Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: SH2 superbinder exhibited excellent security by targeting lung fibroblasts based on pY levels in vivo. A , Schematic showing progression of SH2 superbinder treatment in control mice. B , Percent survival during last 7 days after SH2 superbinder injection (n = 8). C , HE staining after SH2 superbinder challenge. Images showing the panoramic and partial view of lungs. D , Neutrophil accumulation measured by MPO assay in lung tissues after GST, GST-SH2 WT, GST-SH2 TrM or SH2-TrM treatment in saline groups (n = 6). E , Total cell counting in BALF (n = 6). F-G , Changes in different kinds of cells by Giemsa and Diff-quick staining in BALF (n = 6). H , Inflammatory cytokines in BALF measured by ELISA (n = 6). I , The determination of liver function index in serum (n = 6). J , Schematic showing progression of SH2 superbinder treatment in BLM-treated mice. K , Western blot of GST tag of different tissues in BLM group treated with GST-SH2 TrM. L , The process of FACS for mice lungs. M , Western blot of pY levels in different lung cells which were isolated by FACS of BLM treated 14 days mice. N , The fluorescence images of SH2 superbinder entering into activated fibroblasts, epithelial cells (EpCAM + ), leukocytes (CD45 + ) and endothelial cells (CD31 + ). O-R , Representative images of SH2 superbinder in myofibroblast (α-SMA + ) ( O ), epithelial cells (EpCAM + ) ( P ), leukocytes (CD45 + ) ( Q ) and endothelial cells (CD31 + ) ( R ) of BLM-treated mice.

Article Snippet: The amounts of IL-1β, IL-6, IL-10 and TNF-α in BALF of mice were measured by using mouse ELISA kits from R&D Systems (Minneapolis, MN, USA).

Techniques: In Vivo, Injection, Staining, MPO Assay, Saline, Cell Counting, Diff-Quik, Enzyme-linked Immunosorbent Assay, Western Blot, Isolation, Fluorescence

Journal: Stroke

Article Title: Phosphatidylinositol-3-Kinase Gamma Plays a Central Role in Blood–Brain Barrier Dysfunction in Acute Experimental Stroke

doi: 10.1161/strokeaha.110.601369

Figure Lengend Snippet: Figure 5. PI3K deficiency reduces neutrophil infiltration and neutrophil-associated MMP-9. A–B, Immunostaining for neutrophils (A) and MMP-9 (B) in the ischemic cortex in WT and PI3K KO mice at the indicated times after tMCAO. The number of the cells positively stained for neutrophils and MMP-9 was calculated in the 3 predefined regions of interest shown in adjoining coronal section. Bar50 m. n5 per time point per group; *P0.05 vs WT control. C, Neutrophil infiltration was quantified by myeloperoxidase (MPO) assay. Enzyme-linked immunosorbent assay (ELISA) assay of MPO protein in the ischemic cortex (region of interest shown in adjoining coronal section) 24 hours after tMCAO. n5 for each group. *P0.05 vs WT control. In A–C, samples from sham-operated animals served as controls (Ctrl). D, Double immunostaining showing the colocalization of MMP-9 with the specific neutrophil marker (PMN), but not with microglial (Iba-1), astrocytic (GFAP), or neuronal (NeuN) markers in the ischemic cortex at 24 hours after tMCAO. Bar50 m. PI3K indicates phosphatidylinositol-3- kinase-gamma; MMP-9, metallopeptidase-9; WT, wild-type; KO, knockout; tMCAO, transient occlusion of the middle cerebral artery; Iba-1, ionized calcium-binding adapter molecule 1; GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclei.

Article Snippet: Enzyme-Linked Immunosorbent Assays Enzyme-linked immunosorbent assays of brain cortice homogenates were performed to detect total MMP-9 protein using a Quantikine mouse MMP-9 (total) immunoassay kit (R&D Systems), myeloperoxidase using the mouse Enzyme-linked immunosorbent assay kit (HK210; Hycult Biotechnology), and malondialdehyde (MDA) using the NWLSS MDA assay kit (Northwest Life Science Specialties, LLC) according to the instructions by the manufacturers.

