Journal: Journal of Translational Medicine

Article Title: Immunoreactivity of anti-gelsolin antibodies: implications for biomarker validation

doi: 10.1186/1479-5876-8-137

Figure Lengend Snippet: Immunorecognition of hGSN by three antibodies in Western blot assay . (A) The location of epitope specific to the mouse, goat and rabbit anti-hGSN antibodies are provided in reference to the full-length pGSN. Numbers above each epitope correspond to the amino acid sequence from the full-length (with signal sequence intact) pGSN containing peptides used as antigens. (B) Summary of Western blot analyses revealing that immunoreactivity of pGSN depends on not only antibodies but also source of antigen. Total protein loaded per source per lane: 25 μg of human plasma from HIV-infected individuals, 10 μg of human CSF from HIV-infected individuals, 20 μg of cell supernate from both HIV-infected and non-infected cells, 25 μg of monkey plasma from pre- and 10 days post-infection of rhesus macaques with SIV, and 2 μg each of commercially available gelsolin. Membranes from each source were probed with mouse anti-hGSN, goat anti-hGSN, and rabbit anti-hGSN (all 1:1000) and corresponding HRP-conjugated secondary antibodies (1:20,000) diluted in PBS supplemented with 10% Tween-20 and 10% (w/v) skim milk.

Article Snippet: Human recombinant plasma gelsolin protein was obtained from Cytoskeleton, Inc. (Denver, CO).

Techniques: Western Blot, Sequencing, Infection

Journal: Journal of Translational Medicine

Article Title: Immunoreactivity of anti-gelsolin antibodies: implications for biomarker validation

doi: 10.1186/1479-5876-8-137

Figure Lengend Snippet: LC/ESI-MS/MS identification of immunoaffinity purified forms of gelsolin

Article Snippet: Human recombinant plasma gelsolin protein was obtained from Cytoskeleton, Inc. (Denver, CO).

Techniques: Purification, Sequencing

Journal: Journal of Translational Medicine

Article Title: Immunoreactivity of anti-gelsolin antibodies: implications for biomarker validation

doi: 10.1186/1479-5876-8-137

Figure Lengend Snippet: Forms of immunoaffinity purified gelsolin . A and B shows 1DE analysis of gelsolin immunoaffinity purified from plasma and CSF respectively. A total of 15 μg immunoaffinity purified gelsolin was loaded per lane and gels were stained with Coommasie Brilliant Blue. Eight bands (labeled in lane A) were selected for mass spectrometric identification of proteins. A total of 2 μg recombinant gelsolin was used for analysis via Western blot; banding pattern differences were seen between goat anti-hGSN (lane C) and mouse anti-hGSN (lane D). The high molecular weight band - which is identified by an asterisk - was found to contain fibronectin, a protein known to bind gelsolin. All other bands contained gelsolin, which is further discussed in Table 1.

Article Snippet: Human recombinant plasma gelsolin protein was obtained from Cytoskeleton, Inc. (Denver, CO).

Techniques: Purification, Staining, Labeling, Recombinant, Western Blot, Molecular Weight

Journal: Journal of Translational Medicine

Article Title: Immunoreactivity of anti-gelsolin antibodies: implications for biomarker validation

doi: 10.1186/1479-5876-8-137

Figure Lengend Snippet: Schematic model of full-length hGSN and proposed forms of gelsolin isolated from serum/plasma and CSF . Band 1 represents the full-length hGSN molecule and includes its functional and structural features. This form shows electrophoretic mobility corresponding to approximately 86 kDa. hGSN was identified by tandem mass spectrometry analysis in bands 2 to 8. Based on their electrophoretic mobility and identified peptides resulting from trypsin digestion (see Table 1 for details) we estimated their approximate molecular weight and amino acid sequence coverage. Gelsolin peptides identified in each band by LC/ESI-MS/MS are colored.

Article Snippet: Human recombinant plasma gelsolin protein was obtained from Cytoskeleton, Inc. (Denver, CO).

Techniques: Isolation, Functional Assay, Mass Spectrometry, Molecular Weight, Sequencing, Tandem Mass Spectroscopy

Journal: Journal of Translational Medicine

Article Title: Immunoreactivity of anti-gelsolin antibodies: implications for biomarker validation

doi: 10.1186/1479-5876-8-137

Figure Lengend Snippet: Peptide candidates for MRM based quantitation . Two peptides: (1) DSQEEKTEALTSAK and (2) EVQGFESATFLGYFK, derived from pGSN by trypsin digestion and representing N-terminal and C-terminal regions respectively, are well ionized and fragmented by ESI-MS/MS. As such they are excellent potential candidates for MRM based quantitation. Note that peptide (1) was identified in only 2 out of 8 bands and peptide (2) was identified in 7 out of 8 bands of pGSN circulating in plasma/CSF. Therefore, neither of these two peptides will reflect accurately levels of pGSN; additionally, use of these peptides in MRM may not validate pGSN as potential biomarker.

