Journal: Frontiers in Immunology

Article Title: Propionic Acid, Induced in Gut by an Inulin Diet, Suppresses Inflammation and Ameliorates Liver Ischemia and Reperfusion Injury in Mice

doi: 10.3389/fimmu.2022.862503

Figure Lengend Snippet: Intraperitoneal pretreatment with propionic acid ameliorates liver IRI in mice. (A) WT mice were divided into two groups. Mice in the PA group were intraperitoneally pretreated with PA (5 mmol/kg in saline) 2 h prior to liver ischemia, whereas mice in the saline group were pretreated with saline. Then, mice in both groups were performed liver IRI model. (B) The sALT level after 6 h of reperfusion (n = 8 mice/group; ****P < .0001) (Power calculation: 1.000). (C) Macroscopic findings after 6 h of reperfusion in saline- and PA -pretreated mice. Regions (bleeding and swelling) of macroscopic changes induced by IR insult are circled by dotted lines. (D) Representative liver histology (H&E staining) after IR insult (magnification ×400). Congestion area was shown in circle by yellow dot. (E) Suzuki’s histological grading in each group (n = 8 mice/group; ****P < .0001) (Power calculation: 1.000). (F) Quantitative RT-PCR detection of inflammatory cytokines (TNF-α, IL-6, CXCL-1, CXCL-2, IL-1β, and IL-10) at 6 h of reperfusion. Data were normalized to β-actin gene expression (n = 6 mice/group; *P < .05, **P < .01) (Power calculation: TNF-α; 0.998, IL-6; 0.980, CXCL-2; 1.000, IL-1β; 0.996). The reduction of CXCL-1 and IL-10 in PA-treated group compared to saline-treated group was not significant. (G) The levels of serum HMGB-1 at 6 h after reperfusion in saline- and PA -pretreated mice (n = 8 mice/group; **P < .01) (Power calculation: 1.000).

Article Snippet: Then, bovine HMGB-1 (Chondrex, Redmond, WA, USA) (1 µg/mL) was added to the macrophages.

Techniques: Staining, Quantitative RT-PCR, Expressing

Journal: Frontiers in Immunology

Article Title: Propionic Acid, Induced in Gut by an Inulin Diet, Suppresses Inflammation and Ameliorates Liver Ischemia and Reperfusion Injury in Mice

doi: 10.3389/fimmu.2022.862503

Figure Lengend Snippet: PA suppressed HMGB-1-mediated activation of peritoneal macrophages. Peritoneal macrophages from WT mice stimulated by HMGB-1 (1 µg/mL) were cultured with or without the pretreatment with PA (1, 2, 5, and 10 mM, respectively) for 48 h. (A) TNF-α release was measured by ELISA (n = 3 samples/group; ****P < .0001) (Power calculation: 1.000). (B) Western blot assisted analyses of NF-κB signaling and MAPK signaling molecules in the macrophages stimulated by HMGB-1 (1 µg/mL) 30 min after the pretreatment with PA (5 mM). β-actin was used as the internal control.

Article Snippet: Then, bovine HMGB-1 (Chondrex, Redmond, WA, USA) (1 µg/mL) was added to the macrophages.

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: ACS Nano

Article Title: Two-Stage SN38 Release from a Core–Shell Nanoparticle Enhances Tumor Deposition and Antitumor Efficacy for Synergistic Combination with Immune Checkpoint Blockade

doi: 10.1021/acsnano.2c09788

Figure Lengend Snippet: OxPt/SN38 induces ICD and upregulates PD-L1. CLSM images (a) and flow cytometry (b) showing DNA DSBs of MC38 cells after treatment with OxPt plus IRI or OxPt/SN38. Scale bar: 20 μm. Cellular morphology was visualized by F-actin (green) staining. (c) Western blot of γ-H2AX, IRF1, and PD-L1 of MC38 cells. (d) Schematic showing PD-L1 upregulation induced by DNA DSBs. DSBs activate STAT signaling through ATM/ATR/Chk1 kinases, which in turn induces IRF1 activation and downstream PD-L1 upregulation. Flow cytometry (e) with statistical analysis (f) of PD-L1 expression in MC38 cells, n = 3. CLSM images (g) and flow cytometry (h) with statistical analysis (i) of ROS generation in MC38 cells, n = 3. CLSM images (j) and flow cytometry with statistical analysis (k) of CRT expression in MC38 cells, n = 3. (l) HMGB-1 release from MC38 cells detected by ELISA, n = 3. In all treatments, MC38 cells were incubated with drugs at 5 μM OxPt equiv and 9 μM SN38 equiv for 24 h. Data are expressed as means ± SD, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, by one-way ANOVA with Tukey’s multiple comparisons test. MFI denotes mean fluorescent intensity.

Article Snippet: Two ×10 5 cells/well of MC38 cells were seeded in 6-well plates and then cultured with PBS, OxPt, IRI, OxPt plus IRI, ZnP/SN38, OxPt NCP, or OxPt/SN38 for 24 h. The medium was collected to detect HMGB-1 release by ELISA (Chondrex, Redmond, WA).

Techniques: Flow Cytometry, Staining, Western Blot, Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Incubation

Journal: American Journal of Transplantation

Article Title: The lectin‐like domain of thrombomodulin is a drug candidate for both prophylaxis and treatment of liver ischemia and reperfusion injury in mice

doi: 10.1111/ajt.16269

Figure Lengend Snippet: The levels of serum HMGB‐1 and expression of TLR‐4. A, The levels of serum HMGB‐1 at 1 and 6 h after reperfusion in IR‐treated and rTMD1‐pretreated mice (n = 8 mice/group; ** P < .01). B, The expression of TLR‐4 in liver tissues at 6 h after reperfusion in IR‐treated and rTMD1‐pretreated mice. HMGB‐1, high‐mobility group box 1 protein; IR, ischemia and reperfusion; rTMD1, the domain 1 of thrombomodulin; TLR‐4, Toll‐like receptor 4

Article Snippet: After 3 hours, wells were washed 3 times with phosphate‐buffered saline to remove nonadherent cells, which were then incubated with or without bovine HMGB‐1 (Chondrex, Redmond, WA) (1 µg/mL) and various concentrations of rTM domain proteins (n = 3 in each group).

Techniques: Expressing

Journal: American Journal of Transplantation

Article Title: The lectin‐like domain of thrombomodulin is a drug candidate for both prophylaxis and treatment of liver ischemia and reperfusion injury in mice

doi: 10.1111/ajt.16269

Figure Lengend Snippet: rTMD1 suppressed HMGB‐1‐mediated activation of peritoneal macrophages. A, Peritoneal macrophages from WT or TLR‐4 KO mice were cultured with HMGB‐1 (1 µg/mL) alone or with the mixture of HMGB‐1 (1 µg/mL) and rTMD1 (5, 10, and 30 µg/mL, respectively) for 48 h. TNF‐α release was measured by ELISA (n = 3 samples/group; * P < .05, ** P < .01). B, Peritoneal macrophages from WT mice were cultured with HMGB‐1 (1 µg/mL) alone or with the mixture of HMGB‐1 (1 µg/mL), and each rTM domain protein (rTM, rTMD1, rTMD2, rTMD3; 5 and 30 µg/mL, respectively) for 48 h. TNF‐α release was measured by ELISA (n = 3 samples/group; ** P < .01, *** P < .001). ELISA, enzyme‐linked immunosorbent assay; HMGB‐1, high‐mobility group box 1 protein; rTM, recombinant human thrombomodulin; rTMD1, the domain 1 of thrombomodulin; TLR‐4 KO, tlr‐4 gene–deleted; TNF‐α, tumor necrosis factor‐α; WT, wild‐type

Article Snippet: After 3 hours, wells were washed 3 times with phosphate‐buffered saline to remove nonadherent cells, which were then incubated with or without bovine HMGB‐1 (Chondrex, Redmond, WA) (1 µg/mL) and various concentrations of rTM domain proteins (n = 3 in each group).

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: American Journal of Transplantation

Article Title: The lectin‐like domain of thrombomodulin is a drug candidate for both prophylaxis and treatment of liver ischemia and reperfusion injury in mice

doi: 10.1111/ajt.16269

Figure Lengend Snippet: rTMD1 can suppress TNF‐α production even after the addition of HMGB‐1. A, Peritoneal macrophages from WT mice were divided into 5 groups. Nonstimulated group, macrophages were cultured with culture fluid alone; Control group, macrophages were cultured with HMGB‐1 (1 µg/mL); Group A, rTMD1 (30 µg/mL) was added to the macrophages 30 min prior to the addition of HMGB‐1 (1 µg/mL); Group B, rTMD1 (30 µg/mL) and HMGB‐1 (1 µg/mL) were first mixed in a tube and incubated for 30 min, and then mixtures were added together to macrophages; Group C, rTMD1 (30 µg/mL) was added to the macrophages 30 min after the addition of HMGB‐1 (1 µg/mL). All cells were cultured for 48 h. B, TNF‐α release was measured by ELISA (n = 3 samples/group; * P < .05, ** P < .01, *** P < .001). ELISA, enzyme‐linked immunosorbent assay; HMGB‐1, high‐mobility group box 1 protein; rTMD1, the domain 1 of thrombomodulin; TNF‐α, tumor necrosis factor‐α ; WT, wild‐type

Article Snippet: After 3 hours, wells were washed 3 times with phosphate‐buffered saline to remove nonadherent cells, which were then incubated with or without bovine HMGB‐1 (Chondrex, Redmond, WA) (1 µg/mL) and various concentrations of rTM domain proteins (n = 3 in each group).

Techniques: Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Immunostimulatory nanomedicines synergize with checkpoint blockade immunotherapy to eradicate colorectal tumors

doi: 10.1038/s41467-019-09221-x

Figure Lengend Snippet: Immunostimulatory effects in colorectal cancer cells. a , b CRT cell surface expression upon treatment with OxPt/DHA, determined by flow cytometry ( a ) and CLSM ( b ). c HMGB-1 release from tumour cells treated with OxPt/DHA, detected by ELISA. d – f Uptake of treated MC38 cells by bone-marrow-derived dendritic cells ( d ) and macrophages ( e ). f Priming of T-cell responses triggered by OxPt/DHA. MC38 tumour cells were treated with OxPt/DHA, and injected into the right footpads of C57BL/6 mice to determine the capacity of draining lymph node cells to produce IFN-γ in response to MC38 lysates. g , h In vivo anticancer vaccination of OxPt/DHA in immunocompetent C57BL/6 mice ( g ) and immunodeficient Rag 2−/− mice ( h ). The antitumour response was measured by immunizing mice with OxPt/DHA-treated tumour cells in one flank and challenging mice with untreated, live tumour cells in the opposite flank 7 days later. Data are expressed as means ± SD. One of three repetitions with similar results is shown here for ( a )−( e ). The result was obtained without repetition for ( f )−( h ) ( n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by Student’s two-tailed t test. CRT calreticulin, OxPt oxaliplatin, DHA dihydroartemisinin, CLSM confocal laser scanning microscopy

Article Snippet: CT26 cells seeded in six-well plates (2 × 10 5 cells/well) were cultured with free drugs or nanoparticles at a dose of 10 μM OxPt or/and 5 μM DHA for 24 h. The medium was collected for detection of HMGB-1 release by ELISA according to the manufacturer’s instructions (Chondrex, Redmond, WA).

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Derivative Assay, Injection, In Vivo, Two Tailed Test, Confocal Laser Scanning Microscopy