.05 vs. Veh. b Expression levels of Runx2 and ALP in 50 ng/ml CCL3 treated cells from a were measured by qPCR. Fold-changes were calculated by dividing the values with those from Veh. * p < 0.05 vs. Veh. c CCL3+ B cells (yellow) were detected in subchondral bone (SB) area in frozen sections of proximal tibiae from TNF-Tg mice and their WT littermates by double IF staining with anti-B220 (red) and anti-CCL3 (green) Abs. Bar = 25 μm. d , e BM B cells from WT or TNF-Tg mice were purified and stimulated (S) with 2.5 μg/ml anti-CD40 Ab +10 ng/ml IL4 +10 μg/ml LPS or vehicle (U) for 4 h. CCL3 ( d ) and TNF ( e ) protein expression levels were assessed in the culture media by ELISA. f – h WT MPCs were transplanted with BM B cells from WT, TNF-Tg, TNF-Tg/CCL3-KO, TNF-Tg/TNF-KO, and TNF-Tg/CCL3-KO/TNF-KO mice by subcutaneous surgical implantation into recipient SCID mice. 4 weeks later, the implants were harvested and H&E staining ( f ) and Goldner’s Trichrome ( g ) staining were performed. B bone. G GelFoam. Bar = 50 μm. h A histomorphometric analysis of bone volume to tissue volume in H&E-stained sections. i MPCs from GFP transgenic mice were transplanted with BM B cells from mTmG mice. Representative images show mTmG+ B cells (red) and GFP+ MPCs (green) in the implants. White dashed lines indicate the bone surface. Bar = 25 μm. * p < 0.05 as indicated groups. Each experiment was performed 3 to 5 times. Representative images and quantifications are shown from one independent experiment. All error bars represent s.e.m. Two-tailed unpaired Student’s t -test was performed for b . One way ANOVA followed by Dunnett’s post-hoc multiple comparisons was performed for all the others

Article Snippet: After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA.

Techniques: In Vivo, Expressing, Staining, Purification, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Two Tailed Test

Journal: Nature Communications

Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation

doi: 10.1038/s41467-018-07626-8

Figure Lengend Snippet: B cells from RA patients inhibit OB differentiation. a–c Peripheral blood (PB) B cells were purified, as in Fig. and stimulated (S) with CpG2006+anti-Ig (A+G+M) Ab or vehicle (U) for 4 h. a CCL3 and TNF mRNA expression was detected by qPCR. * p < 0.05. b CCL3 and TNF protein expression levels were assessed in the culture media by ELISA. * p < 0.05. c Conditioned medium (40% by volume) from B cell culture were co-cultured with human MSCs ± anti-CCL3 neutralizing Ab ± anti-TNF neutralizing Ab in OB-inducing medium for 3 days. ALP+ area was measured. * p < 0.05 vs. U, # p < 0.05 vs. S-IgG. d PB B cells from RA patients and healthy controls (HC) were co-cultured with human MSCs in OB-inducing medium for 3 days. ALP staining was performed. * p < 0.05 vs. HC. e The expression levels of ALP and Runx2 in hMSCs in d were measured by qPCR. Fold-changes were calculated by dividing patient values by the value from HC cells. Values represent individual HC/RA. *p < 0.05 vs. HC. f RA synovium was stained with Abs to CD20 (B cells), CCL3 and TNF. White arrows indicate the cells with dual staining. Bar = 25 μm. 3 or 6 patients and their controls were included in each experiment. All of these experiments were repeated at least once with representative images and quantifications shown. All error bars represent s.e.m. One way ANOVA followed by Dunnett’s post-hoc multiple comparisons was performed for c . Two-tailed unpaired Student’s t -test was performed for all the others

Article Snippet: After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA.

Techniques: Purification, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, Two Tailed Test

Journal: Particle and Fibre Toxicology

Article Title: Translocation of particles and inflammatory responses after exposure to fine particles and nanoparticles in an epithelial airway model

doi: 10.1186/1743-8977-4-9

Figure Lengend Snippet: TNF-α release in triple cell co-cultures upon particle incubation. TNF-α levels in the supernatants (upper chamber, lower chamber) were measured by ELISA. TNF-α release in cells exposed to LPS, 1 μm, and 0.078 μm polystyrene particles, TiO 2 , and gold NP. Values are means ± SD of 3 experiments. * indicates a statistical difference to the levels in the supernatants in the control of the upper chamber, § indicates a statistical difference to the levels in the supernatants in the control of the lower chamber.

Article Snippet: After centrifugation, TNF-α was quantified by a commercially available DuoSet ELISA Development kit (R&D Systems, Catalogue Number: DY 210, Oxon, UK) according to the manufacturer's recommendations.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Mucosal Immunology

Article Title: TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability

doi: 10.1038/s41385-022-00506-x

Figure Lengend Snippet: A Humanized TNF colitis model. Rag1 −/− or hTNF‐KI x Rag1 −/− mice were reconstituted with naive T cells from either WT or hTNF‐KI mice. Mice were treated once they had lost > 5 % of their initial weight twice per week for two or three weeks with various anti-TNF agents or respective controls (10 mg/kg; i.p.). B Weight of hTNF‐KIxRag1 −/− mice reconstituted with naive T cells from hTNF‐KI mice and treated with infliximab ( n = 6) or Fc control ( n = 5) (10 mg/kg) twice per week. Data are representative of two independent experiments. C Colitis inflammation scores of hTNF‐KIxRag1 −/− mice reconstituted with naive T cells from hTNF‐KI mice upon 2 weeks treatment with infliximab ( n = 4) or isotype control ( n = 8). Data are representative of two independent experiments. D Colitis inflammation scores of hTNF‐KIxRag1 −/− mice reconstituted with naive T cells from hTNF‐KI mice upon 3 weeks treatment with infliximab ( n = 6) or isotype control ( n = 5). Frequencies of CD11b + (CD45 + CD11b + ) E , Th1 (CD45 + CD4 + TCRβ + IFNγ + ) F , Th17 (CD45 + CD4 + TCRβ + IL-17A + ) G and Th1/17 (CD45 + CD4 + TCRβ + IL-17A + IFNγ + ) H cells after 3 weeks of infliximab ( n = 6) or Fc control treatment ( n = 5). All data in D – H are representative of two independent experiments. Data represent mean values ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, as calculated by Student’s t‐test, ns not significant.

Article Snippet: Once mice had lost >5% of initial weight, they were treated with anti-human TNF antibody (Infliximab), anti-mouse IL-22 (R&D Systems; AF582) or human Fc-IgG1 isotype control (BioXCell; Cat # BE0096) in PBS.

Techniques: Control

Journal: Mucosal Immunology

Article Title: TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability

doi: 10.1038/s41385-022-00506-x

Figure Lengend Snippet: Naive hTNF-KI T cells were transferred to Rag1 −/− recipients, anti-TNF (infliximab; 10 mg/kg; i.p. twice per week) was administered once mice had lost 5% of their initial weight. A Weight changes 2 weeks after treatment of colitic mice with either Fc control or infliximab (both 10 mg/kg; i.p, twice per week). Representative images of Hematoxylin/Eosin stained tissue sections B and inflammation score C of the colon in mice treated with either Fc control of infliximab for 2 weeks. Scale bar is equal to 100 µm. A – C Data from three independent experiments are shown. D Weight changes 3 weeks after treatment of colitic mice with either Fc control or infliximab (both 10 mg/kg; i.p, twice per week). Representative images of Hematoxylin/Eosin stained tissue sections E and inflammation score F of the colon in mice treated with either Fc control of infliximab for 3 weeks. Scale bar is equal to 100 µm. D – F Data from two independent are shown. Naive WT T cells were transferred to hTNFK-KI×Rag1 −/− recipients, anti-TNF (infliximab; 10 mg/kg; i.p. twice per week) was administered once mice had lost 5% of their initial weight. G Weight changes 2 weeks after treatment of colitic mice with either Fc control or infliximab (both 10 mg/kg; i.p, twice per week). Representative images of Hematoxylin/Eosin stained tissue sections H and inflammation score ( I ) of the colon in mice treated with either Fc control or infliximab for 2 weeks. Scale bar is equal to 100 µm. Data are representative of two independent experiments. Data represent mean values ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, as calculated by Student’s t-test; ns not significant.

Article Snippet: Once mice had lost >5% of initial weight, they were treated with anti-human TNF antibody (Infliximab), anti-mouse IL-22 (R&D Systems; AF582) or human Fc-IgG1 isotype control (BioXCell; Cat # BE0096) in PBS.

Techniques: Control, Staining

Journal: Mucosal Immunology

Article Title: TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability

doi: 10.1038/s41385-022-00506-x

Figure Lengend Snippet: Naive hTNF-KI T cells were transferred to Rag1 −/− recipients, anti-TNF (infliximab; 10 mg/kg; i.p. twice per week) was administered once mice lost 5% of their initial weight. A Representative images of colon tissue sections stained immunohistochemically for proliferation (Ki67, brown) and for CD3 expression (red) depict increased proliferation of colonic epithelial cells after 2 weeks of anti-TNF therapy. Scale bar is equal to 100 µm in x100 magnification and 25 µm in x400 magnification. B Increased frequencies of Ki67-positive epithelial cells and mean numbers of epithelial cells per crypt after 2 weeks of anti-TNF therapy as described in A . C Representative images of colon tissue sections stained immunohistochemically for proliferation (Ki67, red) and for CD3 expression (brown) show proliferation of colonic epithelial cells after 3 weeks of anti-TNF therapy. Scale bar is equal to 100 µm in x100 magnification and 25 µm in x400 magnification. D Frequencies of Ki67-positive epithelial cells and mean numbers of epithelial cells per crypt after 3 weeks of anti-TNF therapy as described in C . All data are representative of two independent experiments. Data represent mean values ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, as calculated by Student’s t-test; ns not significant.

Article Snippet: Once mice had lost >5% of initial weight, they were treated with anti-human TNF antibody (Infliximab), anti-mouse IL-22 (R&D Systems; AF582) or human Fc-IgG1 isotype control (BioXCell; Cat # BE0096) in PBS.

Techniques: Staining, Expressing

Journal: Mucosal Immunology

Article Title: TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability

doi: 10.1038/s41385-022-00506-x

Figure Lengend Snippet: A Numbers of goblet cells in the colon as revealed by PAS staining after 2 weeks of anti-TNF therapy. Data are representative of two independent experiments. B Numbers of apoptotic EC (Casp3 + ) per 100 EC in the colon after 2 weeks of anti-TNF therapy. Data collected using two independent experiments are shown. C , D . Representative images of colon sections stained immunohistochemically for pSTAT3 (brown) show increased pSTAT3 + epithelial cells upon anti-TNF therapy. B – D Data collected using two independent experiments are shown. Naive hTNF-KI T cells were transferred to Rag1 −/− recipients, anti-TNF (infliximab; 10 mg/kg; i.p. twice per week) was administered once mice lost 5% of their initial weight. Scale bar is equal to 100 µm. E Expression of STAT3 dependent genes ( Reg3β , Reg3γ , survivin , smoothened ) in the colon after 2 weeks of anti-TNF therapy. Data are representative of two independent experiments. F Gene sets upregulated in colonic EC (sorted live EpCAM1+ cells) 24 h after infliximab treatment, when compared to Fc-control treated group. G Gene sets downregulated in colonic EC (sorted live EpCAM1 + cells) 24 h after infliximab treatment, when compared to Fc-control treated group. F , G Data are from one experiment using epithelial cells from individual mice. Data represent mean values ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, as calculated by Student’s t-test; ns not significant.

Article Snippet: Once mice had lost >5% of initial weight, they were treated with anti-human TNF antibody (Infliximab), anti-mouse IL-22 (R&D Systems; AF582) or human Fc-IgG1 isotype control (BioXCell; Cat # BE0096) in PBS.