Article Snippet: Human recombinant plasma gelsolin protein was obtained from Cytoskeleton, Inc. (Denver, CO).

Techniques: Quantitation Assay, Derivative Assay, Tandem Mass Spectroscopy, Biomarker Assay

Journal: Journal of Translational Medicine

Article Title: Plasma gelsolin levels are associated with diabetes, sex, race, and poverty

doi: 10.1186/s12967-023-04026-5

Figure Lengend Snippet: HANDLS longitudinal subcohort for plasma gelsolin measurements

Article Snippet: EK, SLL and MJD are employed by and own stock in BioAegis Therapeutics, which is developing recombinant human plasma gelsolin for clinical use.

Techniques: Clinical Proteomics

Journal: Journal of Translational Medicine

Article Title: Plasma gelsolin levels are associated with diabetes, sex, race, and poverty

doi: 10.1186/s12967-023-04026-5

Figure Lengend Snippet: Association of plasma gelsolin, diabetes status and demographic variables. Plasma gelsolin (pGSN) levels were measured in a cohort of 104 euglycemic (Eu) individuals and 104 individuals with diabetes at 2 different time points ~ 5 years apart. Linear mixed model regression accounting for repeated measurements was used to analyze the relationship of pGSN, diabetes status, sex ( A ), race ( B ), and poverty status ( C ). AfrAm: African American

Article Snippet: EK, SLL and MJD are employed by and own stock in BioAegis Therapeutics, which is developing recombinant human plasma gelsolin for clinical use.

Techniques: Clinical Proteomics

Journal: Journal of Translational Medicine

Article Title: Plasma gelsolin levels are associated with diabetes, sex, race, and poverty

doi: 10.1186/s12967-023-04026-5

Figure Lengend Snippet: Plasma proteins that differ significantly by diabetes status and their correlation with plasma gelsolin

Article Snippet: EK, SLL and MJD are employed by and own stock in BioAegis Therapeutics, which is developing recombinant human plasma gelsolin for clinical use.

Techniques: Clinical Proteomics, Control, Protease Inhibitor

Journal: Journal of Neuroinflammation

Article Title: Recombinant human plasma gelsolin reverses increased permeability of the blood–brain barrier induced by the spike protein of the SARS-CoV-2 virus

doi: 10.1186/s12974-022-02642-4

Figure Lengend Snippet: Plasma gelsolin (pGSN) significantly reduces disruption of the blood–brain barrier caused by the SARS-CoV-2 Spike protein S1 subunit in the 2D Transwell permeability assay. Confluent monolayer of human cerebral microvascular endothelial cells (hCMEC/D3) seeded on a Transwell semi-permeable membrane exposed to tested compounds added to the upper chamber (blood) (Panel A i). The functional state of the cells as a barrier was evaluated with transendothelial electrical resistance (TEER) measurement and Dextran-FITC permeability assay (Panel A ii). The dextran-FITC intensity was measured in the lower chamber (brain) that migrated from the upper chamber (blood) in time (Panels B , D ). Change in TEER was measured using Epithelial Voltohmeter EVOM2 (Panels C and D ). The data represent the mean ± SEM of four ( n = 4, with 2 inserts used per condition each time) independent experiments. * and ^ indicate statistical significance at p ≤ 0.05 compared to CT and S1, respectively, by one-way ANOVA and Tukey post hoc test

Article Snippet: Recombinant human plasma gelsolin (pGSN) used in our study was produced in E. coli and provided by BioAegis Therapeutics (North Brunswick, USA).

Techniques: Clinical Proteomics, Disruption, Permeability, Membrane, Functional Assay

Journal: Journal of Neuroinflammation

Article Title: Recombinant human plasma gelsolin reverses increased permeability of the blood–brain barrier induced by the spike protein of the SARS-CoV-2 virus

doi: 10.1186/s12974-022-02642-4

Figure Lengend Snippet: Plasma gelsolin reverses the destructive effect of SARS-CoV-2 Spike protein S1 subunit on blood–brain barrier function in the 3D flow model. Confocal images of hCMEC/D3 cells (panel A ), tight junction protein ZO-1 (red), and nuclei (blue). Permeability coefficient measured from dextran experiments for endothelial channels exposed to S1 and S1 + pGSN (Panel B ). TEER measurement results (Panel C ). Images demonstrating the measurement of vessel permeability using 4 kDa FITC-dextran (green) and the effects of the S1 and S1 + pGSN (Panel D ). Barrier permeability and TEER measurement were performed after 4 h of perfusion with 10 nM of S1 and S1 + pGSN. The data represent the mean ± SEM of four independent experiments ( n = 4). * and ^ indicate statistical significance at p ≤ 0.05 compared to CT and S1, respectively, by one-way ANOVA and Tukey post hoc test

Article Snippet: Recombinant human plasma gelsolin (pGSN) used in our study was produced in E. coli and provided by BioAegis Therapeutics (North Brunswick, USA).

