Journal: The Journal of Headache and Pain

Article Title: Expression of miR-155 in monocytes of people with migraine: association with phenotype, disease severity and inflammatory profile

doi: 10.1186/s10194-024-01842-y

Figure Lengend Snippet: Percentage of events recorded for the different monocytic sub-populations among the three study groups. Legend: Panel A : percentage of M1 monocytes (CD80 +) sub-populations. Panel B : percentage of M2 monocytes (CD163 +) sub-populations. CM-MO: chronic migraine with medication overuse, EM: episodic migraine, HCs: healthy controls, classical: “classical” monocytes (CD14 + /CD16– expression), non-classical: “non classical-intermediate” monocytes (CD14 + /CD16 + expression), M1: pro-inflammatory “M1” monocytes (CD80 + expression), M2 : anti-inflammatory “M2” monocytes (CD163 + expression). Box-plot: the range between the upper and lower border of the box indicates the interquartile range (IQR), spanning from the 25 th to the 75 th percentile. Within the box, the line indicates the median, and the cross denotes the mean. The upper and lower whiskers extend to the maximum and minimum values, excluding outliers. Symbols positioned above the upper whisker represent outliers, defined statistically as values beyond the 75 th percentile plus 1.5 times the IQR. Kruskal–Wallis Test was used for intergroup comparisons

Article Snippet: Subsequently, they were incubated for 30 min at 4 °C in the dark with all of the following monoclonal antibodies: Peridinin Chlorophyll Protein Complex (PerCP)-conjugated anti-human CD14 (BD Biosciences, 20µL per 1 × 10 6 cells), R-phyco-erythrin-cyanine7 (PE-Cy7)-conjugated anti-human CD16 (BD Biosciences, 5µL per 1 × 106 cells), and R-phycoerythrin (PE)-conjugated CD163 (BD Biosciences, 5µL per 1 × 106 cells) or R-phycoerythrin (PE)-conjugated CD80 (BD Biosciences, 5µL per 1 × 106 cells).

Techniques: Expressing, Whisker Assay

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: Screening for CD14 incorporation by lab-adapted HIV-1 isolates. ( A ) Schematic describing the virion capture assay used throughout. Briefly, antibodies or LPS (not depicted) were adsorbed onto 96-well plates and blocked before adding virus samples to allow precipitation of virus particles bearing the antigen of interest. Unbound virions were washed away and captured virions were lysed for quantitation of HIV-1 by Gag p24 ELISA. ( B ) Virion capture assay was performed on four HIV-1 isolates (IIIB, NL4-3, BaL, and SF162) produced by infected PBMCs using anti-CD14 (orange) and anti-gp120 (blue). Bars are isotype control-subtracted mean ± SD Gag p24 of duplicate assays and are representative of two independent experiments. ( C ) Western blot corroborating the incorporation of CD14. HIV-1 grown in monocyte-derived macrophages (MDMs) (BaL and SF162, two donors each) and PBMCs (IIIB, two donors; NL4-3, four donors) were concentrated, lysed, and resolved by SDS-PAGE before blotting for p24 (loading control), gp120 (positive control for viral surface protein), and CD14. Data are representative of three independent blots.

Article Snippet: While others have described the impact of incorporated immunoregulatory proteins on cell signaling ( ), our work highlights a pivotal role for CD14 in allowing virions to function as a shuttle for bioactive molecules like LPS and eliciting inflammatory responses.

Techniques: Virus, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Produced, Infection, Control, Western Blot, Derivative Assay, SDS Page, Positive Control

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: HIV-1 pseudovirus reliably models virion incorporation of CD14. ( A ) Schematic depicting the process of transfecting 293T cells with plasmids encoding the HIV-1 backbone (pSG3ΔEnv) and CD14 to produce PV bearing CD14 on the surface. ( B ) Confirming the transfection efficiency of 293T cells ectopically expressing CD14. Cells were transfected with backbone only (black histogram) or backbone plus CD14 (orange histogram) to produce WT or CD14 + PVs, respectively. Cells were collected and stained with PE-conjugated anti-CD14 to confirm cell-surface expression of CD14 available for incorporation. ( C ) Gating strategy to identify PV populations by FVM. Phosphate-buffered saline (PBS) or WT virions were acquired, and data are displayed on pseudocolored dot plots. Fluorescence data (y-axis) were calibrated to produce quantitative units of MESF (molecules of equivalent soluble fluorophore), which describe the number of fluorophores and, indirectly, the number of antigens detected. Side scatter data (x-axis) were calibrated to produce arbitrary units of scattering cross section (nm 2 ), which relates to the size and refractive index of particles. Gates to the far left (PBS and WT PV) represent the instrument’s optical noise and are present of every FVM plot. Virions are identified as the dense, monodispersed population (right gate) adjacent to the optical noise. ( D ) WT (top row) and CD14 + (bottom row) virions were stained with PE-conjugated isotype control (left column) or anti-CD14 (right column) (0.4 µg/mL) and analyzed by FVM. Gates were positioned above the unstained PV populations, identifying PE + events, and were set respective to each isotype control stain. PE MESF values are enumerated where PE + events are present (mean ± SD, n = 3 independent stains), describing the number of fluorophores detected in each PE + gate. Virus stocks used in C and D had a titer of 187 ng/mL (WT) and 114 ng/mL (CD14 + ) of p24 as measured by ELISA. ( E ) Virion capture assay was performed on WT (teal) and CD14 + (orange) PVs to corroborate CD14 incorporation. “Nil” represents PBS buffer, “mIgG2a” is an isotype-matched control, and “αCD14” is anti-CD14 mAb M5E2. Bars are mean ± SD p24 of four independently produced virus stocks and are representative of at least two independent assays.

Article Snippet: While others have described the impact of incorporated immunoregulatory proteins on cell signaling ( ), our work highlights a pivotal role for CD14 in allowing virions to function as a shuttle for bioactive molecules like LPS and eliciting inflammatory responses.

Techniques: Transfection, Expressing, Staining, Saline, Fluorescence, Refractive Index, Control, Virus, Enzyme-linked Immunosorbent Assay, Produced

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: CD14 remains functional in the viral envelope and binds to LPS. ( A ) FVM was used to read out a checkboard titration of LPS-bio and PE-SA on CD14 + virions to identify optimal staining concentrations. LPS-bio was incubated overnight at 4°C with virus samples and PE-SA was added for 4 h at 4°C the next day before acquisition. Gates to the left identify optical noise used for ( C ), and gates to the right identify PE + events that were set respective to each negative control PE-SA stain (top row). CD14 + virus stock shown in FVM plots had a titer of 57 ng/mL p24 as measured by ELISA. ( B ) The number of particles in the PE + gates of ( A ) were tabulated and heat mapped with blue, yellow, and red identifying low, intermediate, and high values, respectively. Higher particle counts identify LPS-bio and PE-SA stains that support CD14 + virions binding. ( C ) SI was calculated and plotted for each PE-SA concentration using the PE MESF measured in the PE + gates of ( A ). Peak SI identifies optimal staining pairs (orange stars and blue inverted triangles). ( D ) Incubation times for optimal PE-SA pairs from ( C ) were evaluated over a 16-h time course. Particle counts in PE + gates (right gates) are enumerated on each plot. CD14 + virus stock shown in FVM plots had a titer of 73 ng/mL p24 as measured by ELISA. ( E ) SI of PE MESF measured in ( D ) were plotted over time. All titration data were reproducible across two independent experiments using two independently produced virus stocks. All LPS-bio and PE-SA concentrations in A–E are in units of ng/mL and µg/mL, respectively.

Article Snippet: While others have described the impact of incorporated immunoregulatory proteins on cell signaling ( ), our work highlights a pivotal role for CD14 in allowing virions to function as a shuttle for bioactive molecules like LPS and eliciting inflammatory responses.

Techniques: Functional Assay, Titration, Staining, Incubation, Virus, Negative Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Produced

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: LPS binds to HIV-1 in a CD14-specific manner and does not passively associate with the lipid envelope. ( A ) Optimized LPS-bio (300 ng/mL, overnight, 4°C) and PE-SA (0.2 µg/mL, 4 h, 4°C) staining on CD14 + virions. Particle counts in PE + gates are enumerated on each plot. ( B ) Virion capture assay using immobilized untagged LPS. Ninety-six-well plates were coated with buffer (PBS) or LPS and virions with (CD14 + , orange bars) or without CD14 (WT, teal bars) were added. Captured virions were quantified by Gag p24 ELISA. Bars are mean ± SD of duplicate capture assays and are representative of three independent experiments. ( C ) FVM evaluating the contribution of recombinant LPS-binding protein (LBP) on LPS-bio binding to CD14 + virions. Varying amounts of recombinant LBP (rLBP) were mixed with LPS-bio prior to incubating with WT (top row) and CD14 + (bottom row) virus samples and staining with PE-SA. Gates define PE + events based on negative control (PE-SA only). WT and CD14 + virus stocks shown in FVM plots for ( A ) and ( C ) had a respective titer of 76 and 57 ng/mL p24 as measured by ELISA. ( D ) Particle counts in PE + gates from ( C ) were plotted as a function of PE-SA concentrations for WT (teal triangles) and CD14 + virions (orange circles). Data are mean ± SD of two independent virus stocks. ( E ) FVM measuring neutralization of LPS-bio binding to virions. Anti-CD14 (M5E2) was added to WT (top row) and CD14 + (bottom row) virions before incubation with LPS-bio and PE-SA. Gates are as described for ( C ). WT and CD14 + virus stocks shown in FVM plots had a respective titer of 368 and 141 ng/mL p24 as measured by ELISA. ( F ) Particle counts in PE + gates from ( E ) were plotted over the range of anti-CD14 concentrations tested for WT (teal triangles) and CD14 + (orange circles) virions. Data are representative of two independent virus stocks.

