Journal: Pharmaceutical Biology

Article Title: Methamphetamine leads to the alterations of microRNA profiles in the nucleus accumbens of rats

doi: 10.1080/13880209.2020.1803366

Figure Lengend Snippet: The effects of METH on neuron differentiation by immunofluorescence. The NSE positive cells decreased (A) while GFAP positive cells (B) increased in the striatum, hippocampus, and NAc after METH treatment.

Article Snippet: The primary antibody of NSE (neuron specific enolase, catalog No. AF5169) and GFAP (glial fibrillary acidic protein, catalog No. AF2594) was obtained from the R&D system (Minneapolis, MN, USA).

Techniques: Immunofluorescence

Journal:

Article Title: Functional Evaluation of DC-SIGN Monoclonal Antibodies Reveals DC-SIGN Interactions with ICAM-3 Do Not Promote Human Immunodeficiency Virus Type 1 Transmission

doi: 10.1128/JVI.76.12.5905-5914.2002

Figure Lengend Snippet: HIV-1 transmission mediated by DC is blocked by DC-SIGN MAbs. Transmission of R5-tropic HIV-Luc/JRFL using DC as donor cells and Hut/CCR5 as target cells was performed as described for Fig. ​Fig.3.3. DC cocultured with Hut/CCR5 cells not exposed to HIV-1 were used as a mock-infected control. Mouse IgG was used as a nonspecific antibody control. Anti-DCS(D), cocktail containing the DC-SIGN-specific MAbs 507(D), 516(D), and 531(D) (10 μg/ml combined). Anti-DCS(X): cocktail containing the cross-reactive MAbs 518(X), 526(X), and 612(X) (10 μg/ml combined). The L-SIGN-specific MAb 604(L) was used at 10 μg/ml; mannan was used at 20 μg/ml. DC alone were incubated with the virus, washed to remove unbound virus, and then cultured without Hut/CCR5 target cells. One representative experiment out of two is shown. cps, counts per second.

Article Snippet: To inhibit DC-SIGN and ICAM-3 interactions, fresh DC-SIGN MAb 531(D) and anti-human ICAM-3 MAb (R&D Systems, antibody clone 76205.11) were added daily.

Techniques: Transmission Assay, Infection, Control, Incubation, Virus, Cell Culture

Journal:

Article Title: Functional Evaluation of DC-SIGN Monoclonal Antibodies Reveals DC-SIGN Interactions with ICAM-3 Do Not Promote Human Immunodeficiency Virus Type 1 Transmission

doi: 10.1128/JVI.76.12.5905-5914.2002

Figure Lengend Snippet: Expression of ICAM-3 in GHOST/R5 target cells does not enhance HIV-1 transmission mediated by DC-SIGN. (A) Direct infection of GHOST/R5 and GHOST/R5/ICAM-3 cells. Cells were infected with different amounts of HIV-Luc/JRFL pseudotyped virus as indicated. Luciferase activity was measured 2 days after infection. Cells not exposed to virus were used as a mock-infected control. (B) HIV-1 transmission to ICAM-3-positive or -negative GHOST/R5 cells mediated by DC-SIGN. THP-1 or THP-1/DC-SIGN donor cells were incubated with DC-SIGN-specific MAb 531(D) or mouse IgG control (10 μg/ml) for 30 min at 37°C ([prior]) and then pulsed with HIV-Luc/JRFL for 3 h at 37°C. The washed cells were then added to GHOST/R5 or GHOST/R5/ICAM-3 target cells, respectively. The DC-SIGN-specific MAb 531(D) and anti-ICAM-3 (10 μg/ml) were kept in the cocultivation for 5 h ([post]), and then donor cells were removed and target cells were washed with medium and cultured in 1 ml of fresh medium in the presence of MAb 531(D) or anti-ICAM-3 (refreshed daily) for 2 days before luciferase activity was measured ([post]). Each data set represents the mean of three separate wells of infected cells. One representative experiment out of two is shown. cps, counts per second.

