Searching for the Evironmental Cause of Endemic Brazilian Pemphigus Foliaceus (Fogo Selvagem)
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1 Searching for the Evironmental Cause of Endemic Brazilian Pemphigus Foliaceus (Fogo Selvagem) Luis A. Diaz, M.D. Professor & Chairman Department of Dermatology University of North Carolina
2 RO1 AR32599 with Subcontract to Dermatology UNC and University of Sao Paulo (Over 30 years of funding---->2,018) Diaz L.A.: No disclosures or Conflict of Interest (2013)
3 The Fogo Selvagem (1900- Present) Hospital Ademar de Barros, Sao Paulo (1940 s) Meu Deus, Seja Feita Vossa Vontade (Not my will, but God s) Tocantins Mato Grosso do Sul Goias Brasilia Sao Paulo w High mortality w 10% spontaneous remission Parana Diaz el al: J Invest Dermatol, 92:4,1989 Vector / parasite, tropical disease w Etiology of Leishmaniasis, 1901 w Chagas Disease, 1909 (Brazil) w Carrion s disease, 1905
4 The Changing Prevalence of Fogo Selvagem State of Sao Paulo - Brazil Diaz LA, et al, J Invest Dermatol. 129:4-12-, FS cases 82 FS cases Adhemar de Barros Hospital Guarulhos, SP Census: ~300 hospitalized FS cases Adhemar de Barros Hospital (Padre Bento) Guarulhos, SP Census: Sporadic hospitalized FS cases
5 Endemic Pemphigus Foliaceus (Fogo Selvagem) Vieira, 1937 Dsg1 IgG Beutner, 1968
6 Anhalt, G.J. FS & PF IgG are Pathogenic Roscoe, J Dsg1 IgG Roscoe, Diaz & Anhalt, J Invest Dermatol: 85:538, 1985
7 Pemphigus Foliaceus (FS) Anti-Dsg1 autoantibodies Desmosomal Antigens IgG Pemphigus Vulgaris MucosaI PV (anti-dsg3 antibodies) The Desmosome Cell 1 Cell 2 Plaque:Plakins Desmoplakin 1 (245 kd) Desmoplakin 2 (215 kd) Plakoglobin (85 kd) Plakophilin 1,2,3 (75 kd) Keratocalmin (250 kd) IgG Mucocutaneous PV (anti-dsg1 + anti-dsg3 antibodies) E-Cadherin Core: Cadherins Desmoglein 1 (165 kd) Desmoglein 2 (Colon) Desmoglein 3 (130 kd) Desmoglein 4 (Hair, skin) Desmocollins: 1,2,3 (a,b)
8 Relevant Epitopes on Dsg1 Keratinocyte Rock et al, N Engl J Med 320:1463, 1989 B Cells-IgG4 Pathogenic abs EC5 EC4 EC2 EC1 EC3 Dsg1 T Cells Lin et al, J Clin Invest, 105:207, 2000
9 Etiology of Fogo Selvagem Epidemiology Serology Immunogenetics Population at Risk Risk Factors Antigen Exposure Autoantibody Response IgG IgG4 IgE IgM HLA alleles Normal Subject at risk Incubation Time FS Patient
10 Endemic Pemphigus Foliaceus (Fogo Selvagem) Epidemiology Humoral Autoantibody Response Dsg1 Epitopes bound by FS IgG4 Putative Environmental antigens
11 Anti-Dsg1 Autoantibody Responses in FS u IgG4 anti-dsg1 Response u IgE anti-dsg1 Response u IgM anti-dsg1 Response
12 IgM, IgE and IgG4 anti-dsg1 in FS and healthy controls % Positive IgM, IgE, & IgG4 individuals FS Endemic Brazil n=143 HC Endemic Brazil n=161 HC Non-endemic Brazil n=57 IgM IgE IgG4 HC USA-Japan n=99
13 Anti-Dsg1 Autoantibody Responses in FS u The IgG4 Response u The IgE Response u IgM Response
14 The IgG Subclasses Autoantibodies in FS IgG1 Dsg1 IgG IgG2 IgG3 IgG4
15 Correlation of IgG1 and IgG4 anti-dsg1 autoantibodies and disease activity in FS Warren et al, 2001 ELISA OD IgG4 IgG1 (Log Scale) Cutoff Remission Active Disease Evolution in Time
16 FS IgG4 Autoantibodies are Pathogenic IgG4 Serun IgG4 B. Rock Rock et al, N Engl J Med 320:1463, 1989
17 Domain- swapped strategy IgG4 anti-dsg1-ec1 ELISA % of Posi2ve Sera Dsg1 EC1 His-tag EC2 EC3 EC4 EC5 Desmocollin 1 FS Brazil-NHS UNC-NHS (n=55) (n=23) (n=22)
18 Purification of IgG4 from FS serum FS serum Llama anti-higg4 Sepharose BOUND (Eluted) UNBOUND (FT) IgG4 anti-dsg1 (97%) Non- IgG4 anti-dsg1 (92% IgG1) 100 ug 400 ug
19 Predicted Homodimeric Adhesive Conformation of the Dsg1 Ectodomain Dsg1 monomer EC-1 Dsg1 monomer CELL 1 EC-2 EC-3 EC-4 EC-5 CELL 2 EC-5 EC-4 EC-3 EC-2 EC-1 EC- 1 Epitope (95% FS IgG4) Dsg1 monomer Dsg1 monomer EC-5 EC-4 EC-3 EC-2 EC-1 CELL 2 CELL 1 EC-1 EC-2 EC-3 EC-4 EC-5 EC- 2 Epitope (85% FS IgG4) Ca2+ binding domains R of RAL adhesive mobf N- glycosilabon site
20 Immunological Stages in Fogo Selvagem "People at risk to develop FS are exposed to environmental antigen(s) & generate anti-dsg1 autoantibodies" IgM IgG Immunization: Unknown Antigen Nonpathogenic IgG years Pathogenic Disease Birth Preclinical Stage Autoantibodies Incubation? Clinical Stage w Blisters w Anti-Dsg1 autoab w Anti-Dsg1 T cells
21 Endemic Pemphigus Foliaceus (Fogo Selvagem) Epidemiology Humoral Autoantibody Response Dsg1 Epitopes bound by FS IgG4 Putative Environmental antigens
22 Hypothesis u Hematophagous insect bites, prevalent in endemic areas of FS, may deliver the environmental antigen(s) that induce cross-reactive anti-dsg1 antibodies. Ye Qian, Ph.D. u To test this hypothesis we have began to test the reactivity of FS autoantibodies to salivary gland proteins of sand fly (Lutzomyia longipalpis, a vector of Leishmaniasis)
23 FS serum X Black fly (Simuliid) Anti-Filarial Ab Anti-Simuliid Ab Ant-Dsg1 Ab? Filaria volvulus Onchocerciasis FS serum X Sand Fly (Lutzomyia l) Anti-Leish Ab Anti-Lutzomya Ab Ant-Dsg1 Ab? Leishmania sp Leishmaniasis FS serum X Kissing bug (Reduvii) Anti-Trypanos Ab Anti-Reduvii Ab Ant-Dsg1 Ab? Trypanosoma cruzi Trypanosomiasis (Chagas)
24 Anti-Dsg1 Antibodies in Onchocerciasis, Leishmaniasis & Chagas 400 Diaz, et al. J Invest Dermatol, 123:1045, 2004 Kissing bug (Reduvii) Black fly (Simuliid) Dsg1 ELISA (index value) Onchocerciasis Sand Fly (Lutzomyia l) Leishmaniasis Trypanosomiasis (Chagas) N o FS/PF Onchocerciasis Leishmania Chagas SA-Blastomycosis Leprosy NHS 40/40 34/41 38/88 18/31 7/28 14/83 0/57 % 95% CI P-value < <0.001 <0.001 <
25 In Search of the Etiology of Pemphigus Black fly (Simuliid) Lutzomyia longipalpis Kissing bug (Reduvii) Ribeiro, NIAID Filaria Leishmania sp Trypanosoma cruzi Onchocerciasis Leishmaniasis Chagas Disease 76% Anti-dsg1 ab 51% Anti-dsg1 ab 19% Anti-dsg1 ab Sensitization by vector saliva may lead to FS
26 Three major components of sand fly salivary gland antigens u There are two major immunogenic proteins from SGLL, i.e. LJM11 and LJM17. Antibodies to these proteins are specific markers of human exposure to LL bites u A third component, LJL143, is also immunogenic and elicit antibodies that are considered specific markers of exposure to LL bites in dogs.
27 FS: Hematophagous Insect Hypothesis Sand Fly (Lutzomyia l) IgG4 Anti-Dsg1 Antibodies? Antigen Exposure Normal Subject at risk Incubation Time B Lymphocytes Hybridoma : IgG4 monoclonal abs Phage Display: ScFv monoclonal abs FS Patient
28 Sand Fly Salivary Gland Antigens Valenzuela & Ribeiro, NIAID, Bethesda Xu et al, J Biol Chem, 286, 32383, 2011 Sand Fly (Lutzomyia l) Salivary Glands mrna cdna Library Ribeiro, NIAID Leishmaniasis is endemic in LV Homogenization in PBS Recombinant Proteins Soluble Antigens (SGLL) LJM11 LJM17 LJL143
29 Binding of Monoclonal IgG4 anti-dsg1 Antibodies to LJM11 is Inhibited by rdsg1 & EDTA Qian et al: J Invest Dermatol, 129: , 2009 Qian et al: J Immunol, 189: , 2012 O.D. (450 nm) Binding 4E4 & 2D11 Abs 1.0 4E D LJM11 LJM17 LJL143 Inhibition by hdsg1 1.0 No Dsg µg/ml Dsg E4 2D11 Inhibition by EDTA 1.0 5mM Ca mM EDTA E4 2D11
30 Antibodies from sera of mice immunized with LJM11 also recognize human Dsg1 Dsg1 ELISA O.D. (450 nm) Dsg1 ELISA (Plates coated with hdsg1) 0 m1- anb- LJM11 m2- anb- LJM11 m3- anb- LJM11 m4- anb- LJM17 m5- anb- LJL143 Ctrl Reaction of mouse antisera with human Dsg1 by IP
31 Endemic Pemphigus Foliaceus (Fogo Selvagem) Lutzomyia longipalpis 5 1 Ag 2 T B Dsg1 Autoantibodies 3 4
32 To the Members of The Cooperative Group on Fogo Selvagem Research * For their unselfish support and friendship for more than 25 years * Rosicler Rocha Alvarez: The Cooperative Group on Fogo Selvagem Research. Paper in preparation, 2012
33 The Cooperative Group on FS Research S.A.P. Sampaio, M.D. Former Professor & Chairman Department of Dermatology University of Sao Paulo, Brazil ( ) E. A. Rivitti, M.D. Former Professor & Chairman Department of Dermatology University of Sao Paulo, Brazil
34 The Cooperative Group on FS Research Friendship Loyalty Idealism Hard work Sacrifice V. Aoki D. Eaton H. Friedman G. Hans
35 The Cooperative Group on FS Research Vandir dos Santos, R.N. Joana Katayama Roberto & Maria Elisa Moraes Nurse Supervisor Blood Processing HLA Studies
36 The Cooperative Group on FS Research Nursing Staff-Limao Verde Evandro A. Rivitti, M.D. Valeria Aoiki, M.D.
37 The Terena Leaders and the People of Limao Verde Sr. Isaque Dias
38 Biomedical Research Outcomes 0.01% 99.99%
39 END
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