Article Snippet: While others have described the impact of incorporated immunoregulatory proteins on cell signaling ( ), our work highlights a pivotal role for CD14 in allowing virions to function as a shuttle for bioactive molecules like LPS and eliciting inflammatory responses.

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

Journal: Journal of Virology

Article Title: Virion-incorporated CD14 enables HIV-1 to bind LPS and initiate TLR4 signaling in immune cells

doi: 10.1128/jvi.00363-24

Figure Lengend Snippet: CD14 + HIV-1 can trigger TLR4 signaling and NF-κB activation by delivering LPS to human monocytes. ( A ) Schematic describing how virions were “loaded” with LPS before overlaying onto TLR4-expressing cells. ( B ) NF-κB was proxied by measuring SEAP activity from TLR4-expressing (Tlr4 +/+ ) THP1-Dual cells induced after 24-h stimulation with samples. Bars are mean ± SD across four to five independent experiments, and where indicated, at least four different virus stocks were tested each time. Statistical significance was evaluated with one-way analysis of variance (ANOVA) (α = 0.05) and Tukey’s correction for multiple comparisons. From left to right: n = 21, n = 11, n = 12, n = 21, n = 16, n = 22, and n = 22. ns, non-significant; **** P < 0.0001. ( C ) Experiment as in ( B ) but done on TLR4-KO (Tlr4 -/- ) THP1-Dual cells. Data are mean ± SD of two independent experiments using four different virus stocks. Statistical significance was evaluated as in ( B ). From left to right: n = 7, n = 6, n = 7, n = 4, n = 4, n = 6, and n = 8. ns, non-significant; ** P = 0.0014; **** P < 0.0001. ( D ) Secreted TNF-α was measured by ELISA from stimulated (24 h) THP-1 cells. Data are mean ± SD across two independent experiments with four different virus stocks. Statistical significance was evaluated with one-way ANOVA (α = 0.05) and Tukey’s correction for multiple comparisons. From left to right: n = 8, n = 8, n = 8, n = 4, n = 4, n = 8, and n = 8. ns, non-significant; ** P = 0.0052; **** P < 0.0001.

Article Snippet: While others have described the impact of incorporated immunoregulatory proteins on cell signaling ( ), our work highlights a pivotal role for CD14 in allowing virions to function as a shuttle for bioactive molecules like LPS and eliciting inflammatory responses.

Techniques: Activation Assay, Expressing, Activity Assay, Virus, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Metabolomic and immune alterations in long COVID patients with chronic fatigue syndrome

doi: 10.3389/fimmu.2024.1341843

Figure Lengend Snippet: Elevated levels of proinflammatory biomarkers in LC patients. (A) Cumulative data comparing the plasma IL-1α, (B) IL-6, (C) TNF-α, (D) IP-10, (E) CRP, (F) SAA, (G) IL-13, and (H) IL-1β, (I) Flt-1, and (J) soluble CD14 measured by ELISA in the plasma of R, LC, and HC groups. (K) Comparing the Anti-CaSR antibody levels in plasma samples of HC, R, and LC group. (L) Soluble CaSR levels in plasma samples of HC, R, LC and acute COVID-19 patients. (M) Cumulative data of the plasma ATP in HC, acute, R, and LC groups. Kruskal–Wallis analysis with Dunn’s multiple comparisons test. ns, not significant. Each dot represents a study subject. * p < 0.5, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: The plasma was subjected to ELISA kit sCD14 (R&D, 383CD-050) ( ) and Anti-CaSR (EAGLE Biosciences).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of microbiology and biotechnology

Article Title: Anti-Inflammatory Efficacy of Human-Derived Streptococcus salivarius on Periodontopathogen-Induced Inflammation.

doi: 10.4014/jmb.2302.02002

Figure Lengend Snippet: Fig. 6. Binding inhibition of LPS or LTA of periodontopathogens to CD14 and LBP by S. salivarius LTA. EIA plates were coated with rhCD14 (A-C) and rhLBP (D-F). The plates were incubated with biotinylated P. gingivalis LPS (100 ng/ml), T. forsythia LPS (100 ng/ml), and F. alocis LTA (100 ng/ml) in the presence or the absence of S. salivarius G7 LTA and type strain LTA. The bound LPS and LTA were detected using HRP-labelled streptavidin and TMB solution. The experiments were performed three times in duplicate, and data are represented as means and standard deviations. Asterisk (*) indicates statistically significant differences compared with non-treated group (p < 0.05).

Article Snippet: Recombinant human LBP (rhLBP, R&D Systems) and human CD14 (rhCD14, R&D Systems) were dissolved in DPBS at a concentration of 2 μg/ml and were added into suitable Ab-coated well.

Techniques: Binding Assay, Inhibition, Incubation