Techniques: Immunostaining, Staining, Control, MPO Assay, Enzyme-linked Immunosorbent Assay, Double Immunostaining, Marker, Knock-Out, Binding Assay

Journal: Stroke

Article Title: Phosphatidylinositol-3-Kinase Gamma Plays a Central Role in Blood–Brain Barrier Dysfunction in Acute Experimental Stroke

doi: 10.1161/strokeaha.110.601369

Figure Lengend Snippet: Figure 6. PI3K deficiency reduces MMP-9 activity and protein content. A, Gelatin zymogram. Note that MMP-9 (92 and 82 kDa) and MMP-2 (63 kDa) bands were detected in the homogenates of ischemic (left [L]) and contralateral (right [R]) cortex 24 hours after tMCAO. The region of interest is shown in adjoining coronal section. B, Densitometric analysis of the bands shown in A. Data represent meanSD from 3 separate experiments. *P0.05 vs WT (saline-treated) control. C, Enzyme-linked immunosorbent assay (ELISA) assay of MMP-9 protein in ischemic (left) and contralateral (right) cortex (region of interest shown in adjoining coronal section) 24 hours after tMCAO. n5 for each group. **P0.01 vs WT control. PI3K indicates phosphatidylinositol-3-kinase-gamma; MMP-9, metallopeptidase-9; tMCAO, transient occlusion of the middle cerebral artery; WT, wild-type.

Article Snippet: Enzyme-Linked Immunosorbent Assays Enzyme-linked immunosorbent assays of brain cortice homogenates were performed to detect total MMP-9 protein using a Quantikine mouse MMP-9 (total) immunoassay kit (R&D Systems), myeloperoxidase using the mouse Enzyme-linked immunosorbent assay kit (HK210; Hycult Biotechnology), and malondialdehyde (MDA) using the NWLSS MDA assay kit (Northwest Life Science Specialties, LLC) according to the instructions by the manufacturers.

Techniques: Activity Assay, Saline, Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Neutrophils remain detrimentally active in hydroxyurea-treated patients with sickle cell disease

doi: 10.1371/journal.pone.0226583

Figure Lengend Snippet: (A) Purified and rested neutrophils from healthy donors (H) and SCD patients in steady state (SS) were left untreated in RPMI as control or treated with 20 μM hemin. Median Fluorescence Intensity of F-Actin (upper panel) and CD63 (lower panel) in F-Actin/CD63 double positive neutrophils with normal multi-lobulated nuclei is shown. (Healthy N = 7; SCD N = 6). (B) Z-stack stills of SCD neutrophils left in RPMI for 30 minutes showing neutrophils with roughed cell membranes (yellow arrows, upper panel). Staining for DNA (blue), F-Actin (green) and CD63 (red) is presented (lower panel). (C) MPO release was assayed by ELISA following 30 minutes incubation with or without the hemin stimulus (H (Healthy) N = 4; SS (SCD) N = 4). Degranulation response was assayed in terms of MPO ng/ml plasma (upper panel), or fold increase over healthy neutrophils (lower panel). Data presented as dot plots±S.D., significance calculated with an unpaired Mann-Whitney test.

Article Snippet: The supernatants were then assayed with a Human Myeloperoxidase Quantikine ELISA Kit from R&D Systems according to the manufacturer’s instruction.

Techniques: Purification, Control, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, Incubation, Clinical Proteomics, MANN-WHITNEY

Journal: Chinese Medicine

Article Title: Baitouweng decoction modulates gut microbial production of indole-3-propionic acid and epithelial necroptosis to alleviate DSS-induced colitis in mice

doi: 10.1186/s13020-025-01143-9

Figure Lengend Snippet: BD relieves the inflammation in colitis mice. A – D Concentration of MPO ( A ), IL-6 ( B ), TNF-α ( C ), and CXCL1 ( D ) in the serum (n = 5). E The heatmap of the relative mRNA expression analysis of the inflammatory cytokines Il1β , Il6 , Nos2 , Il17 , Ccl2 , Tnfa , and Mpo in the colon tissue of mice. β-actin was used for normalization and the value is expressed as fold changes compared to the Ctrl group (n = 7). F Concentration of IL-1β, IL-6, TNF-α, and MPO in the colonic tissue mice (n=6). Data are expressed as Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA with Tukey’s post hoc analysis

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse IL-1β (#EK201B), IL-6 (#EK206), TNF-α (#EK282), MPO (#EK2133), and CXCL1 (#EK296) were purchased from MULTI SCIENCES (Hangzhou, China).

Techniques: Concentration Assay, Expressing

Journal: Chinese Medicine

Article Title: Baitouweng decoction modulates gut microbial production of indole-3-propionic acid and epithelial necroptosis to alleviate DSS-induced colitis in mice

doi: 10.1186/s13020-025-01143-9

Figure Lengend Snippet: The protective effect of BD depends on the gut microbiota. A Schematic illustration of the experimental design of antibiotics-mediated gut microbiota depletion. Mice received DSS in drinking water for 7 days to induce colitis. BD treatment groups were orally administered with BD (6.75 g/kg) once daily from day 14 to day 21. B Body weight change of mice during colitis progression. The weight change was expressed as a percent of the initial body weight (n = 6–8). C Colon length of mice (n = 5). D Representative H&E staining images of the colon. Scale bar: 50 μm. E Concentration of IL-1β, IL-6, TNF-α, and MPO in the colon tissue (n = 5). F Schematic illustration of the experimental design of Fecal microbiota transplantation. G Body weight change of mice during colitis progression. The weight change was expressed as a percent of the initial body weight (n = 8). H Colon length of mice (n = 6–7). I Representative H&E staining images of the colon. Scale bar: 50 μm. J Concentration of IL-1β, IL-6, TNF-α, and MPO in the colon tissue (n = 5). Data are expressed as Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA with Tukey’s post hoc analysis

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse IL-1β (#EK201B), IL-6 (#EK206), TNF-α (#EK282), MPO (#EK2133), and CXCL1 (#EK296) were purchased from MULTI SCIENCES (Hangzhou, China).

Techniques: Staining, Concentration Assay, Transplantation Assay

Journal: Chinese Medicine

Article Title: Baitouweng decoction modulates gut microbial production of indole-3-propionic acid and epithelial necroptosis to alleviate DSS-induced colitis in mice

doi: 10.1186/s13020-025-01143-9

Figure Lengend Snippet: IPA alleviates DSS-induced colitis in mice. A Schematic illustration of the experimental design. Mice received DSS in drinking water for 7 days to induce colitis. B Body weight change of mice during colitis progression (n = 6–8). The weight change was expressed as a percent of the initial body weight. C The disease active index score on day 10 (n = 6–7). D Colon length of mice. E Representative H&E staining images of the colon . Scale bar: 50 μm. F Intestinal leakage was measured by FITC-Dextran concentration in serum (n = 4). G Relative mRNA expression analysis of Il1β , Il6 , Tnfa , and Mpo in the colon tissue of mice (n = 6). β-actin was used for normalization and the value is expressed as fold changes compared to the Ctrl group. H The concentration of IL-1β, IL-6, TNF-α, and MPO in the serum (n = 6). Data are expressed as Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA with Tukey’s post hoc analysis

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for mouse IL-1β (#EK201B), IL-6 (#EK206), TNF-α (#EK282), MPO (#EK2133), and CXCL1 (#EK296) were purchased from MULTI SCIENCES (Hangzhou, China).

Techniques: Staining, Concentration Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Didymin Suppresses Microglia Pyroptosis and Neuroinflammation Through the Asc/Caspase-1/GSDMD Pathway Following Experimental Intracerebral Hemorrhage

doi: 10.3389/fimmu.2022.810582

Figure Lengend Snippet: Didymin ameliorated microglial pyroptotic molecules and inflammatory cytokines after ICH. (A) Representative Western blot bands and densitometric quantification of Rkip, Asc, Nlrp3, Caspase-1, and GSDMD. n = 6 for each group. * p < 0.05 vs. sham; # p < 0.05 vs. ICH+vehicle. (B) IL-1β, TNF-α, and MPO production were detected by ELISA assay kit. n = 6 for each group. * p < 0.05 vs. sham; # p < 0.05 vs. ICH+vehicle. (C) Caspase-1/TMEM119 double immunofluorescence staining and quantitative analysis of Caspase-1-positive microglia. Scale bar = 50 μm. n = 3 for each group. * p < 0.05 vs. sham; # p < 0.05 vs. ICH+vehicle.

Article Snippet: A Mouse DuoSet ELISA Kit to IL-1β, TNF-α, and MPO was purchased from R&D Systems and performed as instructed by the manufacturer.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Double Immunofluorescence Staining

Journal: Frontiers in Immunology

Article Title: Didymin Suppresses Microglia Pyroptosis and Neuroinflammation Through the Asc/Caspase-1/GSDMD Pathway Following Experimental Intracerebral Hemorrhage

doi: 10.3389/fimmu.2022.810582

Figure Lengend Snippet: Locostatin abolished the neuroprotective effects of Didymin. (A–C) Neurological deficits, brain water content, and EB extravasation were tested at 24 h after ICH. (D, E) Representative Western blot bands and densitometric quantification of Asc, Nlrp3, Caspase-1, and GSDMD. n = 6 for each group. * p < 0.05 vs. sham; # p < 0.05 vs. ICH+vehicle. & p < 0.05 vs. ICH+Didymin. (F) IL-1β, TNF-α, and MPO production was detected by ELISA assay kit. n = 6 for each group. * p < 0.05 vs.sham; # p < 0.05 vs. ICH+vehicle. & p < 0.05 vs. ICH+Didymin.

Article Snippet: A Mouse DuoSet ELISA Kit to IL-1β, TNF-α, and MPO was purchased from R&D Systems and performed as instructed by the manufacturer.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Didymin Suppresses Microglia Pyroptosis and Neuroinflammation Through the Asc/Caspase-1/GSDMD Pathway Following Experimental Intracerebral Hemorrhage

doi: 10.3389/fimmu.2022.810582

Figure Lengend Snippet: VX-765 ameliorated microglial pyroptosis and brain injury after Locostatin-treated ICH. (A–D) Neurological deficits, brain water content, EB extravasation, and hematoma size were tested at 24 h after ICH. (E) Representative Western blot bands and densitometric quantification of Asc, Nlrp3, Caspase-1, and GSDMD. n = 6 for each group. * p < 0.05 vs. sham; # p < 0.05 vs. ICH+vehicle. (F) IL-1β, TNF-α, and MPO production was tested by ELISA assay. n = 6 for each group. * p < 0.05 vs. sham; # p < 0.05 vs. ICH+vehicle. (G) GSDMD/TMEM119 double immunofluorescence staining and quantitative analysis of GSDMD-positive microglia. Scale bar = 50 μm. n = 3 for each group. * p < 0.05 vs. sham; # p < 0.05 vs. ICH+vehicle.

Article Snippet: A Mouse DuoSet ELISA Kit to IL-1β, TNF-α, and MPO was purchased from R&D Systems and performed as instructed by the manufacturer.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Double Immunofluorescence Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pterostilbene 4′- β -Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1

doi: 10.1155/2018/2747018

Figure Lengend Snippet: 4-PG and PTER exhibit therapeutic effects in LPS- or P. aeruginosa -induced acute lung injury. (a–g) C57BL/6 mice were challenged with LPS (2.5 mg/kg) by intranasal exposure for 24 h. 4-PG (10 mg/kg per day, i.p.) and PTER (10 mg/kg per day, i.p.) were administered 5 h after LPS challenge. (b) Representative lung histology was shown by hematoxylin and eosin- (H&E-) stained lung sections from six experimental groups (left). Quantitative analysis of histologic lung section by lung injury score for six experimental groups. The score generates the average of two independent investigators (right). (c and d) BAL fluid and lung tissues were harvested after treatment with LPS for 24 h and then (c) analyzed for total cells in BAL fluid. (d) Neutrophil infiltration into lung tissues was measured by activity of myeloperoxidase (MPO) in lung tissues. (e and f) The secreted levels of TNF- α and IL-6 in BAL fluid were analyzed by ELISA, respectively. (g) The mRNA levels of TNF- α , IL-6, IL-1 β , CXCL1, and CXCL2 in lung tissues were detected by RT-PCR. (h–m) C57BL/6 mice were instilled P. aeruginosa (1 × 10 7 CFU/mouse) by intranasal exposure for 24 h. After instillation of P. aeruginosa , mice were post-treated with 4-PG (10 mg/kg, i.p.) at 6, 12, and 18 h, respectively. After 24 h, BAL fluid and lung tissues were collected and then (i) analyzed for total cells in BAL fluid. (j) Neutrophil infiltration into lung tissues was analyzed by MPO in lung tissues. (k and l) The levels of TNF- α and IL-6 in BAL fluid were analyzed by ELISA, respectively. (m) The wet-to-dry weight ratio of whole lungs was determined on 24 h after the stimulation of P. aeruginosa . Data were expressed as means ± SD, n = 3. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: To measure neutrophil infiltration into lung tissues, MPO enzyme activity in lung tissues was measured using the Mouse Myeloperoxidase DuoSet kit (R&D Systems, Minneapolis, MN).

Techniques: Staining, Activity Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Phytomedicine

Article Title: Re-Du-Ning injection ameliorates LPS-induced lung injury through inhibiting neutrophil extracellular traps formation

doi: 10.1016/j.phymed.2021.153635

Figure Lengend Snippet: RDN reduced LPS-induced cytokine secretion. Cytokine levels in BALF (A) and serum (B) were measured by using ELISA. Values are shown as the mean ± SEM of 3 mice. (C) The cytokine mRNA expression was measured in lung homogenates by using qPCR. Data are expressed as the mean ± SEM of 6 mice. * p < 0.05, ** p < 0.01 vs . the LPS group.

Article Snippet: ELISA kits for mouse IL-1β, IL-6 and TNF-α were purchased from Dakewei (Beijing, China); Human MPO ELISA kit was bought from Lianke (Hangzhou, China); Mouse NE and MPO kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Phytomedicine

Article Title: Re-Du-Ning injection ameliorates LPS-induced lung injury through inhibiting neutrophil extracellular traps formation

doi: 10.1016/j.phymed.2021.153635

Figure Lengend Snippet: RDN reduced the release of neutrophil extracellular traps. (A) Mice were intraperitoneally administered by Cl-amidine (20 mg/kg) and GSK484 (4 mg/kg) for day 0. The animals were euthanized 24 h after LPS instillation. H&E staining of lung sections from each group, respectively. Scale bar, 100 μm. Concentration of MPO (B) and NE (C) in BALF, serum and lung tissue were determined by ELISA. (D) Immunofluorescence staining of lung tissue sections from mice was performed, with imaging for cit H3 (green) and myeloperoxidase (MPO; red) and staining with DAPI for DNA-blue. Scale bar, 50 μm. (E) Immunoblot analysis of the PAD4 protein levels from lung tissues of each group. Data are expressed as the mean ± SEM of 5-6 mice. * p < 0.05, ** p < 0.01 vs . the LPS group.

Article Snippet: ELISA kits for mouse IL-1β, IL-6 and TNF-α were purchased from Dakewei (Beijing, China); Human MPO ELISA kit was bought from Lianke (Hangzhou, China); Mouse NE and MPO kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Imaging, Western Blot

Journal: Phytomedicine

Article Title: Re-Du-Ning injection ameliorates LPS-induced lung injury through inhibiting neutrophil extracellular traps formation

doi: 10.1016/j.phymed.2021.153635

Figure Lengend Snippet: RDN injection reduced MAPK pathway in vitro . (A) Human neutrophils were stimulated with PMA (50 nM) for 3 hours. After 3 h, supernatants were collected (B) and the level of MPO was measured by using ELISA. (C) Western blot analysis of ERK 1/2, ERK 1/2 phosphorylation and PAD4. (D) The quantitative results of Western blot were shown. Data are shown as the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 vs . the PMA group.

Article Snippet: ELISA kits for mouse IL-1β, IL-6 and TNF-α were purchased from Dakewei (Beijing, China); Human MPO ELISA kit was bought from Lianke (Hangzhou, China); Mouse NE and MPO kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Injection, In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics

Journal: Mediators of Inflammation

Article Title: Lipopolysaccharide-Binding Protein Downregulates Fractalkine through Activation of p38 MAPK and NF- κ B

doi: 10.1155/2017/9734837

Figure Lengend Snippet: SB203580 and SC-514 ameliorated LPS-induced lung injury and inflammation. Hematoxylin and eosin staining was used to determine the LPS-induced lung injury and inflammatory response in the lung tissue of the ARDS rat model. (a) The control group rats (A) had a distinct framework with complete alveolar walls and interstitium without exfiltration. The LPS group rats (B) showed edema, neutrophil infiltration, hemorrhage, moderate bronchiole epithelial desquamation, and minimal hyaline membrane formation. Histotological analysis also show that rats receiving LBPK95A, SB203508, or SC514 injection had substantially less inflammatory cell infiltration, edema, hemorrhage, and thickness of alveolar walls in comparison to rats treated with LPS ((C), (D), and (E), resp.). The photomicrographs are at 400x magnification. ((b) and (c)) The total number of cells, neutrophil number, and neutrophil ratio in BALF were higher in the LPS group than that in the control group and lower in the LPS+LBPK95A, LPS+SB, and LPS+SC groups than that in the LPS group ( p < 0.05, n = 10, in all cases). The MPO of lung tissues and BALF was determined by ELISA. The results show that the level of MPO concentration in the LPS group was significantly higher than that in the control group ( p < 0.05). (d) However, the MPO concentration level in the LPS+LBPK95A, LPS+SB, and LPS+SC groups was significantly lower than that in the LPS group ( p < 0.05 in all cases). (e) The LPS group had a significantly higher W/D ratio than the control group, and the W/D ratio in the LPS+LBPK95A, LPS+SB, and LPS+SC groups were significantly lower than that in the LPS group ( p < 0.05 in all cases). The LPS group had a lower PaO 2 /FiO 2 ratio than the control group. (f) The LPS+LBPK95A, LPS+SB, and LPS+SC groups had higher PaO 2 /FiO 2 ratios than the LPS group ( p < 0.05 in all cases). ∗ represents p < 0.05 when compared with that in the CTL group of rats; # represents p < 0.05 when compared with that in the LPS group of rats; △ represents p < 0.05 when compared with that in the LPS+LBPK95A group of rats ( n = 10).

Article Snippet: The levels of myeloperoxidase activity were measured by the rat MPO ELISA kit (HK105-01, Hycult Biotech, USA) according to the manufacturer's instructions.

Techniques: Staining, Injection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Nature Communications

Article Title: The role of platelets in mediating a response to human influenza infection

doi: 10.1038/s41467-019-09607-x

Figure Lengend Snippet: Characterization of human blood from influenza-infected patients. a – d Blood from influenza-infected patients was fixed after intravenous collection and stained as described in Methods. In all cases DNA was assessed by DAPI. a Many platelets appeared similar to those observed in control blood from healthy donors with a size of 2–5 µm. b Some platelets from influenza-infected patients had undergone spreading with a distinctive distribution of CD41. c Platelets and platelet microparticles associated with DNA (arrow). d Spread platelets were found surrounding released DNA with distinct DNA content in their center. Of note, blood from influenza-infected patients and controls in ( a – d ) was not permeabilized. Since formaldehyde can cause certain levels of permeabilization as a function of cross-linking positive staining in control samples for H4 and MPO do not necessarily indicate activation. e – g Levels of proteins related to DNA release in the plasma of influenza-infected patients assessed by ELISA. Source data are provided as a file. e neutrophil elastase, f myeloperoxidase (MPO), and g histone nucleosome core. The graphs represent the average ± SD of healthy donors ( n = 15) and influenza-infected patients ( n = 18); significance for ( e – g ) was assessed by Mann–Whitney U test, star symbol (*) indicates p < 0.0001. h , i To synchronize time of influenza presence as a function of infection and quantify the released DNA, we treated blood from human donors for 30 min with sucrose-purified infectious influenza (WSN/33) at constant rotation and 37 °C. Influenza was used at 1 pfu to 100 platelets. h Representative images of blood from 3 donors with influenza and one (out of 3) healthy control and i their quantitation. Of note, in certain cases the DNA is not entirely covered with platelets, suggesting differences in kinetics of interaction and/or a physiological relationship that needs further in vivo characterization. Data in graph is represented as average ± SD of n = 3 different donors; significance was assessed by unpaired t -test (two-tailed value) and star symbol (*) indicates p = 0.0019, df = 4