Article Snippet: To inhibit DC-SIGN and ICAM-3 interactions, fresh DC-SIGN MAb 531(D) and anti-human ICAM-3 MAb (R&D Systems, antibody clone 76205.11) were added daily.

Techniques: Expressing, Transmission Assay, Infection, Virus, Luciferase, Activity Assay, Control, Incubation, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) P2X7R and NLRP3 immunoprecipitation (IP) in human CD4+ T cells. Expression of NLRP3 (top blot) and P2X7R (bottom blot) is shown. Lane 1: Total protein. Lane 2: IP with NLRP3 Ab. Lane 3: IP with P2X7R Ab. Lane 4: IP with Ab alone (NLRP3 and P2X7R). Lane 5: IP with control IgG (for NLRP3 Ab in top blot, for P2X7R Ab in bottom blot). The experiment was run in triplicate (representative blot shown). (B and C) Confocal microscopy analysis (B, scale bar: 5 μm, ×100 original magnification; C, scale bars: 20 μm, ×40 original magnification) depicting baseline colocalization of P2X7R (green) and NLRP3 (red) in human CD4+ T cells. Cells were stained with DAPI (blue) and immunolabeled with anti-P2X7R (green) and anti-NLRP3 Abs (red) (n = 3). (D–F) Bar graphs depicting expression of NLRP3 mRNA by qRT-PCR (D), and protein by flow cytometry (E) and ELISA (F), evaluated in human CD4+ T cells activated with benzoyl ATP (BzATP) and treated with CE-224,535, a P2X7R inhibitor. Experiments were run in duplicate (n = 5). (G) Bar graph representing expression of NLRP3 on human CD4+P2X7R+ cells analyzed by flow cytometry upon BzATP stimulation (n = 5). (H) Representative flow dot plots of NLRP3 expression upon gating on human BzATP-stimulated CD4+P2X7R+ cells. (I) Confocal analysis (scale bar: 5 μm; ×100 original magnification) depicting colocalization of P2X7R (green) and NLRP3 (red) in CD4+ T cells upon in vitro stimulation of P2X7R with BzATP (n = 3). (J–M) Bar graphs comparing expression of NLRP3 downstream signaling Th2-related factors IL-4 (J), IRF4 (K), GATA-3 (L), and IL-10 (M) by qRT-PCR using mRNA isolated from human CD4+ T cells activated with BzATP and treated with the P2X7R inhibitor CE-224,535. Experiments were run in triplicate (n = 5). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA with Bonferroni’s post hoc test or Student’s t test. mRNA expression was normalized to β-actin (ACTB).

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Immunoprecipitation, Expressing, Control, Confocal Microscopy, Staining, Immunolabeling, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay, In Vitro, Isolation

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) A 3D representation of the full-length structure of P2X7R, highlighting the putative location of the P2X7R mutation in the C-terminal intracellular portion. (B and C) Quantification of P2X7R total protein (B, ELISA, n = 3) and of P2X7R mRNA (C, qRT-PCR, n = 10) on CD4+ T cells of carrier and noncarrier patients. Samples were run in duplicate (B) or in triplicate (C) and normalized to expression level of β-actin (ACTB). (D) Transcriptome profiling of immune-relevant genes (see also Supplemental Table 3) examined in CD4+ T cells of carrier and noncarrier cardiac-transplanted patients (n = 5). (E–G) Expression of NLRP3 mRNA using qRT-PCR (E) and NLRP3 protein using flow cytometry (F) and ELISA (G) in CD4+ T cells of carrier and noncarrier patients (n = 5). (H and I) Flow cytometric expression of NLRP3 on CD4+P2X7R+ cells of carrier patients stimulated with BzATP (n = 5). (J) Percentage of P2X7R+NLRP3+ cells of carrier and noncarrier patients analyzed by immunofluorescence (Figure 1C and Supplemental Figure 2G) (n = 3). (K) Confocal microscopy analysis (×100 original magnification) of P2X7R (green) and NLRP3 (red) coexpression in CD4+ T cells of carrier patients (n = 3). Scale bar: 5 μm. (L) Subcellular localization of NLRP3 in CD4+ T cells of carrier and of noncarrier patients (n = 3). (M and N) IL-4 (M) and IRF4 (N) gene expression detected after ChIP with NLRP3 antibody in CD4+ T cells. (n = 3). (O) Quantification of NLRP3 protein measured in CD4+ T cells treated with the ubiquitin/protease inhibitor MG132 (n = 3). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 2-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Gene Expression, Ubiquitin Proteomics, Protease Inhibitor

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) Percentage of in vitro–generated Th17 cells obtained from CD4+ T cells of carrier and noncarrier patients (n = 8). (B and C) Representative flow zebra plots (B) and quantitative histogram (C) depicting the percentage of peripheral CD4+IL-17+ cells (n = 8). (D) IL-17 plasma levels of carrier and noncarrier patients (n = 10). (E) IL-17 levels (Luminex) measured in the supernatants of unstimulated 24-hour-cultured CD4+ T cells of carrier and noncarrier patients (n = 5). (F) Table summarizing the secretome profile (Luminex, n = 5) and primary phenotypic characteristics (flow cytometry, n = 4) of carrier and noncarrier polarized Th17 cells. (G and H) Normalized mRNA expression of Th2-related factors IL-4 (G) and GATA-3 (H) measured in noncarrier CD4+ T cells exposed to transient knockdown of NLRP3 using silencing RNA (siRNA), before and after anti-CD3-Ig/anti-CD28-Ig stimulation (n = 3). (I–K) Normalized mRNA expression of the Th2-related factors IL-4 (I), IL-10 (J), and GATA-3 (K) measured in noncarrier CD4+ T cells exposed to transient knockdown of NLRP3 (siRNA), upon BzATP exposure (n = 4). (L and M) Normalized mRNA expression of the Th2-related factors IL-4 (L) and GATA-3 (M) measured in carrier CD4+ T cells, in which NLRP3 was overexpressed, before and after anti-CD3-Ig/anti-CD28-Ig stimulation (n = 3). (N) Effects of various treatments (anti–IL-17 antibody, RMT1-10, cyclosporin A [CsA] and rapamycin [Rapa]) on in vitro–generated Th17 cells (n = 5). Experiments were run in triplicate (D, G, H, and N) or in duplicate (F and I–M). mRNA expression was normalized to ACTB. Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 1-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: In Vitro, Generated, Clinical Proteomics, Luminex, Cell Culture, Flow Cytometry, Expressing, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) P2X7R–/– mice receiving bm12 heart transplantation demonstrated reduced graft survival as compared with B6 recipients (**P < 0.01), which was significantly prolonged by anti–IL-17 treatment (murine IL-17–depleting antibody) (*P < 0.05 vs. P2X7R–/–) (n = 10 mice per group). (B–D) Semiquantification of graft infiltration (B), coronary vasculopathy (C), and myocyte necrosis (D) confirmed accelerated allograft rejection in P2X7R–/– mice (n = 3). (E) Representative H&E staining (x20 original magnification) showing graft cell infiltration (top panels), vasculopathy (middle panels), and myocyte necrosis (bottom panels) in B6 and P2X7R–/– mice. Scale bars: 200 μm (middle panels), 300 μm (top and bottom panels). (F and G) Numbers of IFN-γ–producing (F) and IL-4–producing (G) cells (ELISPOT) measured in cardiac-transplanted mice (n = 3). (H–M) Percentage of CD4+IL-17+ (H), CD4+IFN-γ+ (I), CD4+IL-10+ (J), CD4+CD44hiCD62Llo (K), CD8+CD44hiCD62Llo (L), and CD4+CD25+Foxp3+ (M) cells detected by flow cytometry in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (N) Serum IL-17 level (Luminex) measured in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (O) Percentage of CD4+NLRP3+ cells analyzed by flow cytometry in P2X7R–/– and B6 mice (n = 3). (P) Number of IL-4–producing cells (ELISPOT) in P2X7R–/– and B6 mice upon allostimulation (n = 3). (Q) Serum IL-4 level (Luminex), measured in B6 and P2X7R–/– cardiac-transplanted mice (n = 5). Samples were run in duplicate (Luminex) and in triplicate (ELISPOT). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; log-rank (Mantel-Cox) test (A), Wilcoxon’s and Student’s t test (2 groups), 1-way ANOVA with Bonferroni’s post hoc test (3 groups).

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Transplantation Assay, Staining, Enzyme-linked Immunospot, Flow Cytometry, Luminex

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 102) with an MIT change greater than 0.5 mm (early cardiac allograft vasculopathy, in black) at 1 year after transplantation in the CTOT-05 cohort. (B) Bar graph depicting the number of acute rejection episodes in cardiac-transplanted patients who carry the WT (black) or mutant (white) P2X7R allele (n = 181) within the first year after transplant in the NIT-Bergamo cohort. (C) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 130) with major adverse cardiac events (MACEs, in black) at 10 years of follow-up in the AIRT-Bologna cohort. In A and C: black, percentage of patients who experienced the event; white, percentage who were free from events. (D) Line graph depicting the estimated odds ratio (OR) for clinical outcomes recorded in the 3 cohorts of cardiac-transplanted patients who carry the WT or mutant P2X7R allele. In the NIT-Bergamo cohort, the OR was calculated based on the requirement of medical intervention for acute rejection episodes with a frequency of greater or less than 3 episodes. *P < 0.05; **P < 0.01. Supplemental Tables 7–9 report detailed analyses. Fisher’s exact and Student’s t tests. (E and F) A stable connection between P2X7R and NLRP3 is necessary to establish a physiological NLRP3-mediated Th2 program (E), while alteration in the P2X7R intracellular domain induces NLRP3 displacement and retains NLRP3 in the cell membrane, thus preventing its nuclear activity and accelerating ubiquitination of NLRP3 (F). This shifts the balance of the immune response toward Th17 cells and favors the development of immune-related events, such as allograft rejection and vasculopathy. Ub, ubiquitin; eATP, extracellular ATP.

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Mutagenesis, Transplantation Assay, Membrane, Activity Assay, Ubiquitin Proteomics

Journal: World Journal of Stem Cells

Article Title: Patient-derived induced pluripotent stem cells with a MERTK mutation exhibit cell junction abnormalities and aberrant cellular differentiation potential

doi: 10.4252/wjsc.v16.i5.512

Figure Lengend Snippet: Generation and identification of the human induced pluripotent stem cells derived from the retinitis pigmentosa patient. A: Timeline of human induced pluripotent stem cell generation; B: Imaging by phase-contrast microscopy. Scale bar = 200 μm; C: Karyotype analysis of the healthy control (left) and retinitis pigmentosa patient (right); D: Flow cytometry of pluripotency markers SSEA-4 and TRA-1-81; E and F: Immunostaining of pluripotency markers OCT4, NANOG, SOX2, and SSEA4 of the healthy control and retinitis pigmentosa patient. Scale bar = 20 μm; G and H: In vitro differentiation of control (G) and patient (H) induced pluripotent stem cells into three germ layers, endoderm (AFP+), mesoderm (α-SMA+) and ectoderm (GFAP+). Scale bar = 20 μm. PB: Peripheral blood; PBMC: Peripheral blood mononuclear cell; iPSC: Induced pluripotent stem cell.

Article Snippet: Following primary antibodies were used: OCT4 (Cat. #ab18976; Abcam), SOX2 (Cat. #sc-365823; Santa Cruz), NANOG (Cat. #ab80892; Abcam), SSEA4 (Cat. #ab16287; Abcam), GFAP (Cat. #HPA056030; Sigma), α-SMA (Cat. #A5228; Sigma), AFP (Cat. #MAB1368; R&D System).

Techniques: Derivative Assay, Imaging, Microscopy, Control, Flow Cytometry, Immunostaining, In Vitro