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Rubella persistence in epidermal keratinocytes and granuloma M2 macrophages in patients with primary immunodeficiencies  Ludmila Perelygina, PhD, Stanley.

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Presentation on theme: "Rubella persistence in epidermal keratinocytes and granuloma M2 macrophages in patients with primary immunodeficiencies  Ludmila Perelygina, PhD, Stanley."— Presentation transcript:

1 Rubella persistence in epidermal keratinocytes and granuloma M2 macrophages in patients with primary immunodeficiencies  Ludmila Perelygina, PhD, Stanley Plotkin, MD, Pierre Russo, MD, Timo Hautala, MD, PhD, Francisco Bonilla, MD, PhD, Hans D. Ochs, MD, Avni Joshi, MD, MSc, John Routes, MD, Kiran Patel, MD, MS, Claudia Wehr, MD, Joseph Icenogle, PhD, Kathleen E. Sullivan, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 138, Issue 5, Pages e11 (November 2016) DOI: /j.jaci Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Cutaneous granulomas in patients. A, Hematoxylin and eosin staining of a cutaneous granuloma from case 1. A well-formed granuloma is centrally located. B and C, Cutaneous skin lesions from case 3. Acute and chronic ulcers are observed. D-F, Distribution of RV-infected cells in skin samples of patients with PID. Histological immunofluorescent staining showing focal (Fig 1, D, case 1) or widespread (Fig 1, E, case 2) distribution of RV capsid in granulomas and focal capsid localization in the epidermis (Fig 1, F, case 5). Activation status of macrophages in granulomas (G-I, case 6). Double immunofluorescent staining of granulomas with RV capsid antibody (red) and M2 macrophage-specific antibodies, CD206 (Fig 1, G, green) or CD163 (Fig 1, H, green). I, RV antigen expression in the suprabasal cell layer of skin epidermis (case 1). Double immunofluorescent staining with RV capsid antibody (red) and keratinocyte-specific antibody (cytokeratin, green). Nuclei were counterstained with DAPI. DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride; RV-C, rubella virus capsid. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig E1 The genetic relationships between the full rubella genome sequences were inferred using the Neighbor-Joining method. A, The tree is composed of 76 rubella viruses, including sequences of RA27/3 vaccine (bold) and RVs/Oulu.FIN/22.15/PID (red). Sequences were obtained from the GenBank or from Dr Min-hsin Chen. The genetic distances were computed using the maximum composite likelihood method. The scale bar indicates the number of base substitutions per site. The recovered virus sequence is clearly related to the vaccine strain RVI RA27.USA 64 1a vac. B, RV genome organization. The rubella genome is a single-strand RNA virus of positive polarity. The 2 open reading frames are denoted by blue and green boxes. Individual proteins after processing of the 2 polyproteins are shown below. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E2 RV-positive control slides containing a mixture of 4 human tissues and RV-infected A459 cell double immunostained with capsid mAb and pan-keratin polyclonal antibody and counterstained with DAPI. The slides were either untreated (A) or treated (B) with 0.3% Sudan black solution before mounting. Note the reduction in autofluorescence after Sudan black treatment (Fig E2, B). DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E3 Histological immunofluorescent staining of positive control slides with either rabbit RV polyclonal antibody (CDC) (A), mouse capsid mAb (Abcam) (B), or mouse capsid mAb (CDC) (C), which were preabsorbed with human proteins. Anti-rabbit IgG Alexa Flour-555 (Fig E3, A), anti-mouse IgG Alexa Flour-555 (Fig E3, B), or Alexa Flour-488 (Fig E3, C) served as secondary antibody; nuclei were counterstained with DAPI. DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E4 Specificity of rabbit RV polyclonal antibody (CDC). Immunofluorescent staining of Vero cells infected with rubella virus (A and B) with either RV polyclonal or RV polyclonal preabsorbed with purified RV virions. C, Mock-infected Vero cells. Note the lack of specific staining with the antibody preabsorbed with the specific antigen. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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