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Malattie Autoimmuni

Malattie Autoimmuni. Rappresentano ancora oggi uno dei problemi più spinosi dell’immunologia, sia sul piano sperimentale che su quello clinico. Le conoscenze attuali sui meccanismi coinvolti sono ancora incomplete e di conseguenza l’eziologia è spesso ignota.

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Malattie Autoimmuni

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  1. MalattieAutoimmuni • Rappresentano ancora oggi uno dei problemi più spinosi • dell’immunologia, sia sul piano sperimentale che su quello • clinico. • Le conoscenze attuali sui meccanismi coinvolti sono ancora • incomplete e di conseguenza l’eziologia è spesso ignota. • Inoltre sono per lo più malattie multifattoriali, in quanto nella • loro patogenesi intervengono fattori di predisposizione genetica • e fattori ambientali.

  2. Manifestazioni Autoimmuni Diabete mellito di tipo I Tiroidite Anemia emolitica Enteropatia Dermatite • Alopecia

  3. CD4+ CD25+ Regulatory T cells The immune system Protection from microorganisms Tolerance to the SELF

  4. Delezione clonale (interazione Fas e Fas-L) Anergia clonale Attività delle T regolatorie Principali meccanismi di eliminazione (periferici) di linfociti autoreattivi Indifferenza clonale

  5. Generazione delle Treg Il timo produce la maggior parte delle Treg CD4+CD25+, (5-10% linfociti CD4+T circolanti) come sottopopolazione distinta e funzionalmente matura Lo sviluppo di T reg puo’ avvenire a partire da cellule convenzionali, sotto specifiche condizioni di attivazione

  6. suppressing the proliferation or function • of autoreactive T cells • possibility that Treg cells may play a central role in immune homeostasis • and regulation of immune responses toward foreign antigens CD4+ CD25+ Regulatory T cells in the immune system • maintenance of immunological • self-tolerance

  7. Sindrome X-linked recessiva • Mortalità elevata entro il primo anno di vita. CASO CLINICO IPEX: Immune Dysregulation, Polyendocrinopathy, Entheropathy, X-linked. → Patologia da difetto della tolleranza immune.

  8. Presents most commonly in early childhood: • IDDM • Severe enteropathy • Skin disorders • Variable autoimmune • phenomena Autoantibodies against: Thyroid Kidney Pancreatic islets Small Intestine Platelets and others CD4+CD25+ Regulatory T cells are involved in I P E X mmune dysregulation olyendocrinopathy nteropathy -linked

  9. FoxP3 in regulatory T cells C NFAT and NF-kappaB (nuclear factors) C blocks their ability to induce the endogenous expression of their target genes, including key cytokine genes • Foxp3 is a regulatory T cell-specific transcriptional factor – FKH family • master regulator of the development and function of regulatory T cells As trascrptional factor: • Probably regulates/suppresses cytokine expression

  10. FOXP3 as proteic product N C PRR Zn FKH 1 2 3 • 11 esoni – 431 aa • functional domains: • Proline rich region in N-terminal zone • Zinc finger domain • Forkhead winged-helyx domain: necessary of DNA-binding and nuclear location

  11. Human IPEX: 13 mutations identified until now  • In the FKH domain • in leucine zipper domain • removing of stop codon  products with C-terminal extensions Every patient with these mutations has classic IPEX, and symptomatic differences are unremarkable

  12. Molecular and proteomical study 1 . Identification and estimation of FOXP3 expression 2 .Identification and estimation of other important and related genes expression 3 . Identification of specific protein-protein interactions of FOXP3 and its different domains

  13. Steps of the study Recombinant protein Tagged products Cloning of FoxP3 gene and its domains N PRR Zn FKH C 1 2 3 Cloning into expression vector

  14.  pTrcHis  Production in  Purification bacterial cells by His Tag Recombinant  protein  Policlonal sera Expression of FoxP3 in procariotic cells

  15. Expression in culture cells: - HEK293 - T lymphocytes (if possible) Expression of FoxP3 in eukaryotic cells Cloning of FoxP3 in fusion with GFP - N-terminal with StrepTag - C-terminal FoxP3/Dom GFP FoxP3/Dom TAG

  16. MICROSCOPY  Confocal  Detection of GFP Use of anti-His (or anti-GFP) antibodies on  Nuclear extracts  Cytoplasmatic extracts Localization of FoxP3 Under different conditions of stimulation

  17. FOXP3 interactions • Characterization of co-precipitated proteins (mass spectrophotometry…) • Immunoprecipitation of FoxP3 with anti-TAG (anti-His / anti-GFP / Strep-Tag vs Strep-Tactin)

  18. Interagent molecule 2D SDS-PAGE IEF detection Mass spectometry identification

  19. FOXP3 interactions Use of Bacterial Two-Hybrid System • Easy in vivo screening and selection offunctioninteractions between two proteins • Analysis of different cDNA libraries of T cells subpopulations under different conditions of stimulation • Informations about methabolic pathways in which FoxP3 is involved

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