Techniques: Clinical Proteomics, Permeability

Journal: Journal of Neuroinflammation

Article Title: Recombinant human plasma gelsolin reverses increased permeability of the blood–brain barrier induced by the spike protein of the SARS-CoV-2 virus

doi: 10.1186/s12974-022-02642-4

Figure Lengend Snippet: Plasma gelsolin improves migration of hCMEC/D3 upon SARS-CoV-2 Spike protein S1 subunit treatment in wound-healing assay. Images of endothelial cells in a wound healing setting (Panel A ), the yellow color indicates wound width, which is quantitatively shown in Panel B . Western blot quantitative analysis of VEGRF2 expression in hCMEC/D3 cells after 24 h stimulation with pGSN, S1, and S1 + pGSN (Panel C ). The data represent the mean ± SEM of four independent experiments ( n = 4). * and ^ indicate statistical significance at p ≤ 0.05 compared to CT (100%) and S1, respectively, by one-way ANOVA and Tukey post hoc test

Article Snippet: Recombinant human plasma gelsolin (pGSN) used in our study was produced in E. coli and provided by BioAegis Therapeutics (North Brunswick, USA).

Techniques: Clinical Proteomics, Migration, Wound Healing Assay, Western Blot, Expressing

Journal: Journal of Neuroinflammation

Article Title: Recombinant human plasma gelsolin reverses increased permeability of the blood–brain barrier induced by the spike protein of the SARS-CoV-2 virus

doi: 10.1186/s12974-022-02642-4

Figure Lengend Snippet: Secretion of inflammatory mediators by hCMEC/D3 cell line stimulated with SARS-CoV-2 Spike protein S1 subunit and S1 + pGSN after 6 and 24 h. Expression of IL-2 ( A , G ), IL-6 ( B , H ), IL-8 ( D , I ), TNF-α ( D , J ), INF-γ ( E , K ), and GM-CSF ( F , L ). Protein expression after 6 h of treatment is presented on Panels A – F , and expression after 24 h is on Panels G – L . Alternation of inflammatory response was monitored using a magnetic bead-based assay. The data represent the mean ± SEM of three independent experiments ( n = 3). * and ^indicate statistical significance at p ≤ 0.05 compared to CT and S1, respectively, by one-way ANOVA and Tukey post hoc test

Article Snippet: Recombinant human plasma gelsolin (pGSN) used in our study was produced in E. coli and provided by BioAegis Therapeutics (North Brunswick, USA).

Techniques: Expressing, Bead-based Assay

Journal: Journal of Neuroinflammation

Article Title: Recombinant human plasma gelsolin reverses increased permeability of the blood–brain barrier induced by the spike protein of the SARS-CoV-2 virus

doi: 10.1186/s12974-022-02642-4

Figure Lengend Snippet: Plasma gelsolin (pGSN) inhibits NF-κB activation by the SARS-CoV-2 Spike protein S1 subunit in hCMEC/D3 cells. Panel A shows a Log 2 fold change heat map for genes involved in VEGF signaling and activation of blood–brain barrier endothelial cells upon 6 h stimulation with pGSN [250 µg/mL], S1 [10 nM] and S1 [10 nM] + pGSN [250 µg/mL]. Log 2 fold change was calculated based on delta Ct values compared to the control samples. Warmer colors imply increased expression, while cold reflects decreased expression. The assay was performed twice in quadruplicate; data within frames shows Log2FC for every tested gene. Panel B shows the schematic representation of signaling pathways triggered by the SARS-CoV-2 Spike protein S1 subunit in hCMEC/D3 cells. (1) Spike protein interacts with the given receptor on a cell membrane, which (2) activates the catalytic effect of PI3K on PIP2, (3) enzymatically transforming it to PIP3, which is possibly inhibited by plasma gelsolin given its direct binding to PIP2. (4) PIP3 binds to AKT, promoting its phosphorylation and activation. (5, 6) Activated AKT regulates transcriptional activity of MAPK kinases and NF-κB by inducing phosphorylation and degradation of inhibitor of κB (IκB). (7) MAPK initiates the downstream induction of NF-κB and its translocation (8) to the nucleus. (9, 10) NF-κB, after activation, triggers the transcription of various genes and thereby regulates inflammation

Article Snippet: Recombinant human plasma gelsolin (pGSN) used in our study was produced in E. coli and provided by BioAegis Therapeutics (North Brunswick, USA).

Techniques: Clinical Proteomics, Activation Assay, Control, Expressing, Protein-Protein interactions, Membrane, Binding Assay, Phospho-proteomics, Activity Assay, Translocation Assay

Journal: Frontiers in immunology

Article Title: Gelsolin Attenuates Neonatal Hyperoxia-Induced Inflammatory Responses to Rhinovirus Infection and Preserves Alveolarization.

doi: 10.3389/fimmu.2022.792716

Figure Lengend Snippet: FIGURE 1 | In neonatal mice, gelsolin blocks hyperoxia-induced pro-inflammatory responses to RV infection. Two-day old wild type mice were exposed to hyperoxia or normoxia for 10 days and treated with recombinant human plasma gelsolin (GSN, 0.5 mg/kg) or equal volume BSA-PBS, administered intranasally under anesthesia daily. On day of life 14 the mice were inoculated with RV. Whole lung mRNA expression of Il12p40, Tnfa and Ifng (A) and protein expression of Il12p70, TNF-a and IFN-g (B) were measured 2 days later. (N = 4-6, mean ± SEM, *p < 0.05, **p < 0.01, NS, nonsignificant, ANOVA). One of two independent experiments is shown.

Article Snippet: In selected experiments, neonatal mice were treated with 10ul of PBS (Sigma D6662) or recombinant human plasma gelsolin (Cytoskeleton Inc., HPG6) 0.5mg/kg intranasally daily during exposure to hyperoxia.

Techniques: Infection, Recombinant, Clinical Proteomics, Expressing

Journal: Frontiers in immunology

Article Title: Gelsolin Attenuates Neonatal Hyperoxia-Induced Inflammatory Responses to Rhinovirus Infection and Preserves Alveolarization.

doi: 10.3389/fimmu.2022.792716

Figure Lengend Snippet: FIGURE 2 | Gelsolin inhibits hyperoxia-induced CD103+ DC expansion and inflammation. Two-day old wild type mice were exposed to hyperoxia or normoxia for 4 or 10 days and treated with recombinant human plasma gelsolin (GSN, 0.5 mg/kg) or equal volume BSA-PBS, administered intranasally under anesthesia daily. Lungs were enzymatically digested, and a single cell suspension was incubated and stained with specific cell-surface antibodies. Lung CD103+ DCs were distinguished from other lung cells based on expression of CD45, F4/80, CD11c, CD103 and CD11b. (A) Gating strategy to identify lung DCs. (B) Conventional lung DC populations are distinguished based on the expression of CD103 and CD11b. Following hyperoxia, two distinct populations of CD103+ DCs, CD103lo and CD103hi DCs are observed. GSN treatment during hyperoxia decreases CD103lo, but not CD103hi DCs. (C) Quantification of the CD103lo and CD103hi DCs. (D) Both CD103lo and CD103hi DC populations are absent in the lungs of 4-day old Batf3 null mice compared to age-matched wild type (WT) mice. *p < 0.05, **p < 0.01, ***p < 0.001, NS, nonsignificant (ANOVA). These results are representative of three independent experiments. (E) RNA was extracted from the whole lung tissue on day of life 16 after 10 days exposure to normoxia or hyperoxia with and without daily GSN treatment. GSN attenuated hyperoxia-induced mRNA expression of Il12p40, Myd88, Cd207, Cd103 and Clec9a. *p < 0.05, **p < 0.01 (ANOVA). N = 4 per groups. One of three independent experiments is shown.

Article Snippet: In selected experiments, neonatal mice were treated with 10ul of PBS (Sigma D6662) or recombinant human plasma gelsolin (Cytoskeleton Inc., HPG6) 0.5mg/kg intranasally daily during exposure to hyperoxia.

Techniques: Recombinant, Clinical Proteomics, Suspension, Incubation, Staining, Expressing

Journal: Frontiers in immunology

Article Title: Gelsolin Attenuates Neonatal Hyperoxia-Induced Inflammatory Responses to Rhinovirus Infection and Preserves Alveolarization.

doi: 10.3389/fimmu.2022.792716

Figure Lengend Snippet: FIGURE 3 | Gelsolin attenuates hyperoxic BALF-induced inflammatory response to RV. Two-day old mice were exposed to hyperoxia or normoxia for 14 days. BALF was collected on day of life 16. F-actin and gelsolin (GSN) protein levels were measured in BALF supernatant using ELISA and the ratio of F-actin to GSN was calculated and compared between samples from hyperoxia- and normoxia-exposed mice, unpaired t-test. *p < 0.05 (A). (B) 14-day old mice were exposed to hyperoxia for 4 days and BALF was collected (H-BALF). Cell-free H-BALF was incubated with GSN (5mg) in vitro for 10min and F-actin levels were analyzed by ELISA (paired t-test p < 0.001). (C) 14-day old mice were inoculated with H-BALF, RV, GSN, or appropriate controls and whole lung mRNA expression of Il12p40, Ifng and Tnfa were analyzed 2 days later. *p < 0.05, ***p < 0.001 (ANOVA). N = 3-6 per group. One of two independent experiments is shown. ns, not significant

Article Snippet: In selected experiments, neonatal mice were treated with 10ul of PBS (Sigma D6662) or recombinant human plasma gelsolin (Cytoskeleton Inc., HPG6) 0.5mg/kg intranasally daily during exposure to hyperoxia.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, In Vitro, Expressing

Journal: Frontiers in immunology

Article Title: Gelsolin Attenuates Neonatal Hyperoxia-Induced Inflammatory Responses to Rhinovirus Infection and Preserves Alveolarization.

doi: 10.3389/fimmu.2022.792716

Figure Lengend Snippet: FIGURE 5 | Gelsolin prevents neonatal hyperoxia-induced hypoalveolarization. Two-day old wild type mice were exposed to hyperoxia or normoxia for 10 days and treated with recombinant human plasma gelsolin (0.5 mg/kg) or equal volume BSA-PBS, administered intranasally under anesthesia daily. Lung histology was assessed on day of life 16. (A) Representative lung sections were stained with hematoxylin and eosin. Hyperoxia induced enlargement of the alveolar spaces (hypoalveolarization) in BSA-PBS-treated mice. In contrast, gelsolin treatment preserved the alveolar architecture of hyperoxia-exposed mice. (B) Alveolar chord length was significantly increased in hyperoxia-exposed, BSA-PBS-treated mice, consistent with hypoalveolarization. In contrast, alveolar chord length of hyperoxia- exposed, gelsolin-treated mice was similar to that of normoxia-exposed mice, indicating protective effect of gelsolin on alveolarization during neonatal hyperoxia. ****p < 0.0001 (ANOVA). N = 5-12 per group.

Article Snippet: In selected experiments, neonatal mice were treated with 10ul of PBS (Sigma D6662) or recombinant human plasma gelsolin (Cytoskeleton Inc., HPG6) 0.5mg/kg intranasally daily during exposure to hyperoxia.

Techniques: Recombinant, Clinical Proteomics, Staining

Journal: Frontiers in immunology

Article Title: Gelsolin Attenuates Neonatal Hyperoxia-Induced Inflammatory Responses to Rhinovirus Infection and Preserves Alveolarization.

doi: 10.3389/fimmu.2022.792716

Figure Lengend Snippet: FIGURE 6 | F-actin/Gelsolin ratio in tracheal aspirates of mechanically ventilated human preterm infants with respiratory distress and FiO2 on day of sample collection. Tracheal aspirates were collected in the first week of life. F-actin and gelsolin levels were measured by ELISA in the supernatant. The association between the ratio of F-actin to gelsolin and the FiO2 on the day of sample collection was determined by Pearson’s correlation analysis.

Article Snippet: In selected experiments, neonatal mice were treated with 10ul of PBS (Sigma D6662) or recombinant human plasma gelsolin (Cytoskeleton Inc., HPG6) 0.5mg/kg intranasally daily during exposure to hyperoxia.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: Gelsolin Attenuates Neonatal Hyperoxia-Induced Inflammatory Responses to Rhinovirus Infection and Preserves Alveolarization.

doi: 10.3389/fimmu.2022.792716

Figure Lengend Snippet: FIGURE 7 | In tracheal aspirates of human preterm infants with respiratory distress, gelsolin concentrations decrease during the first two weeks of mechanical ventilation. For preterm infants who remain endotracheally intubated receiving mechanical ventilation tracheal aspirates were obtained during week 1 and week 2 of mechanical ventilation and gelsolin levels in the supernatant were quantified. Statistical significance was pointed by paired t-test.

Article Snippet: In selected experiments, neonatal mice were treated with 10ul of PBS (Sigma D6662) or recombinant human plasma gelsolin (Cytoskeleton Inc., HPG6) 0.5mg/kg intranasally daily during exposure to hyperoxia.

Techniques: