Journal: Frontiers in Immunology

Article Title: Head and neck dermatitis is exacerbated by Malassezia furfur colonization, skin barrier disruption, and immune dysregulation

doi: 10.3389/fimmu.2023.1114321

Figure Lengend Snippet: Immunohistochemistry of HND and non-HND facial skin specimens. (A) Representative histological image at 100x magnification (left) and 200x magnification (right). Quantified staining intensity of (B) SDF-1α, (C) IL-1β, (D) TGF-β, (E) TNF-α, and (F) VEGF. (Unpaired two-tailed t-test, SDF-1α; p = 0.0014, IL-1β; p = 0.0374, TGF-β; p = 0.0058, TNF-α; p = 0.0035, VEGF; p = 0.0002) HND, head and neck dermatitis; SDF-1 α , stromal cell-derived factor-1-alpha; IL-1 β , Interleukin-1-beta; TGF- β , transforming growth factor-beta; TNF- α , tumor necrosis factor-alpha; VEGF, vascular endothelial growth factor. * : p<0.05; ** : p<0.01.

Article Snippet: Immunohistochemical staining was performed using paraffin-embedded sections with antibodies against factor VIII-related antigen (1:100, ab236284, Abcam), stromal cell-derived factor-1-alpha (SDF1-α) (1:100, ab25117, Abcam, Cambridge, United Kingdom), Interleukin-1-beta (IL-1-β) (1:100, ab2105, Abcam), tumor necrosis factor-alpha (TNF-α) (1:50, ab1793, Abcam), transforming growth factor-beta (TGF-β) (1:100, ab66043, Abcam), and vascular endothelial growth factor (VEGF) (1:200, ab1316, Abcam).

Techniques: Immunohistochemistry, Staining, Two Tailed Test, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Head and neck dermatitis is exacerbated by Malassezia furfur colonization, skin barrier disruption, and immune dysregulation

doi: 10.3389/fimmu.2023.1114321

Figure Lengend Snippet: In vitro experiments showing (A) increased expression levels of VEGF, VEGFR, IL-31, and IL-33 in keratinocytes cultured with M. furfur , supplemented with (B) TSLP or (C) IL-4 . M. furfur induced increased expression levels of VEGFR, TGF-β, TNF-α, and IL-1β in (D) endothelial cells supplemented with IL-4. (three replicates for each column, Unpaired t-test, (B) VEGF; p = 0.0044, VEGFR; p = 0.0019, IL-31; p = 0.0145, IL-33; p = 0.0381, (C) VEGF; p = 0.1244, VEGFR; p = 0.0244, IL-31; p =0.0382, IL-33; p = 0.0117, (D) VEGFR; p-value 0.0266, TGF-β; p-value 0.0002, TNF-α; p-value 0.0009, IL-1β; p-value 0.0009). * : p<0.05; ** : p<0.01; *** : p<0.001; ns, Not Significant (p>0.05).

Article Snippet: Immunohistochemical staining was performed using paraffin-embedded sections with antibodies against factor VIII-related antigen (1:100, ab236284, Abcam), stromal cell-derived factor-1-alpha (SDF1-α) (1:100, ab25117, Abcam, Cambridge, United Kingdom), Interleukin-1-beta (IL-1-β) (1:100, ab2105, Abcam), tumor necrosis factor-alpha (TNF-α) (1:50, ab1793, Abcam), transforming growth factor-beta (TGF-β) (1:100, ab66043, Abcam), and vascular endothelial growth factor (VEGF) (1:200, ab1316, Abcam).

