Journal: Scientific Reports

Article Title: Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

doi: 10.1038/s41598-025-92016-6

Figure Lengend Snippet: Adjuvant effect on IFN-γ production in conjunction with anti-CD3/CD28 mAb stimulation. Samples were stimulated with anti-CD3/28 mAb with and without either anti-PD-1 mAb (Nivolumab; NIVO) or IL-7. Anti-CD/CD28 mAb (CD3) increased IFN-γ SFU (upper panels), SS (upper-middle panels) and TWI (lower-middle panels) in samples from both Healthy donors (left, n = 46) and Septic patients (right, n = 63) as compared to unstimulated spontaneous (SPON) production. NIVO did not increase CD3 induced IFN-γ SFU, SS, or TWI, while IL-7 did increase CD3 induced IFN-γ SFU, SS, and TWI. SFU (Top panels) were normalized to the number of lymphocytes plated in the ELISpot well (Bottom panels, n = 34 healthy and n = 59 septic). Lymphocyte number per well was determined by multiplying the draw specific ALC (K/cumm) by 1000 (converting K/cumm to cells/cumm) then by the volume of blood added to the well (5µL). Pairwise statistical relationships of SFU/Lymphocyte remain largely the same as SFU. Red bars represent Median with Interquartile Ranges (IQR) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by Friedman Test with multiple comparisons.

Article Snippet: Immunomodulatory agents included LPS (1 ng/mL, ENZO Life Sciences, Farmingdale, NY, USA), anti-CD3 and anti-CD28 mAb (0.5 and 5 ug/mL respectively, or 0.125 and 1.25 ng/mL for low dose, BioLegend, San Diego, CA, USA), recombinant human IL-7 (50 ng/mL, R&D Systems, Minneapolis, MN, USA), anti-PD1 mAb (1 ug/mL, Nivolumab, Bristol Myers Squibb, New York, NY, USA), Dexamethasone (1 ug/mL, Viatris, formerly Mylan, Canonsburg, PA, USA), and Resiquimod (1 ug/mL, Invivogen, San Diego, CA, USA), using IFN-γ and TNF-α ELISpot kits (CTL).

Techniques: Adjuvant, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

doi: 10.1038/s41598-025-92016-6

Figure Lengend Snippet: Difference in relative IFN-γ response to IL-7 between healthy donors and septic patients. A. IL-sevenfold-changes were calculated for SFU, SS, and TWI for all samples; anti-CD3/CD28 mAb (CD3) + IL-7 values were divided by CD3 alone to yield fold-change. Samples from healthy donors displayed a higher responsiveness to IL-7 in the SFU (Left) and TWI (Right) measurements, based on fold-change, than did those from septic patients. B. Visualization of paired data points for CD3 alone or with IL-7 for samples from healthy donors (Left, n = 46) and septic patients (Right, n = 63). The bulk of samples show an increase of SFU and SS with IL-7 treatment. Red bars represent Median with IQR. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by ( A. ) Mann–Whitney or ( B. ) Wilcoxon tests.

Article Snippet: Immunomodulatory agents included LPS (1 ng/mL, ENZO Life Sciences, Farmingdale, NY, USA), anti-CD3 and anti-CD28 mAb (0.5 and 5 ug/mL respectively, or 0.125 and 1.25 ng/mL for low dose, BioLegend, San Diego, CA, USA), recombinant human IL-7 (50 ng/mL, R&D Systems, Minneapolis, MN, USA), anti-PD1 mAb (1 ug/mL, Nivolumab, Bristol Myers Squibb, New York, NY, USA), Dexamethasone (1 ug/mL, Viatris, formerly Mylan, Canonsburg, PA, USA), and Resiquimod (1 ug/mL, Invivogen, San Diego, CA, USA), using IFN-γ and TNF-α ELISpot kits (CTL).

Techniques: MANN-WHITNEY

Journal: Scientific Reports

Article Title: Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

doi: 10.1038/s41598-025-92016-6

Figure Lengend Snippet: Septic patient samples produce greater IFN-γ SFU and SFU/Lymphocyte than Healthy donor samples. Comparison between Healthy donor (n = 46) and Septic patient (n = 63) whole blood samples shows septic blood samples have higher IFN-γ SFU (top) than healthy donor samples in response to anti-CD3/CD28 mAb (CD3) alone (left), CD3 + anti-PD-1 mAb (Nivolumab; NIVO) (middle), and CD3 + IL-7 (right). To account for high variability in cell counts among septic patients and between septic and healthy samples, SFU were normalized to number of lymphocytes plated in ELISpot wells (bottom, n = 34 healthy and n = 59 septic). When SFU were normalized to lymphocyte numbers, the differences between septic and healthy IFN-γ production became more apparent. As no significant differences were discerned between CD3 treated samples and CD3 + NIVO treated samples, the differences displayed in the “CD3 + NIVO” column between healthy and septic are a consequence of the greater responsiveness of septic samples to CD3 treatment. Red bars represent Median with IQR. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by Mann–Whitney test.