Techniques: Staining, Expressing, Control

Journal: Mucosal Immunology

Article Title: TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability

doi: 10.1038/s41385-022-00506-x

Figure Lengend Snippet: A Levels of IL-22 in ex vivo colonic explants from mice treated with Fc control or infliximab for 2 weeks. B IL-22 mRNA levels in the colons of mice treated with Fc control or infliximab for 2 weeks. C IL-22BP mRNA levels in the colons of mice treated with Fc control or infliximab for 2 weeks. D Serum IL-22BP concentration in sera, E Ratio of IL-22 and IL-22BP mRNA levels in the colons of mice treated with Fc control or infliximab for 2 weeks. Naive hTNF-KI T cells were transferred to Rag1 −/− recipients, anti-TNF (infliximab; 10 mg/kg; i.p. twice per week) was administered once mice lost 5% of their initial weight. F Representative images of colon sections stained with Hematoxylin/Eosin and immunohistochemically stained for Ki67 (brown) and CD3 (red), G weight changes, H corresponding inflammation scores, I percentage of proliferating colonic epithelial cells after treatment of colitic mice with either Fc control, infliximab, anti-IL-22 or infliximab/anti-IL-22 (Fc control and infliximab - 10 mg/kg; anti-IL-22 - 10 mcg/mouse; i.p, twice per week) for 2 weeks. Naive hTNF-KI T cells were transferred to Rag1 −/− recipients, antibodies were administered once mice had lost 5% of their initial weight. Scale bar is equal to 100 µm in x100 magnification and 25 µm in x400 magnification. J Colonic inflammation score at various time points after start of anti-mTNF treatment. Percentage of proliferating colonic epithelial cells K , IL-22BP mRNA levels L in the colons of mice treated with Fc control or anti-mTNF for 2 weeks. All data are representative of two independent experiments. Data represent mean values ± SEM. * p < 0.05, ** p < 0.01,*** p < 0.001, as calculated by Student’s t-test; ns not significant.

Article Snippet: Once mice had lost >5% of initial weight, they were treated with anti-human TNF antibody (Infliximab), anti-mouse IL-22 (R&D Systems; AF582) or human Fc-IgG1 isotype control (BioXCell; Cat # BE0096) in PBS.

Techniques: Ex Vivo, Control, Concentration Assay, Staining

Journal: Mucosal Immunology

Article Title: TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability

doi: 10.1038/s41385-022-00506-x

Figure Lengend Snippet: A TNF blockade increases levels of bioactive colonic IL-22 that can be inhibited by recombinant IL-22BP. Supernatants derived from colonic explants of Rag1 −/− mice which received naive hTNF-KI T cells and treated for 2 weeks with anti-TNF, were added to human colonic Colo205 cell line with or without rmIL-22BP (1,25 mg/ml). Production of human IL-10 as an indicator of bioactive IL-22 has been determined 48 h later. B Expression of IL-22BP mRNA in sorted CD45 + CD11c + MHCII + CD103 + and CD45 + TCRβ + cells from the colon of mice treated with Fc control or infliximab for 2 weeks. Naive hTNF-KI T cells were transferred to Rag1 −/− recipients, anti-TNF (infliximab; 10 mg/kg; i.p. twice per week) was administered once mice had lost 5% of their initial weight. C Expression of IL-22BP in colon and spleen from naive WT and TNF −/− mice. D Expression of IL-22BP in spleen from naive WT, T-TNF −/− , B-TNF −/− , and M-TNF −/− mice. E Expression of IL-22BP in sorted CD4 + CD11c + MHCII + or CD8 + CD11c + MHCII + cells isolated from spleen of naïve WT and TNF −/− mice. CD4 + CD11c + MHCII + cells from TNF −/− mice were incubated with rmTNF (100 ng/ml; 4 h). F Levels of human IL-22BP in human monocyte-derived DCs (moDCs) stimulated with TNF (100 ng/ml), LPS (100 ng/ml), PAM 3 Cys (5 mcg/ml) for indicated times. G Correlation between hTNF and hIL-22BP levels in sera of IBD patients. All mouse data are representative of two independent experiments. Data represent mean values ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, as calculated by Student’s t-test; ns not significant. The Pearson correlation was used for correlative analyses. The significance level was set to p ≤ 0.05.

Article Snippet: Once mice had lost >5% of initial weight, they were treated with anti-human TNF antibody (Infliximab), anti-mouse IL-22 (R&D Systems; AF582) or human Fc-IgG1 isotype control (BioXCell; Cat # BE0096) in PBS.

Techniques: Recombinant, Derivative Assay, Expressing, Control, Isolation, Incubation

Journal: Mucosal Immunology

Article Title: TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability

doi: 10.1038/s41385-022-00506-x

Figure Lengend Snippet: A Levels of hTNF and mTNF in ex vivo colonic explants from Rag1 −/− mice following transfer of hTNF-KI T cells into Rag1 −/− mice and treated with Fc control or infliximab for 2 weeks. B Levels of hTNF and mTNF in ex vivo colonic explants from hTNF-KIxRag1 −/− mice that received T cells isolated from WT mice and were treated with Fc control or infliximab for 2 weeks. C hTNF and mTNF levels in sera of colitic Rag1 −/− mice transferred with hTNF-KI naive T cells before anti-TNF therapy. D Analysis of hTNF expression on the surface of blood T cells. Rag1 −/− mice were reconstituted with naive T cells from WT or hTNF-KI mice. Blood cells were analysed once mice had lost 5% of their initial weight. E Expression of IL-22BP in CD4 T cells isolated from WT and hTNF KI spleen. All data are representative of two independent experiments. Data represent mean values ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, as calculated by Student’s t-test; ns not significant.

Article Snippet: Once mice had lost >5% of initial weight, they were treated with anti-human TNF antibody (Infliximab), anti-mouse IL-22 (R&D Systems; AF582) or human Fc-IgG1 isotype control (BioXCell; Cat # BE0096) in PBS.

Techniques: Ex Vivo, Control, Isolation, Expressing

Journal: Mucosal Immunology

Article Title: TNF hampers intestinal tissue repair in colitis by restricting IL-22 bioavailability

doi: 10.1038/s41385-022-00506-x

Figure Lengend Snippet: A Weight of hTNFRp75‐KIxRag1 −/− mice reconstituted with naive T cells from hTNF‐KI mice and treated with Infliximab or Fc control (10 mg/kg) twice per week. IL-22 levels in colonic explants B and expression of IL-22BP mRNA in colon C of hTNFRp75‐KIxRag1 −/− mice reconstituted with naive T cells from hTNF-KI mice and treated with-infliximab for 2 weeks. Frequencies of CD11b + (CD45 + CD11b + ) D of granulocytes (CD45 + CD11b + Ly6G + Ly6C + ) and inflammatory monocytes (CD45 + CD11b + Ly6G - Ly6C + ) E , Th1 (CD45 + CD4 + TCRβ + IFNγ + ) F , Th17 (CD45 + CD4 + TCRβ + IL-17A + ) G and Th1/17 (CD45 + CD4 + TCRβ + IL-17A + IFNγ + ) H cells 2 weeks after infliximab or Fc control therapy. All data are representative of two independent experiments. Data represent mean values ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, as calculated by Student’s t-test; ns not significant.

Article Snippet: Once mice had lost >5% of initial weight, they were treated with anti-human TNF antibody (Infliximab), anti-mouse IL-22 (R&D Systems; AF582) or human Fc-IgG1 isotype control (BioXCell; Cat # BE0096) in PBS.

Techniques: Control, Expressing

Journal: Frontiers in Neuroscience

Article Title: Autism-Like Behavior in the Offspring of CYP11A1 -Overexpressing Pregnant Rats

doi: 10.3389/fnins.2021.774439

Figure Lengend Snippet: Primary microglial transcriptome and primary culture indicates immune activation in offspring from CYP11A1 -overexpressing dams. (A) Microglial cells from the offspring of AD-CYP11A1-treated pregnant rats were identified by their Cd11b positivity. (B) GO analysis revealed the activation of immune responses in primary microglia from the CYP11A1 overexpression group. (C) KEGG analysis indicated that GABAergic synapse related genes were enriched from the CYP11A1 overexpression group (over_represented p = 0.033). N = 3 for each group, and only male rats were used for the transcriptomic analysis. (D,E) Expression of the inflammatory tumor necrosis factor-alpha (TNF-α) was increased in the primary microglia in the CYP11A1 adenovirus infected cells. ELISA revealed TNF-α in the adenovirus infected culture medium (D) , and real-time PCR detected TNF-α mRNA in the microglia (E) (* P < 0.05).

Article Snippet: TNF-α levels were measured by rat TNF-α ELISA Kit (D731168, R&D Systems).

Techniques: Activation Assay, Over Expression, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction