Journal: International immunology

Article Title: Regulation of NOD mouse autoimmune diabetes by T cells that recognize a TCR CDR3 peptide.

doi: 10.1093/intimm/11.6.957

Figure Lengend Snippet: Fig. 8. Vaccination to C9 CDR3β induces anti-C9 CDR3β IgG2a antibodies and anti-p277 IgG2b and IgG1 antibodies. Ten-week-old female NOD mice were vaccinated with flagellin (filled shapes) or with recombinant C9 CDR3β–flagellin (open shapes). Four weeks later, the sera of individual mice were assayed by ELISA for antibody isotypes to C9 CDR3β peptide (upper panel) or to p277 peptide (lower panel). The response to C9 CDR3β was significantly of the IgG2a isotype (P , 0.01), while the response to p277 was significantly of the IgGI (P , 0.05) and IgG2b isotypes (P , 0.01).

Article Snippet: The binding of antibodies to the adherent antigens was detected using alkaline phosphatase-conjugated antimouse IgG 1 IgM, or isotype-specific anti-mouse IgG1, IgG2a or IgG2b (Jackson Immunoresearch, West Grove, PA).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: Regulation of NOD mouse autoimmune diabetes by T cells that recognize a TCR CDR3 peptide.

doi: 10.1093/intimm/11.6.957

Figure Lengend Snippet: Fig. 9. Vaccination to C9 CDR3β down-regulates the spontaneous antibody responses to intact hsp60 (A) and to insulin (B). Ten-week- old female NOD mice were vaccinated to flagellin or to recombinant C9 CDR3β–flagellin. Four weeks later, their serum IgG antibodies were assayed by ELISA to intact hsp60 or to insulin. The antibody titers of the C9 CDR3β-vaccinated mice were significantly decreased (*P , 0.01).

Article Snippet: The binding of antibodies to the adherent antigens was detected using alkaline phosphatase-conjugated antimouse IgG 1 IgM, or isotype-specific anti-mouse IgG1, IgG2a or IgG2b (Jackson Immunoresearch, West Grove, PA).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies.

doi: 10.1038/s41598-021-84116-w

Figure Lengend Snippet: Figure 2. NIT211 conjugate is recognized by oligomannose-specific bnAbs and their germline precursors. NIT211 (4.1 glycosides per CRM197) was coated as solid-phase antigen onto ELISA plate wells at 5 µg/ml in PBS and assayed for recognition by the different antibodies. All antibodies were expressed recombinantly as human IgG1. (A) Binding of PGT128/130 bnAb family members PGT125, PGT126, PGT128 and PGT130. (B) Binding of non-PGT128/130 family antibodies BF520.1, BG18, PCDN-33A, PGDM12, PGDM21, PCDN76- 33A, VRC41.01. (C) Binding of inferred gl precursors BF520.1, BG18, PCDN-33A and the PGT128/130 family. Results are from single experiments performed with technical duplicates.

Article Snippet: Total serum IgG was detected with a mixture of equal amounts of alkaline phosphatase conjugated anti-mouse IgG1, IgG2b, IgG2c and IgG3 secondary antibodies (Jackson ImmunoResearch) and nitrophenylphosphate substrate (Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Scientific reports

Article Title: A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies.

doi: 10.1038/s41598-021-84116-w

Figure Lengend Snippet: Figure 4. The relative binding affinities of PGT antibodies for NIT211 increase with increasing ligand density. Binding to NIT211 conjugates was assessed by ELISA. NIT211 (2.6 ligands, 4.1, and 6.2 ligands) was coated as solid-phase antigen onto ELISA plate wells at 5 µg/ml (82, 79, 76 nM respectively) and assayed for antibody binding. (A) Binding of PGT125, PGT126, PGT128 and PGT130 IgG to NIT211 at three different densities of glycoside per CRM197. (B) Binding of PGT125, PGT126, PGT128 and PGT130 Fabs to NIT211 at two different densities of glycoside per CRM197.

Article Snippet: Total serum IgG was detected with a mixture of equal amounts of alkaline phosphatase conjugated anti-mouse IgG1, IgG2b, IgG2c and IgG3 secondary antibodies (Jackson ImmunoResearch) and nitrophenylphosphate substrate (Sigma).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies.

doi: 10.1038/s41598-021-84116-w

Figure Lengend Snippet: Figure 5. Only animals administered GLA-SE-adjuvanted NIT211 mount an IgG response to the oligomannose mimetic that is of the IgG3 subclass. Trianni mice (n = 5 per group) were immunized subcutaneously (days 0, 21, 42 and 105) and sera collected on day 0 prior to immunization and on days 10, 28, 49, and 119 post- immunization. (A) Binding of total IgG antibodies in pre-immune and post-immune sera to BSA-conjugated oligomannose mimetic. Binding curves represent mean values for the five animals in each immunization group, each tested in duplicate, with error bars denoting the standard deviation from the mean. (B) Individual IgG binding curves for NIT211 + GLA-SE immunized mice (Ms1 to 5). The sex of each mouse is also noted. (C) IgG1, IgG2b, IgG2c and IgG3 antibody subclass responses in day 119 post-immune sera of NIT211 + GLA-SE immunized animals for the BSA-conjugated oligomannose mimetic in comparison to the CRM197 protein carrier.

Article Snippet: Total serum IgG was detected with a mixture of equal amounts of alkaline phosphatase conjugated anti-mouse IgG1, IgG2b, IgG2c and IgG3 secondary antibodies (Jackson ImmunoResearch) and nitrophenylphosphate substrate (Sigma).

Techniques: Binding Assay, Standard Deviation, Comparison

Journal: Scientific reports

Article Title: A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies.

doi: 10.1038/s41598-021-84116-w

Figure Lengend Snippet: Figure 6. NIT211 formulated in GLA-SE elicits antibodies with capacity to bind recombinant HIV Env trimers. (A) Binding of total IgG from day 119 sera from unimmunized, and NIT211-immunized animal groups to SOSIP trimers AMC008 (subtype B), Du422 (subtype C), ZM197M (subtype C), BG505 (subtype A), B41 (subtype B), and CZA97 (subtype C). The HIS-tagged trimers were captured on nickel-coated ELISA plates at 5 µg/ml in PBS. (B) Level of IgG1, IgG2, IgG3 antibody subclass binding (1:100 dilution) to B41 SOSIP trimer in day 119 sera of NIT211 + GLA-SE immunized animals. (C) Residual binding of bnAb PGT128 to B41 SOSIP trimer following incubation with sera from NIT211 + GLA-SE immunized animals (day 119) vs sera from KLH + Alum/CpG ODN1826 immunized animals (day 34). (D) Day 119 sera from NIT211 + GLA-SE immunized animals and sera from a group of unimmunized animals were assessed for pseudovirus neutralization using a panel of seven diverse HIV-1 strains (92TH021, 92RW020, 94UG103, 92BR020, 97ZA012, JRCSF and NL4-3). Pseudotyped vesicular stomatitis virus (VSV) was used as a negative control. All graphs depict mean values for the serum samples from all animals (n = 5) in each group. Error bars represent standard error from the mean.

Article Snippet: Total serum IgG was detected with a mixture of equal amounts of alkaline phosphatase conjugated anti-mouse IgG1, IgG2b, IgG2c and IgG3 secondary antibodies (Jackson ImmunoResearch) and nitrophenylphosphate substrate (Sigma).

Techniques: Recombinant, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Neutralization, Virus, Negative Control

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 1 Expression of plac8 and ERK activation in peripheral blood mononuclear cells of septic patients and normal individuals. A Flow cytometry analysis of peripheral blood CD14+ and CD16+ cell subsets. B Flow cytometry analysis of CD14+ and CD16+ cell subsets. C qRT- PCR analysis of gene expression. D ELISA analysis of cytokine expression. E Western blot analysis of protein expression. F Western blot analysis of protein expression. G Flow cytometry analysis of cell cycle. H Cell proliferation index PI = (S + G2M)/(G0/1 + S + G2M). * indicates P < 0.05 compared to the normal group. Data is presented as mean ± standard deviation. Data analysis was performed using an independent samples t-test, and the experiment was repeated three times.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Expressing, Activation Assay, Flow Cytometry, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 2 Effects of plac8 on peripheral blood mononuclear cell proliferation in septic patients. A qRT-PCR analysis of gene expression. B Western blot analysis of protein band. C Western blot analysis of protein expression. D ELISA analysis of cytokine expression. E Cell proliferation measured by CCK-8 assay, cell proliferation (%) = [OD (treatment group) −OD (blank group)] / [OD (control group) −OD (blank group)] × 100%. * indicates P < 0.05 compared to si-Plac8-NC or Plac8-NC group. Data is presented as mean ± standard deviation. An independent samples t-test was used to compare two groups, and two-way ANOVA was used to compare at different time points. The experiment was repeated three times.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Quantitative RT-PCR, Gene Expression, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Control, Standard Deviation

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 5 Effects of plac8 on monocyte cell survival and proliferation in the mouse model. A Flow cytometry analysis of CD14+ and CD16+ cell subsets. B Flow cytometry analysis of peripheral blood CD14+ and CD16+ cell subsets. C Western blot analysis of protein band. D Western blot analysis of protein expression. E ELISA analysis of cytokine expression. F Detection of cell proliferation. G Flow cytometry analysis of cell cycle. H Cell proliferation index PI. * indicates P < 0.05 compared to si-Plac8-NC or Plac8-NC group. Data is presented as mean ± standard deviation. Independent samples t-test was used to compare two groups, and two-way ANOVA was used for comparison at different time points. The experiment was repeated three times.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Flow Cytometry, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison

Journal: Cell death discovery

Article Title: Plac8-ERK pathway modulation of monocyte function in sepsis.

doi: 10.1038/s41420-024-02012-4

Figure Lengend Snippet: Fig. 6 The role and mechanism of Plac8-mediated ERK signaling pathway activation in the proliferation and activation of peripheral blood monocytes in septic patients.

Article Snippet: After blocking with 0.5% BSA, the membranes were incubated overnight with primary antibodies: rabbit anti-CD14 (ab182032, 1:1000, Abcam, UK), CD16 (Cat No. 16559-1-AP, 1:1000, Proteintech), plac8 (Cat No. 12284-1-AP, 1:200, Proteintech), ERK1/2 (ab17942, 1:1000, Abcam, UK), p-ERK1/2 (ab223500, 1:1000, Abcam, UK), and GAPDH (ab8245, 1:1000, Abcam, UK).

Techniques: Activation Assay