Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The neonatal Fc receptor (FcRn) and complement fixation facilitate prophylactic vaccine-mediated humoral protection against viral infection in the ocular mucosa

doi: 10.4049/jimmunol.1700316

Figure Lengend Snippet: (A) Representative confocal images centered above apparent viral lesions at 40× magnification showing C3d (blue) and HSV-1 antigen (red) in healthy or HSV-1 infected corneas at 48 hours PI with phalloidin (green) staining to delineate cell boundaries. “Naive” reflects HSV-infected immunologically naive mice. “0ΔNLS Vacc.” reflects mice that were immunized in the footpad, boosted in the flank with HSV-1 0ΔNLS, and infected 30 days later with 1000 PFU HSV-1 McKrae in the eye. (B) Individual z-slices of representative composite image shown in (A) with colocalization of HSV-1 and C3d calculated using Imaris displayed as white overlay. Images are representative of 4–6 corneas per group. (C) Flow cytometry was utilized to characterize various leukocyte populations in the corneas of immunologically naive and HSV-1 0ΔNLS-vaccinated mice immunized at 48 hours post infection. Populations represented include total CD45+ leukocytes, CD11b+ myeloid cells, Ly6G+ neutrophils, Ly6C+ monocytes, F4/80+ macrophages, CD45+ EpCAM+ Langerhans cells, CD11c+ dendritic cells, CD3− NK1.1+ NK cells, and CD4+ T cells. Data reflect mean ± SEM for 7–8 mice per group; 3 independent experiments. Differences were determined by Student’s T test. Panels (A), (B), and (C) reflect CD-1 mice. Given the evidence of complement activation and presence of ADCC effector cells in the corneas of vaccinated CD-1 mice, C57BL/6 mice were subsequently utilized to assess viral clearance/shedding and the effector mechanism of humoral protection in the tear film of naive and HSV-1 0ΔNLS-vaccinated WT and C3−/− mice (D) or WT and FcγRIII−/− mice (E); data reflect 5 mice per group; 2 independent experiments. Differences were determined by two-way ANOVA with Bonferroni’s multiple comparisons tests.

Article Snippet: Three-dimensional protein fluorescence colocalization analysis was performed using Imaris software (Bitplane USA, Concord, MA).

Techniques: Infection, Staining, Flow Cytometry, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The neonatal Fc receptor (FcRn) and complement fixation facilitate prophylactic vaccine-mediated humoral protection against viral infection in the ocular mucosa

doi: 10.4049/jimmunol.1700316

Figure Lengend Snippet: (A) Representative confocal images of FcRn (green) labeling in the epithelium of healthy uninfected and HSV-1 infected corneas at 48 hours PI. Images were captured at 40× magnification, scale bar = 50 μm. DAPI (blue) and phalloidin (red) staining were utilized to identify tissue boundaries and borders. (B) Representative low magnification confocal images showing the radial patterning of FcRn expression (cyan) in the healthy corneal epithelium (peripheral expression > central) and its widespread elevated expression in mock infected (scratch control) or HSV-1 infected corneas. (C) FcRn expression (cyan) in the deeper stromal keratocyte layer is also upregulated following corneal injury or infection. (D) Colocalization of FcRn (cyan) and HSV-1 antigen (red) was observed within the corneal epithelium of naive CD-1 mice and digitally confirmed using Imaris software to verify pixel overlap (white overlay, bottom right). Images shown in panels (B), (C), and (D) are representative of 4–6 labeled corneas per group and were captured at 20× magnification, scale bars = 100 μm. (E) Western blot confirmation of FcRn antibody specificity showing the p51 subunit of FcRn in digests from healthy and HSV-1 infected corneal digests (48 hours PI). Actin labeling was used as a loading control with 20 μg of total protein input per sample. (F) Gating strategy for FcRn analysis by flow cytometry including scatter profile for doublet discrimination, selection of cell subsets (EpCAM+ CD45− epithelial cells, EpCAM+ CD45+ Langerhans cells, or EpCAM− CD45+ leukocytes), and downstream quantification of FcRn MFI relative to fluorescence minus one (FMO) background controls. Summary of flow cytometry data quantifying the total number of cells and median fluorescence intensity (MFI) of FcRn expression for epithelial cells (G), Langerhans cells (H), and other leukocytes (I) in corneal digests; (n = 5–6 corneas per group, 3 independent experiments). Data in panels G, H, and I reflect mean ± SEM. Statistical differences were determined by one-way ANOVA with Student-Newman-Keuls multiple comparisons tests. Flow cytometry was used to assess FcRn expression in peripheral blood CD45+ CD11b+myeloid cells (J) and CD45+ lymphocytes based on scatter profile (K); data are representative of 3 independent experiments.

Article Snippet: Three-dimensional protein fluorescence colocalization analysis was performed using Imaris software (Bitplane USA, Concord, MA).

Techniques: Labeling, Infection, Staining, Expressing, Control, Software, Western Blot, Flow Cytometry, Selection, Fluorescence

Journal: Journal of Neurology

Article Title: Identification of novel Angiogenin ( ANG) gene missense variants in German patients with amyotrophic lateral sclerosis

doi: 10.1007/s00415-009-5124-4

Figure Lengend Snippet: ANG missense variants identified in German ALS patients and clinical picture

Article Snippet: Missense variants were mapped onto the three-dimensional crystal structure of the mature ANG protein using PyMOL software (Delano Scientific LLC).

Techniques: Variant Assay