Article Snippet: Immunomodulatory agents included LPS (1 ng/mL, ENZO Life Sciences, Farmingdale, NY, USA), anti-CD3 and anti-CD28 mAb (0.5 and 5 ug/mL respectively, or 0.125 and 1.25 ng/mL for low dose, BioLegend, San Diego, CA, USA), recombinant human IL-7 (50 ng/mL, R&D Systems, Minneapolis, MN, USA), anti-PD1 mAb (1 ug/mL, Nivolumab, Bristol Myers Squibb, New York, NY, USA), Dexamethasone (1 ug/mL, Viatris, formerly Mylan, Canonsburg, PA, USA), and Resiquimod (1 ug/mL, Invivogen, San Diego, CA, USA), using IFN-γ and TNF-α ELISpot kits (CTL).

Techniques: Comparison, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

doi: 10.1038/s41598-025-92016-6

Figure Lengend Snippet: Effect of dexamethasone on IFN-γ production. IFN-γ SFU (Top), SS (Middle), and TWI (Bottom), are reduced when dexamethasone (DEX) is added to ELISpot. DEX abrogates anti-CD3/CD28 mAb (CD3) induced IFN-γ production. DEX-induced reduction of IFN-γ production is still present when co-cultured with anti-CD3/CD28 mAb and IL-7, though there is still an IL-7 effect present even with DEX co-treatment. Results were similar in healthy donor (n = 46) and septic patient (n = 63) samples. Red bars represent Median with IQR. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by Friedman Test with multiple comparisons.

Article Snippet: Immunomodulatory agents included LPS (1 ng/mL, ENZO Life Sciences, Farmingdale, NY, USA), anti-CD3 and anti-CD28 mAb (0.5 and 5 ug/mL respectively, or 0.125 and 1.25 ng/mL for low dose, BioLegend, San Diego, CA, USA), recombinant human IL-7 (50 ng/mL, R&D Systems, Minneapolis, MN, USA), anti-PD1 mAb (1 ug/mL, Nivolumab, Bristol Myers Squibb, New York, NY, USA), Dexamethasone (1 ug/mL, Viatris, formerly Mylan, Canonsburg, PA, USA), and Resiquimod (1 ug/mL, Invivogen, San Diego, CA, USA), using IFN-γ and TNF-α ELISpot kits (CTL).

Techniques: Enzyme-linked Immunospot, Cell Culture

Journal: Protein expression and purification

Article Title: Affimers as an alternative to antibodies for protein biomarker enrichment.

doi: 10.1016/j.pep.2020.105677

Figure Lengend Snippet: Figure 1 (A) graphical overview of alternative splicing of the IL-37 gene that yields different IL-37 protein isoforms and (B) the synthesis of preproinsulin into proinsulin by signal peptidases, which then is cleaved into insulin by prohormone convertase-1 and 2 at the B-chain - C-peptide and A-chain - C-peptide junctions.

Article Snippet: To generate affimer candidates against proinsulin, the library was selected against biotinylated recombinant human proinsulin (R&D Systems), with counter-selection performed against insulin and the C-peptide.

Techniques: Alternative Splicing

Journal: Protein expression and purification

Article Title: Affimers as an alternative to antibodies for protein biomarker enrichment.

doi: 10.1016/j.pep.2020.105677

Figure Lengend Snippet: Figure 4 Graphical illustration of the affimer and antibody-based capture experiments of recombinant proinsulin.

Article Snippet: To generate affimer candidates against proinsulin, the library was selected against biotinylated recombinant human proinsulin (R&D Systems), with counter-selection performed against insulin and the C-peptide.

Techniques: Recombinant

Journal: Developmental Cell

Article Title: Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human thymus

doi: 10.1016/j.devcel.2023.08.017

Figure Lengend Snippet:

Article Snippet: HumanKine® recombinant human IL-7 protein , Proteintech , cat# HZ-1281.

Techniques: Recombinant, Modification, Selection, RNA Extraction, Polymer, Cell Culture, Derivative Assay, Software