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MalattieAutoimmuni Malattie Autoimmuni Rappresentano ancora oggi uno dei problemi più spinosi dellimmunologia, sia sul piano sperimentale che su quello.

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Presentazione sul tema: "MalattieAutoimmuni Malattie Autoimmuni Rappresentano ancora oggi uno dei problemi più spinosi dellimmunologia, sia sul piano sperimentale che su quello."— Transcript della presentazione:

1 MalattieAutoimmuni Malattie Autoimmuni Rappresentano ancora oggi uno dei problemi più spinosi dellimmunologia, sia sul piano sperimentale che su quello clinico. Le conoscenze attuali sui meccanismi coinvolti sono ancora incomplete e di conseguenza leziologia 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 The immune system Protection from microorganisms Tolerance to the SELF CD4+ CD25+ Regulatory T cells

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

5 Generazione delle Treg Lo sviluppo di T reg puo avvenire a partire da cellule convenzionali, sotto specifiche condizioni di attivazione Il timo produce la maggior parte delle Treg CD4 + CD25 +, (5-10% linfociti CD4 + T circolanti) come sottopopolazione distinta e funzionalmente matura

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

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

8 CD4+CD25+ Regulatory T cells are involved in IPEX mmune dysregulation olyendocrinopathy olyendocrinopathy nteropathy nteropathy -linked -linked Presents most commonly in early childhood: - IDDM - Severe enteropathy - Skin disorders - Variable autoimmune phenomena phenomena Autoantibodies against: ThyroidKidney Pancreatic islets Small Intestine Platelets and others

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

10 11 esoni – 431 aa11 esoni – 431 aa functional domains:functional domains: FOXP3 as proteic product 1.Proline rich region in N-terminal zone 2.Zinc finger domain 3.Forkhead winged-helyx domain: necessary of DNA-binding and nuclear location ZnFKHNC PRR 1 2 3

11 Human IPEX: 13 mutations identified until now Human IPEX: 13 mutations identified until now In the FKH domain In the FKH domain in leucine zipper domain in leucine zipper domain removing of stop codon products with C-terminal extensions removing of stop codon products with C-terminal extensions Every patient with these mutations has classic IPEX, and symptomatic differences are unremarkable 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 related genes expression 3. Identification of specific protein-protein interactions of FOXP3 and its different domains FOXP3 and its different domains

13 Steps of the study Cloning of FoxP3 gene and its domains ZnFKH Cloning into expression vector N CPRR Recombinant protein Tagged products Tagged products

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

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

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

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

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

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


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