Techniques: In Vitro, Expressing, Cell Culture

Journal: Haematologica

Article Title: SDF1α-induced chemotaxis of JAK2-V617F-positive cells is dependent on Bruton tyrosine kinase and its downstream targets PI3K/AKT, PLCγ1 and RhoA

doi: 10.3324/haematol.2018.201921

Figure Lengend Snippet: Bruton tyrosine kinase mediates stromal cell-derived factor 1α-induced chemotaxis via activation of the small GTPase RhoA. (A, B) 32D JAK2-wild-type (WT) and JAK2-V617F (VF) cells were starved of serum for 2 h and left alone (n=3) or treated with dimethyl sulfoxide (DMSO) and inhibitors against Bruton tyrosine kinase (BTK) (2 mM of ibrutinib: IBR) and JAK2 (1 mM of ruxolitinib: RUX) as indicated for an additional 2 h (n=4) and chemotaxis assays were performed using transwells (pore size: 5 mm). Starvation medium (0.5% fetal bovine serum) containing stromal cell-derived factor 1 alpha (SDF1α: 100 ng/mL) served as the chemoattractant. (C) Active RhoA was determined by the G-LISA RhoA activation kit (Cytoskeleton #BK124) following the manufacturer’s instructions. 32D WT and VF cells were starved of serum for 4 h and left alone (left panel) (n=4) or treated with DMSO and 1 mM IBR as indicated (right panel) (n=4). After 1 h, the cells were stimulated with SDF1α (100 ng/mL) for an additional 2 h and whole cell lysates were analyzed for RhoA activation. (D) 32D VF cells were treated with 2 mM of Rho inhibitor (Rho Inh.I) for 4 h in serum starvation medium as indicated and chemotaxis assays were performed using transwells (pore size: 5 μm) (n=3). Starvation medium (0.5% fetal bovine serum) containing SDF1α (100 ng/mL) served as the chemoattractant. Columns represent mean ± standard error of mean from independent experiments. Statistical significance between different conditions was calculated using the Student t-test; *P<0.05, **P<0.01 and ***P<0.001.

Article Snippet: Starvation medium (0.5% fetal bovine serum) containing stromal cell-derived factor 1 alpha (SDF1α: 100 ng/mL) served as the chemoattractant. (C) Active RhoA was determined by the G-LISA RhoA activation kit (Cytoskeleton #BK124) following the manufacturer’s instructions.

Techniques: Derivative Assay, Chemotaxis Assay, Activation Assay

Journal: Haematologica

Article Title: SDF1α-induced chemotaxis of JAK2-V617F-positive cells is dependent on Bruton tyrosine kinase and its downstream targets PI3K/AKT, PLCγ1 and RhoA

doi: 10.3324/haematol.2018.201921

Figure Lengend Snippet: Stromal cell-derived factor 1 alpha-induced chemotaxis of primary granulocytes isolated from peripheral blood of JAK2-V617F-positive patients with myeloproliferative neoplasms could be impeded by a clinically relevant dose of ibrutinib. Granulocytes were isolated (Ficoll-paque density gradient centrifugation- erythrocyte lysis based method) from peripheral blood of healthy donors (▲: HD), and patients with untreated JAK2-V617F-positive (●: JAK2 V617F-MPN) (A) or CALR-mutated (■: CALR-MPN) (B) myeloproliferative neoplasia (MPN). The protocol was approved by the local ethics committee (protocol n. MD115/08), and all patients signed informed consent. The cells were rested for 30 min in starvation medium (0.5% fetal bovine serum) and left alone (A, left panel and B) or treated with dimethylsulfoxide (DMSO) or 0.5 mM of ibrutinib (IBR) (A, right panel) for an additional 1 h and chemotaxis assays were performed (pore size: 3 mm). Starvation medium (0.5% fetal bovine serum) containing stromal cell-derived factor 1 alpha (SDF1α: 100 ng/mL) served as the chemoattractant. Chemotaxis of IBR-treated cells was normalized to that of DMSO-treated cells. The horizontal lines indicate medians and the statistical significance of differences between control and treated samples was calculated by the Mann-Whitney test; *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

Article Snippet: Starvation medium (0.5% fetal bovine serum) containing stromal cell-derived factor 1 alpha (SDF1α: 100 ng/mL) served as the chemoattractant. (C) Active RhoA was determined by the G-LISA RhoA activation kit (Cytoskeleton #BK124) following the manufacturer’s instructions.

Techniques: Derivative Assay, Chemotaxis Assay, Isolation, Gradient Centrifugation, Lysis, MANN-WHITNEY