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: Diseases (Basel, Switzerland)

Article Title: Cyclosporine A Causes Gingival Overgrowth by Promoting Entry into the S Phase at the G1/S Cell Cycle Checkpoint in Gingival Fibroblasts Exposed to Lipopolysaccharide.

doi: 10.3390/diseases12120322

Figure Lengend Snippet: Figure 4. Protein expression levels of mediators for G1-S cell cycle transition in gingival fibroblasts cultured in the presence or absence of cyclosporine A. After semi-confluent cells were cultured in D- MEM with 1% serum for 24 h, cells were treated with or without (control) 200 ng/mL of cyclosporine A in D-MEM containing 5% serum and 1 µg/mL of LPS for 24 h, after which, Western blot analysis was performed. The fold change from control was calculated. Representative band images from three independent experiments are shown. Data are presented as means ± SEM. * p < 0.05 compared with control using Welch’s t-test (n = 3); (A) G1-S transition-promoter proteins (CDC25A, cell division cycle 25A; CYCLIN D, D-type cyclin; CDK4, cyclin-dependent kinase 4; CDK6, cyclin-dependent kinase 6; pRB, phospho-retinoblastoma protein); (B) G1-S transition-inhibitor proteins (RB, retinoblastoma protein; SMAD3, SMAD family member 3; SMAD4, SMAD family member 4). Cs A, cyclosporine A. Original images of Figure 4 are available in the Supplementary File.

Article Snippet: The rabbit anti-CDC25A antibody (cat. no. 55031-1-AP) and mouse antibodies against CDK4 (cat. no. 66950-1-Ig), CDK6 (cat. no. 66278-1-Ig), and CYCLIN D (cat. no. 60186-1-Ig) were obtained from Proteintech Group Inc. (Rosemont, IL, USA).

Techniques: Expressing, Cell Culture, Control, Western Blot

Journal: Diseases (Basel, Switzerland)

Article Title: Cyclosporine A Causes Gingival Overgrowth by Promoting Entry into the S Phase at the G1/S Cell Cycle Checkpoint in Gingival Fibroblasts Exposed to Lipopolysaccharide.

doi: 10.3390/diseases12120322

Figure Lengend Snippet: Figure 5. This schematic illustrates the process by which cyclosporine A promotes entry into the S phase at the G1/S cell cycle checkpoint in gingival fibroblasts. Cyclosporine A triggers the downregu- lation of SMAD3 and SMAD4, increasing CDC25A levels. Furthermore, it stimulates the upregulation of CYCLIN D, CDK4, and CDK6, which leads to elevated levels of phosphorylated RB. This cascade ultimately facilitates the transition into the S phase from the G1 phase. Additionally, cyclosporine A contributes to the downregulation of RB. The light yellow rectangles represent molecules involved in signaling that promotes the G1/S transition, whereas the light red rectangles indicate those that in- hibit this transition, as analyzed in this study. The large blue arrows signify downregulation whereas the large red arrows indicate upregulation, both as a result of cyclosporine A treatment. BMI1, BMI1 proto-oncogene polycomb ring finger; CDC25A, cell division cycle 25A; CDK4, cyclin-dependent kinase 4; CDK6, cyclin-dependent kinase 6; CYCLIN D, D-type cyclin; GSK-3B, glycogen synthase kinase 3 beta; MYC, MYC proto-oncogene, bHLH transcription factor; pRB, phosphorylated RB tran- scriptional corepressor; P15, cyclin-dependent kinase inhibitor 2B (CDKN2B); P16, cyclin-dependent kinase inhibitor 2A (CDKN2A); P18, cyclin-dependent kinase inhibitor 2C (CDKN2C); P19, CDK inhibitor p19INK4d; RB, RB transcriptional corepressor; SMAD3, SMAD family member 3; SMAD4, SMAD family member 4.

Article Snippet: The rabbit anti-CDC25A antibody (cat. no. 55031-1-AP) and mouse antibodies against CDK4 (cat. no. 66950-1-Ig), CDK6 (cat. no. 66278-1-Ig), and CYCLIN D (cat. no. 60186-1-Ig) were obtained from Proteintech Group Inc. (Rosemont, IL, USA).

Techniques: