Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A and B ) Flow cytometry and statistical analysis of CD8 + CD45 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( C and D ) Flow cytometry and statistical analysis of IFN-γ + CD8 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( E and F ) Flow cytometry and statistical analysis of Granzyme B + CD8 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( G ) The secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in the tail skin of mice in the vehicle, 6-OHDA, and 6-OHDA + NE groups were detected by ELISA ( n =4 per group). All vehicle mice were only treated with 0.1% ascorbic acid in 0.9% sterile NaCl. Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Sterility

Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A ) Volcano plots illustrated the expression of adrenergic receptors on fibroblasts in human skin, including ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, and ADRB3. ( B ) Bubble map illustrated the up-regulation of ADRA2A expression on fibroblasts in the lesional skin of patients with vitiligo and the normal skin of healthy controls. ( C ) Representative immunofluorescence images of fibroblasts, ADRA2A, CXCL9, CXCL10, and CD8 + T cells from the normal skin of the healthy control and the lesional skin of the patient with vitiligo ( n =3 per group). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. ( D ) Immunofluorescence analysis of ADRA2A (red) expression in BJ cells after treatment with 5 μM NE for 48 hours. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( E ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in BJ cells were detected by ELISA with treatment with aposcopolamine (Apos) or NE ( n =3 per group). ( F ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in BJ cells with ADRA2A siRNA or control siRNA were detected by ELISA ( n =3 per group). Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A ) Volcano plots illustrated the expression of adrenergic receptors on keratinocytes in human skin, including ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, and ADRB3. ( B ) Bubble map illustrated the up-regulation of ADRB2 expression on keratinocytes in the lesional skin of patients with vitiligo and the normal skin of healthy controls. ( C ) Representative immunofluorescence images of keratinocytes, ADRB2, CXCL9, CXCL10, and CD8 + T cells from the normal skin of the healthy control and the lesional skin of the patient with vitiligo ( n =3 per group). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. ( D ) Immunofluorescence analysis of ADRB2 (red) expression in keratinocytes after treatment with 5 μM NE for 48 hours. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( E ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in keratinocytes were detected by ELISA with treatment with NE or ICI ( n =3 per group). ( F ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in keratinocytes with ADRB2 siRNA or control siRNA were detected by ELISA ( n =3 per group). Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: CD44 3'-Untranslated Region Functions as a Competing Endogenous RNA to Enhance NK Sensitivity of Liver Cancer Stem Cell by Regulating ULBP2 Expression

doi: 10.7150/ijbs.35216

Figure Lengend Snippet: CSCs derived from liver cancer were susceptible to NK cell mediated cytotoxicity in correlation with CD44 expression. (A) CD44 high iCSC, CD44 int iCSC, HepG2, and Hep3B were used in the 4 hours NK cell cytotoxicity assay with NK-92 cells at different E:T ratios. Data are showed in means ± SD from three independent experiments which are performed in triplicate (ns: not significant, **: p < 0.01). (B) IFN-γ release in the supernatants of cytotoxic assay (Fig. A, E:T ratio = 15:1) was determined by ELISA. Data were presented as mean ± SD (n=3) (*: p<0.05, **: p<0.01). (C) CD44 transcript level of CD44 high iCSC/CD44 int iCSC stably expressing dCas9-KRAB and sgRNA against CD44 (CD44-knockdown, CD44-kd) was analyzed by qRT-PCR. Levels are represented relative to those found in control-infected cells as means ± SD (n=3) (**: p < 0.01). (D) Identical cells (Fig. C) were used in the 4 hours NK cell cytotoxicity assay with NK-92 cells at different E:T ratios. Data are showed in means ± SD from three independent experiments which are performed in triplicate (**: p < 0.01). (E) IFN-γ release in the supernatants of cytotoxic assay (Fig. D, E:T ratio = 15:1) was determined by ELISA. Data were presented as mean ± SD (n=3) (**: p<0.01).

Article Snippet: Concentrations of secreted IFN-γ were determined using Human Interferon gamma ELISA Kit (ab46048; Abcam, Cambridge, MA, USA).

Techniques: Derivative Assay, Expressing, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Knockdown, Quantitative RT-PCR, Control, Infection

Journal: International Journal of Biological Sciences

Article Title: CD44 3'-Untranslated Region Functions as a Competing Endogenous RNA to Enhance NK Sensitivity of Liver Cancer Stem Cell by Regulating ULBP2 Expression

doi: 10.7150/ijbs.35216

Figure Lengend Snippet: CD44 regulated ULBP2 expression, which then further influenced the susceptibility of CSCs to NK cell mediated cytotoxicity. (A ) MICA/B, ULBP1-3, CD48, CD112, CD155, and ICAM1 transcript levels of CD44 high iCSC/CD44 int iCSC stably expressing dCas9-KRAB and sgRNA against CD44 (CD44-knockdown, CD44-kd) were analyzed by qRT-PCR. Levels are represented relative to those found in control-infected cells as means ± SD (n=3) (**: p < 0.01). (B) CD44 and ULBP2 protein levels of identical cells (Fig. A) were analyzed by Western blotting. β-actin served as a loading control. (C) CD44 high iCSC and CD44 int iCSC were used in the 4 hours NK cell cytotoxicity assay with NK-92 cells at different E:T ratios. Assays were performed either in the presence of M311 mAb (anti-ULBP2) or control IgG. Data are showed in means ± SD from three independent experiments which are performed in triplicate (**: p < 0.01). (D) IFN-γ release in the supernatants of cytotoxic assay (Fig. C, E:T ratio = 15:1) was determined by ELISA. Data were presented as mean ± SD (n=3) (**: p<0.01).

Article Snippet: Concentrations of secreted IFN-γ were determined using Human Interferon gamma ELISA Kit (ab46048; Abcam, Cambridge, MA, USA).

Techniques: Expressing, Stable Transfection, Knockdown, Quantitative RT-PCR, Control, Infection, Western Blot, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Veterinary Science

Article Title: Positive effects of porcine IL-2 and IL-4 on virus-specific immune responses induced by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 DNA vaccine in swine

doi: 10.4142/jvs.2014.15.1.99

Figure Lengend Snippet: Humoral immune responses in vaccinated piglets from the different groups detected with a commercial ELISA kit.

Article Snippet: To further evaluate cellular immune responses, IFN-γ levels in peripheral blood isolated from the vaccinated piglets at 35 dpi were measured using a commercial ELISA kit (Pig Interferon-γ, IFN-γ ELISA Kit; CUSABIO, USA) according to the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The veterinary quarterly

Article Title: Holstein × Montbéliarde-sired F1 generation crossbred female calves have an increased cellular immune response potential compared with purebred Holsteins.

doi: 10.1080/01652176.2024.2435982

Figure Lengend Snippet: Figure 2. Gating strategies to define bovine T cell subsets and their IFN-γ expression. Mononuclear cells were isolated from blood of 3 months-old female calf and surface stained with antibody cocktails (CD4, CD8, CD44, IFN-γ) after PMA/ionomycin mixture stimulation. The lymphocytes are chosen with FSC versus SSC (A) and then singlets are gated using FSC-a and FSC-H (B). By outputting CD4 versus CD8, CD4+ CD8-, CD8+ CD4- are identified, respectively (C). The CD4+ CD8- T cells expressing IFN-γ (D). The CD8+ CD4- T cells expressing IFN-γ (G). CD4+ CD8- T cells and CD4+ CD8- T cells can be further subdivided into CD44+ and CD44- subsets (E and F), respectively. The lymphocytes expressing IFN-γ (H).

Article Snippet: Monoclonal antibodies (mAb) specific for bovine TCRγδ (WC1), CD8, CD4 and IFN-γ with different fluorochrome conjugates were purchased from BIO-RAD (BIO-RAD, Shanghai, China) and mAb to human CD27, mouse CD44 were purchased from Biolegend (Biolegend, San Diego, USA) (Table 1).

Techniques: Expressing, Isolation, Staining

Journal: The veterinary quarterly

Article Title: Holstein × Montbéliarde-sired F1 generation crossbred female calves have an increased cellular immune response potential compared with purebred Holsteins.

doi: 10.1080/01652176.2024.2435982

Figure Lengend Snippet: Figure 5. Differences of IFN-γ expression in lymphocytes and their T cells of MH and HO calves. Representative dot-plots depict IFN-γ+ T cell subsets in CD4+ (a), CD8+ (B) T cells and total lymphocytes (C). Representative histograms showed the expressions of IFN-γ in lymphocytes (D), CD4+ (E) and CD8+ (F) T cells in the whole blood of MH and HO calves. Data were shown in LSM ± SEM (n = 25). *p < 0.05, **p < 0.01.

Article Snippet: Monoclonal antibodies (mAb) specific for bovine TCRγδ (WC1), CD8, CD4 and IFN-γ with different fluorochrome conjugates were purchased from BIO-RAD (BIO-RAD, Shanghai, China) and mAb to human CD27, mouse CD44 were purchased from Biolegend (Biolegend, San Diego, USA) (Table 1).

Techniques: Expressing

Journal: The veterinary quarterly

Article Title: Holstein × Montbéliarde-sired F1 generation crossbred female calves have an increased cellular immune response potential compared with purebred Holsteins.

doi: 10.1080/01652176.2024.2435982

Figure Lengend Snippet: Figure 6. The expression of IFN-γ was derived from subsets of CD4+ and CD8− T cells, characterized by CD44+ cells rather than CD44− cells. Representative dot-plots depict CD4+ (A) and CD8- (B) T cells subsets of IFN-γ and CD44 from MH (left panel) and HO (right panel) calves. Numbers represent the percentages of cells in each quadrant. Bar graphs showed the expressions of IFN-γ by CD44+ or CD44− subsets in CD4+ (C) and CD8+ (D) T cells of MH and HO calves. Data were shown in LSM ± SEM (n = 25). Labels (a, b, c and d) of different letters indicated a significant difference between any two different sets of data (p < 0.01).

Article Snippet: Monoclonal antibodies (mAb) specific for bovine TCRγδ (WC1), CD8, CD4 and IFN-γ with different fluorochrome conjugates were purchased from BIO-RAD (BIO-RAD, Shanghai, China) and mAb to human CD27, mouse CD44 were purchased from Biolegend (Biolegend, San Diego, USA) (Table 1).

Techniques: Expressing, Derivative Assay