Article Snippet: 1 μL of supernatant collected after cell mixing treatment was assayed for C3 (Abcam, cat# 108823); human plasma diluted 800× was assayed for C3 (Abcam, cat# 108822); murine plasma was diluted 50,000× (Abcam, cat# 157711); human plasma diluted 100× was assayed for Neutrophil Elastase (Abcam, cat# ab119553), MPO (Abcam, cat# ab119605), and histone nucleosome core (NovusBio cat# KA1091).

Techniques: Infection, Staining, Activation Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Purification, Quantitation Assay, In Vivo, Two Tailed Test

Journal: Nature Communications

Article Title: The role of platelets in mediating a response to human influenza infection

doi: 10.1038/s41467-019-09607-x

Figure Lengend Snippet: Platelet-TLR7mediates release of MPO from neutrophils. Isolated human platelets and neutrophils were incubated together or by themselves for 30 min, at 37 °C and constant rotation, in the presence of TLR agonists [TLR7 (Loxo)—1 mM; TLR2 (Pam)—10 μg/mL] or thrombin (IIa)—0.05 U/mL. Myeloperoxidase (MPO) release from neutrophils was measured in the a absence ( p = 0.7206, F = 0.4493, df = 3) and b presence of platelets by ELISA ( p = 0.0002, F = 10.4, df = 3). The graphs represent the average fold change for each individual of n = 6 (3F; 3M) ± SD. Source data are provided as a file. Significance was measured by ANOVA followed by Bonferroni follow-up test; in all cases star symbol (*) indicates p < 0.05

Article Snippet: 1 μL of supernatant collected after cell mixing treatment was assayed for C3 (Abcam, cat# 108823); human plasma diluted 800× was assayed for C3 (Abcam, cat# 108822); murine plasma was diluted 50,000× (Abcam, cat# 157711); human plasma diluted 100× was assayed for Neutrophil Elastase (Abcam, cat# ab119553), MPO (Abcam, cat# ab119605), and histone nucleosome core (NovusBio cat# KA1091).

Techniques: Isolation, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Pathogens and Disease

Article Title: Impaired cytokine expression, neutrophil infiltration and bacterial clearance in response to urinary tract infection in diabetic mice

doi: 10.1093/femspd/ftv002

Figure Lengend Snippet: Proinflammatory cytokine response to UPEC in the bladder. (a) qRT-PCR. Bladders were harvested from 4-week diabetic mice and age-matched controls at the indicated time points following UPEC inoculation (n = 5 per group). Total RNA was isolated, cDNA was synthesized, and MIP-2, KC, MCP-1 and IL-6 mRNA levels were quantified by qRT- PCR as described in the section ‘Materials and Methods’. Using the comparative CT method, chemokine threshold cycles (CT) were normalized to the corresponding β-actin CT values, and expression levels were calculated relative to the average normalized values in uninoculated (time 0), non-diabetic control mice (set at 1.0). Each bladder sample was assayed in triplicate. qRT- PCR data are expressed as means ± SEM. (b) ELISA. Bladders were harvested from 4-week diabetic mice and age-matched controls at the indicated time points following UPEC inoculation (n = 10 per group). Bladders were homogenized, and chemokine levels were quantified by ELISA. MIP-2, KC, MCP-1 and IL-6 MPO levels were calculated from the corresponding standard curves as pg/mg protein. Each bladder sample was assayed in quadruplicate. ELISA data are expressed as means ± 95% CI. Statistical analyses of qRT-PCR results and ELISA results in DM compared with control mice at each time point were performed by multiple Student's t tests with corrections for multiple comparisons by the Holm–Sidak method (‡P < 0.02, †P < 0.005, §P < 0.001 and *P < 0.0001).

Article Snippet: Bladders were homogenized for 3 min on ice using PowerGen 125 homogenizer (Fisher Scientific, Waltham, MA, USA), and urine and bladder tissue neutrophils were quantified using a MPO ELISA kit from Hycult Biotech (Plymouth Meeting, PA) as described by Haraoka et al. ( 1999 ) and the manufacturer's protocol. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 Proinflammatory cytokine response to UPEC in the bladder. ( a ) qRT-PCR.

Techniques: Quantitative RT-PCR, Isolation, Synthesized, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Pathogens and Disease

Article Title: Impaired cytokine expression, neutrophil infiltration and bacterial clearance in response to urinary tract infection in diabetic mice

doi: 10.1093/femspd/ftv002

Figure Lengend Snippet: Influx of neutrophils into the urine, with confirmatory MPO kinetics in the urine and bladder. Urine was collected aseptically and bladders were harvested from diabetic and control mice at the indicated times after UPEC inoculation. (a) Urine was stained with Turk's solution and neutrophils were counted using a hemacytometer. The initial neutrophil response was markedly attenuated in diabetic mice compared with controls (P < 0.0001 by Student's t test comparison of AUC values over the 3–12 h period in diabetic vs control mice). (b) Results of ELISA quantification of the neutrophil marker MPO in urine, shown as ng MPO per ml of urine. P < 0.0001 by Student's t test comparison of AUC values over the 3–12 h period after UPEC challenge in diabetic vs control mice comparing DM with control at the indicated times. (c) ELISA analysis of MPO concentrations in bladder homogenates, shown as concentrations of MPO per mg total protein. *P < 0.0001 by multiple Student's t tests corrected for multiple comparisons by the Holm–Sidak method. Data in (a)–(c) are expressed as means ± 95% CI.

Article Snippet: Bladders were homogenized for 3 min on ice using PowerGen 125 homogenizer (Fisher Scientific, Waltham, MA, USA), and urine and bladder tissue neutrophils were quantified using a MPO ELISA kit from Hycult Biotech (Plymouth Meeting, PA) as described by Haraoka et al. ( 1999 ) and the manufacturer's protocol. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 Proinflammatory cytokine response to UPEC in the bladder. ( a ) qRT-PCR.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Marker

Journal: Frontiers in Immunology

Article Title: Enhanced Neutrophil Immune Homeostasis Due to Deletion of PHLPP

doi: 10.3389/fimmu.2019.02127

Figure Lengend Snippet: Enhanced neutrophil activation due to PHLPP deficiency. (A) Representative images of immunohistological staining of neutrophils (LY6G+) in colon tissues of DSS-treated WT and PHLPP −/− mice on day 0, 3, and 6 during the process of experimental colitis. (B) Colon tissues were collected on day 0, 3, and 6 cultured overnight. Supernatant was collected for detection of MPO by ELISA. (C) Flow cytometry analysis of peripheral neutrophils (gated on CD11B+ LY6G+) harvested on day 0, 3, and 6 during the process of experimental colitis. (D) FPR2 expression on peripheral neutrophils (gated on LY6G+ CD11B+) from WT and PHLPP −/− mice on day 0, 3, and 6 post DSS challenges via flow cytometry. (E) CXCR2 expression on peripheral neutrophils (gated on LY6G + CD11B + ) from DSS-treated mice on day 0, 3, and 6 post DSS challenges via flow cytometry. For mouse number in each group, n ≥ 4. * p < 0.05, ** p < 0.01.

Article Snippet: Myeloperoxidase (MPO) levels of colon culture supernatants and plasma were assessed using mouse MPO ELISA Kit from R&D system.

Techniques: Activation Assay, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing