Journal: Molecular Medicine Reports

Article Title: Itgb3-integrin-deficient mice may not be a sufficient model for patients with Glanzmann thrombasthenia

doi: 10.3892/mmr.2021.12088

Figure Lengend Snippet: Spleen extramedullary hematopoiesis and iron deficiency of Itgb3 −/− mice. (A) Relative ratio of each organ to whole body weight in different genotype mice. *P<0.01 vs. Itgb3 +/+ mice; ✩ P<0.01 vs. Itgb3 +/− mice. (B) Relative ratio of spleen to whole body weight in Itgb3 −/− , Itgb3 +/− , Itgb3 +/+ and CHM mice. *P<0.01 vs. Itgb3 +/+ mice; ✩ P<0.01 vs. Itgb3 +/− mice. Immunohistochemical study of spleen biopsy from Itgb3 +/+ , Itgb3 −/− mice. Anti-CD3 antibody was used to label T lymphocytes, anti-CD19 antibody to label B lymphocytes and anti-CD71 antibody to label erythrocytes. Magnification (C) ×100 and (D) ×600. (E) Concentration of plasma ferritin in Itgb3 −/− mice. (F) OD value of fecal occult blood in Itgb3 −/− mice using ELISA. CHM, chronic hemorrhagic model; H&E, hematoxylin and eosin; Itgb3, integrin β3; OD, optical density; ns, not significant.

Article Snippet: Rabbit anti-CD3 monoclonal antibody (cat. no. MAB4841) was purchased from R&D Systems China Co., Ltd. and rat anti-transferrin R (CD71) monoclonal antibody (8D3; cat. no. NB 100-64979-0.05mg) was purchased from Novus Biologicals, LLC.

Techniques: Immunohistochemical staining, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

doi: 10.1101/2022.02.01.478701

Figure Lengend Snippet: (a&b) Neutrophils (4 × 10 5 /ml) from healthy donors were incubated with SARS-CoV-2 (MOI = 1) with or without autologous platelets (4 × 10 6 /ml) for 5 h at 37 °C. The scale bar is 10 μm. The detailed structure of SARS-CoV-2-induced NET formation was observed under a confocal microscope (Leica). NET formation was visualized by fluorescent staining of DNA (blue), histone (green), and MPO (red) (a) . NETs level was measured by MetaMorph software and presented as Cit-H3 area (mm 2 ) (b) . (c) Human neutrophils (4 × 10 5 /ml) were pretreated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 (MOI = 0.1 and 1) in the presence or absence of platelets (4 × 10 6 /ml) for 5 h and 20 h. The level of NET formation was determined by histone area (μm 2 ). (d) Neutrophils (4 × 10 5 /ml) from WT, clec5a -/- tlr2 -/- , and clec5a -/- tlr2 -/- mice were incubated with SARS-CoV-2 (MOI = 1) in the presence or absence of WT platelets (4 × 10 6 /ml) for 5 h at 37 °C. (e) Human neutrophils were pre-treated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 spike pseudotyped virus (MOI = 0.1) in the presence or absence of autologous platelets (4 × 10 6 /ml) for 3 h. Data are mean ± SEM and repeats of 3 to 5 independent experiments. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).

Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

Techniques: Incubation, Microscopy, Staining, Software, Virus

Journal: bioRxiv

Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

doi: 10.1101/2022.02.01.478701

Figure Lengend Snippet: (a) EVs from healthy controls (HCs-EVs, n=5) and COVID-19 patients (COVID19-EVs, n=5) were harvested by ultracentrifugation, then lysed in RIPA solution before subjected to mass spectrometry analysis. Proteins expressed in COVID-19 EVs, but not in HCs EVs, were further analyzed using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA) software. Proteins which were expressed in all the COVID19-EVs were displayed. (b&c) HCs-EVs (n=10) and COVID19-EVs (n=10) were analyzed by flow cytometry, and markers highly activated in COVID-19 platelets were expressed as a heat map (b) or by mean fluorescence intensity (c) . (d) Neutrophils were pre-incubated with anti-CLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or both anti-CLEC5A mAb (3E12A2, 100 μg/ml) and anti-TLR2 mAb (# MAB2616, 100 μg/ml), for 30 min at room temperature, followed by incubation with EVs (1 μg/ml) from COVID-19 patients (n=6) at 37°C for 3 h. Data are mean ± sd and repeats of at least three independent experiments. * p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t -test).

Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

Techniques: Mass Spectrometry, Software, Flow Cytometry, Fluorescence, Incubation

Journal: bioRxiv

Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

doi: 10.1101/2022.02.01.478701

Figure Lengend Snippet: C57BL/6 mice (WT) (n=3) and clec5a -/- tlr2 -/- mice (n=3)were inoculated with AAV-hACE2 for 14 days, followed by intranasal inoculation of SARS-CoV-2 (8 × 10 4 PFU/per mice). Tissues were collected at 3 days and 5 days post-infection. (a) The level of proinflammatory cytokines and chemokines were measured by real-time PCR and presented as fold change (compared to AAV-hACE2 uninfected mice/mock). ( b-d) NET structure and thrombus were detected by Hoechst. 33342 (blue), anti-MPO antibody (green), anti-citrullinated histone H3 (red), anti-CD42b antibody (yellow) (b) , and images were captured by a confocal microscope and subjected to determine the area of MPO (c) and CD42b (d) using MetaMorph TM software. (e) Cell infiltrated to lung. Interstitial macrophage (interstitial MΦ) was defined as CD11b + CD64 + F4/80 + cells; monocyte-derived dendritic cell (DC)/macrophage (MΦ) was defined as CD11b + CD64 + Ly6C + ; Ly6C + monocyte was defined as Ly6C + . The cell number of each cell population was calculated using the multiple fluorescent staining image and analyzed by software MetaMorph TM , and the data was presented as cell number/ per 664225 (815 × 815). Scale bar is 200 μm. Data are represented as mean ± SEM. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).

Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

Techniques: Infection, Real-time Polymerase Chain Reaction, Microscopy, Software, Derivative Assay, Staining

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of allogeneic T cells on arterial graft function at 1 week in vivo. Transplanted human arterial segments were recovered from mice injected with saline (open squares) or PBMCs (filled squares) 7–9 days before harvest (n = 5 pairs from four experiments). (A–D) Response curves. Restriction response to various concentrations of PGF2α (A) and relaxation response curves for nitroprusside (B), bradykinin (C), or substance P (D) after preconstriction with PGF2α. *P < 0.05; **P < 0.01; #P < 0.001 vs. saline control. mN, milliNewtons. (E) Immunohistochemistry of graft sections stained for human and murine (inset) CD31, human CD45, and HLA-DR. The staining for human CD45 is indistinguishable from that for human CD3 (data not shown). Original magnification, ×200.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: In Vivo, Injection, Saline, Control, Immunohistochemistry, Staining

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of allogeneic T cells on arterial graft function at 2 weeks in vivo. Transplanted human arterial segments were recovered from mice injected with saline (open squares) or PBMCs (filled squares) for 13–15 days (n = 4–7 pairs from four to six experiments). (A–D) Response curves. Constriction response to various concentrations of PGF2α (A) and relaxation response to bradykinin (B), nitroprusside (C), or YC-1 (D) after preconstriction with PGF2α. *P < 0.05; **P < 0.01; #P < 0.001 vs. saline control. (E) Immunohistochemistry of graft sections stained for human and murine (inset) CD31, human CD3, and iNOS. iNOS expression was highly localized to infiltrating T cells (arrows). Original magnification: CD31 and CD3 panels, ×200; iNOS panels, ×400.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: In Vivo, Injection, Saline, Control, Immunohistochemistry, Staining, Expressing

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of IFN-γ neutralization on T cell–mediated graft dysfunction in vivo. Transplanted human arterial segments were recovered from mice injected with saline alone (open squares) or PBMCs together with anti–IFN-γ mAb (filled triangles) or control mAb (IgG2a) (open triangles) for 2 weeks (n = 5 matched triplicates from three experiments). (A and B) Endothelium-independent responses to various concentrations of PGF2α (A) and nitroprusside (B). (C and D) Endothelium-dependent response to bradykinin before (C) and after (D) treatment with L-NAME. *P < 0.05; **P < 0.01; #P < 0.001 vs. PBMC + IgG2a group, n = 3–5. (E) Immunohistochemistry of graft sections stained for human CD3 and HLA-DR. The luminal HLA-DR–positive cells are also human CD31–positive (data not shown). Goat anti-mouse secondary Ab alone did not stain the grafts. Original magnification: CD3 panels, ×200; HLA-DR panels, ×400. (F and G) RNA transcript levels of iNOS (F) and eNOS (G) in whole tissue sections analyzed by quantitative RT-PCR.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: Neutralization, In Vivo, Injection, Saline, Control, Immunohistochemistry, Staining, Quantitative RT-PCR

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of TNF neutralization on T cell–mediated graft dysfunction in vivo. Transplanted human arterial segments were recovered from mice injected with saline alone (open squares) or PBMCs together with anti-TNF mAb (filled triangles) or control mAb (IgG1) (open triangles) for 2 weeks (n = 4 matched triplicates from two experiments). (A–C) Concentration-dependent responses to PGF2α (A), nitroprusside (B), and bradykinin (C). *P < 0.05; **P < 0.01; #P < 0.001 vs. PBMC + IgG1 group; n = 3–4. (D) Immunohistochemistry of graft sections stained for human CD3 and HLA-DR. Original magnification: CD3 panels, ×200; HLA-DR panels, ×400. (E and F) RNA transcript levels of iNOS (E) and eNOS (F) in whole tissue sections analyzed by quantitative RT-PCR.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: Neutralization, In Vivo, Injection, Saline, Control, Concentration Assay, Immunohistochemistry, Staining, Quantitative RT-PCR

Journal: mBio

Article Title: Reduction of the HIV-1 reservoir in T cells from people with HIV-1 on suppressive antiretroviral therapy using expanded natural killer cells

doi: 10.1128/mbio.02956-25

Figure Lengend Snippet: Expansion of NK cells from PLWH yields NK cells that express high levels of activating receptors. ( A and B ) NK cell phenotypic analysis by flow cytometry before and after expansion with irradiated C9.K562.mbIL21 cells. ( A ) Representative dot plots gated on CD3 − cells showing expression of NK cell markers CD56 and CD16 before and after (pre and post) expansion from representative HIV− donor and PLWH. ( B ) Frequency of CD56 + NK cells expressing NK cell markers before (blue box) and after expansion (red box) from HIV− donor (black symbols) and PLWH (green symbols). In total, n = 3 biological replicates per group. Significant differences after expansion were determined using paired t -tests, * P < 0.05, ** P < 0.01, and ns, non-significant.

Article Snippet: NK cells were isolated from cryopreserved PBMCs by negative selection using EasySep NK cell enrichment kits (StemCell technologies) or by T cell depletion using human CD3 MicroBeads (Miltenyi Biotec) and cultured in SCGM (CellGenix) supplemented with 10% FBS, normocin, and IL-2 (100–120 IU/mL) with γ-irradiated (100 Gy) aAPC (C9.K562-mbIL21 at a 2:1 ratio or NKF at a 5:1 ratio (aAPC:NK cells)).

Techniques: Flow Cytometry, Irradiation, Expressing

Journal: mBio

Article Title: Reduction of the HIV-1 reservoir in T cells from people with HIV-1 on suppressive antiretroviral therapy using expanded natural killer cells

doi: 10.1128/mbio.02956-25

Figure Lengend Snippet: eNK cells from PLWH reduce HIV release from autologous CD4+ T cells after latency reversal. ( A ) Schema of eNK killing assays detected by virus release. CD4+ T cells from ART-treated PLWH were isolated from PBMCs and activated with LRA for 24 h. In parallel, NK cells were enriched from PBMCs by CD3 depletion and expanded into eNK cells using NKF cells. Cocultures of activated CD4+ T cells were mixed with an equal number of autologous eNK cells and incubated for 7 days in growth media containing antiretroviral drugs. After the coculture, supernatants were collected to measure virus release (Donor 1). For Donors 2 and 3, eNK cells were removed at day 7 and incubated for an additional 7 days prior to collection of supernatants. ( B and C ) Virus release measurements in CD4+ T cells from PLWH. Supernatants from CD4+ T cells cultures alone or with eNK cells were collected to quantify HIV release by detecting HIV RNA in the supernatant using the Aptima HIV-1 Quant assay. After complete media change at day 5 (Donor 1) and day 8 (Donors 2–3), de novo virus release was detected at day 7 (Donor 1) and day 14 (Donors 2–3). ( B ) Virus release in CD4+ T cells from PLWH after eNK cell treatment. Bar graphs show HIV RNA copies/μL for unstimulated CD4+ T cells (Unstim) compared to stimulated CD4+ T cells alone (Stim) or with eNK cells (Stim+eNK) from each donor. ( C ) Percent virus release was normalized to stimulated CD4+ T cells (Stim) and compared to stimulated CD4+ T cells with eNK cells (Stim+eNK). CD4+ T cells with no stimulus (Unstim) were also tested as a control for HIV latency in PLWH samples; n = 3 biological replicates. Significant differences were determined using paired t -tests. *** P < 0.001. Donor 1 n = 4 replicates per sample, Donor 2 n = 9 replicates per sample, and Donor 3 n = 3 replicates per sample. Error bars represent STDEV.

Article Snippet: NK cells were isolated from cryopreserved PBMCs by negative selection using EasySep NK cell enrichment kits (StemCell technologies) or by T cell depletion using human CD3 MicroBeads (Miltenyi Biotec) and cultured in SCGM (CellGenix) supplemented with 10% FBS, normocin, and IL-2 (100–120 IU/mL) with γ-irradiated (100 Gy) aAPC (C9.K562-mbIL21 at a 2:1 ratio or NKF at a 5:1 ratio (aAPC:NK cells)).

Techniques: Virus, Isolation, Incubation, Control

Journal: JBMR Plus

Article Title: RNA ‐ seq Analysis of Peri‐Implant Tissue Shows Differences in Immune, Notch, Wnt, and Angiogenesis Pathways in Aged Versus Young Mice

doi: 10.1002/jbm4.10535

Figure Lengend Snippet: 3D immunofluorescence staining of the implant surface harvested from young and aged mice. ( A ) After 1 week post‐implantation, the implants were polished, fixed, and stained using alexa fluor plus 555 donkey anti‐mouse IgG secondary antibody to detect CD3 (green) and DAPI to label nuclei (blue) (10×, z stacks). The white dashed lines represent the outline of the implant. ( B ) Confocal imaging and image orientation. The red frames indicate the image region.

Article Snippet: Subsequently, the primary antibody, CD3 (NBP2‐43674, Novus Biologicals, Littleton, CO, USA), was separately diluted in 5% donkey serum (1:100) and incubated overnight at 4°C.

Techniques: Immunofluorescence, Staining, Imaging