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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
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.
manifestazioni autoimmuni
Manifestazioni Autoimmuni

Diabete mellito di tipo I

Tiroidite

Anemia emolitica

Enteropatia

Dermatite

  • Alopecia
the immune system

CD4+ CD25+ Regulatory T cells

The immune system

Protection from microorganisms

Tolerance to the SELF

slide4

Delezione clonale

(interazione Fas e Fas-L)

Anergia clonale

Attività delle T regolatorie

Principali meccanismi di eliminazione (periferici)

di linfociti autoreattivi

Indifferenza clonale

generazione delle treg
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

slide6

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
slide7

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.

cd4 cd25 regulatory t cells are involved in

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

foxp3 in regulatory t cells
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
slide10

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
slide11

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

slide12

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

slide13

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

slide14

 pTrcHis  Production in  Purification bacterial cells by His Tag

Recombinant  protein

 Policlonal sera

Expression of FoxP3 in procariotic cells

expression of foxp3 in eukaryotic cells

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

localization of foxp3

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

slide17

FOXP3 interactions

  • Characterization of co-precipitated

proteins (mass spectrophotometry…)

  • Immunoprecipitation of FoxP3 with anti-TAG (anti-His / anti-GFP / Strep-Tag vs Strep-Tactin)
slide18

Interagent molecule

2D SDS-PAGE IEF

detection

Mass spectometry identification

slide20

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