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IMUNITATEA = APRARE

Toi ne mbolnvim uneori..., apoi ne


nsntoim

Ce se ntmpl n organism n timpul unei


infecii / afeciuni ?
De ce ne nsntoim ?
SISTEMUL IMUN

Colecie de CELULE i MOLECULE care


protejeaz organismul mpotriva infeciilor,
transformrilor maligne i celulelor proprii
transformate non-self
IMUNITATE
INNASCUTA / nespecifica DOBINDITA / specifica
(innate immunity
immunity)) (adaptive immunity)

raspuns independent de antigen raspuns dependent de antigen


receptori pt. patogeni: receptori pt. patogeni:
codificati in genom generati (rearanjare genica)
specificitate joasa specificitate inalta
raspuns imediat raspuns lent
lipsa memorie memorie imunologica
(expansiune clonala Ly)
prezenta la toate organismele prezenta doar la vertebrate
pluricelulare

SISTEM INTEGRAT
Rspunsuri imune
Barriers Skin & Mucous membranes
Invasion rapidly regenerating surfaces,
peristaltic movement, mucociliary
& infection escalator, vomiting, flow of urine/tears,
coughing

Innate immunity Cellular and humoral defences


+ lysosyme, sebaceous/mucous secretions,
stomach acid, commensal
organisms,complement proteins,
phagocytosis, NK cells

+ Inflammation
Cellular and humoral defences
Adaptive immunity Antibodies, cytokines, T helper cells,
cytotoxic T cells
Imunitatea
- piele
innscut
1. Bariere anatomice - mucoase

Functia de prevenire a intrarii


microorganismelor patogene
motilitate: muco-ciliara, peristaltica
Factori fizici / mecanici mucus
fluxul fluidelor prin organism

pH
Factori chimici molecule antimicrobiene

limfocite T intraepiteliale ()
Factori biologici celule B-1
Imunitatea
- receptori
innscut 2. Componente moleculare
- molecule secretate

Functia de recunoastere
Functii efectorii

Mol. anorganice: HCl, NO, H2O2


Peptide antibacteriene: defensine, cathelicidine, histatine
Proteine antibacteriene: lizozim, lactoferina, transferina
Lectine: colectine, ficoline, receptori pentru Manoza
Complement
Citokine: IFN-/, IL-1, TNF-, CSF
Chemokine: IL-8, MIP, MCP
Receptori:: TLR
Receptori
Imunitatea
innscut
3. Componente celulare

Functia de eliminare a
microorganismelor patogene

neutrofile
monocite/macrofage Fagocitoza
celule dendritice

bazofile
mastocite Inflamatie
eozinofile

celule NK Citotoxicitate
Immune responses

Barriers Skin & Mucous membranes


rapidly regenerating surfaces,
Invasion peristaltic movement, mucociliary
& infection escalator, vomiting, flow of urine/tears,
coughing

Innate immunity Cellular and humoral defences


+ lysosyme, sebaceous/mucous secretions,
stomach acid, commensal
organisms,complement proteins,
phagocytosis, NK cells

+ Inflammation
Cellular and humoral defences
Antibodies, cytokines, T helper cells,
Adaptive immunity
cytotoxic T cells
Inflamaia

Component major a rspunsului imun nespecific/


specific
Rspuns protectiv la injurie, montat s restabileasc
statusul normal
Iniiat de lezarea esuturilor:
mecanic/fizic (ex. arsuri)
chimic ( ex. expunere la ageni corozivi)
biologic (ex. microorganisme)
imunologic (ex. hipersenzitivitate)
Implic fagocite i mediatorii secretai de fagocite,
precum i excretai de celulele secretorii
Inflamatia - Functii

Distrugerea si indeprtarea substantelor


/ particulelor duntoare / strine
Izolarea zonei infectate/inflamate
Stimularea rspunsului imun specific
Vindecare
Rspunsul inflamator celular (1)

Neutrofilele si
monocitele circulante
au o mobilitate foarte
mare.

PMN si Mo au capacitatea sa
se strecoare prin spatiile
inguste interendoteliale,
recrutate chemotactic
(chemokine) la locul
infectiei
Daca infectia se
raspandeste, fagocite
noi din torentul
circulator sunt atrase
la locul infectiei

Fagocitele ingurgiteaza
particulele straine
similar cu amoebele
Leukocyte Adhesion

Phagocytic
neutrophils
respond to an
epithelial
chemokine IL-8

Cells migrate
from the blood
into the tissue
underlying the
infection
Diapedesis
Caracteristicile Inflamaiei
Edemaierea zonei (tumor)
Eritem (rubor)
Creterea temperaturii
(calor)
Durere (dolor)
Pierderea funcionalitii
(functio lesa)

Reacii / interferene
inflamatorii locale:
Activarea coagulrii
Cile formatoare ale
Kininelor
Fibrinoliza
Rspunsul inflamator local

Eliberarea de histamin i prostaglandine determin


vasodilataie local nsoit de:
o increased blood flow redness and warmth
o increased capillary permeability
o edema (swelling) due to fluids seeping from
capillaries
o more WBCs to site
o phagocytes move out of vessels into
extracellular fluid (ECF)
o phagocytes engulf and destroy bacteria
Inflammation
Heat - Swelling - Redness - Pain - Loss of Function
Lymphocyte Adaptive Cells Of The
immunity
Immune System

Macrophage Phagocytosis Common


Monocyte Ag presentation lymphoid
progenitor
Neutrophil Phagocytic
PMN Anti-bacterial
Pluripotent
haemopoietic
Anti-parasite stem cell
Eosinophil
Immunity - Allergy

Myeloid
Basophil ?Protection of progenitor
mucosal surfaces?
- Allergy

Protection of
Mast cell mucosal surfaces
- Allergy
Fagocite profesionale - PMN, MQ (1)
Leucocite Polimorfonucleare (PMN)
Granulocite:
o Neutrofile, eozinofile, basofile, mastocite
Sunt fagocite cu via scurt, continnd lizozomi
Produc ap oxigenat i radicali superoxid
Proteine bactericide lactoferina
PMNs joac un rol major in protectia mpotriva infectiilor
Defecte genetice/dobndite infectii cronice sau recurente
Macrofagele fagocite tisulare derivate din monocitele ciculante,
migreaza din sange in tesuturi se diferentiaza:
o celule Kupffer in ficat
o macrofage alveolare in plaman
o celule mezangiale in rinichi
o macrofage splenice (sinusale) in pulpa alba - Sistemul Reticuloendotelial
(SRE) / Reticulohistiocitar (SRH)
o macrofage peritoneale plutind liber in fluidul peritoneal (seroase)
o celule microgliale in SNC
o celule Langerhans in piele
Inflamatia locala

In tesutul interstitial
exista o populatie
rezidenta de
leucocite.

Mastocitul:
elibereaza amine
biogene (histamina,
serotonina)
secreta
prostaglandine,
leukotriene, citokine
si TNF-a.
PMN:
fagociteaza
agresorul
Neutrophils

Also called polymorphonucelar


leukocytes
4 to 10 million per ml of blood
Infection activates cytokines that
Neutrophil stimulate the bone marrow to produce
up to 20 million neutrophils per ml of
blood.
Main role is to get to site of infection
rapidly and ingest microorganisms.
After taking up microorganisms the
neutrophil will die.
Neutrophil Chemotaxis
Neutrophils chasing yeast

Neutrophil
Monocytes

0.5 to 1 million monocytes per ml of


blood
Migrate into the tissues and
differentiate into Macrophages
Blood monocyte
Phagocytose microorganisms
Present antigens to T cells
The name of monocyte-derived cells
depends upon the tissue they reside in:
Liver - Kupffer cells
Lung - Alveolar macrophages
CNS - Microglial cells
Bone - Osteoclasts
Tissue macrophage Human Macrophage ingesting yeast
Fagocite (2)
Initial, in spatiul interstitial trec
PMN:
se acumuleaza in 30-60 minute de la
aparitia agentului declansator
fagociteaza intrusul si tesuturile
lezate
elibereaza enzime lizozomale
Daca inflamatia persista:
dupa 12-18 ore interstitiul este
infiltrat cu mononucleare
(macrofage si limfocite)
Macrofagele
o suplimenteaza activitatea PMN
o prezinta Ag, Ly T
Daca inflamatia continua, raspunsul
inflamator este suplimentat si
accentuat cu elemente ale
imunitatii dobandite Ac si LyT
(Ac activeaza si sistemul
complementului) Vindecare
Fagocite (3)

Celulele seriei macrofagice au doua functii majore:


Ingereaza si digera microorganisme si particule
straine
Prezinta Antigene (Ag) - componenta imunogena
o Preia Ag, il proceseaza si il prezinta Ly T

* Alte celule prezentatoare de Ag (APC) (precursori


hematopoietici, fagocite mai putin eficiente)
Celule dendritice in splina si ganglioni
Celule interdigitate in timus
Celule Langerhans in piele
Fagocitoza (cont)
Proces activ, initiat de
legarea fagocitului la
patogen prin
intermediul unor
receptori
Agentul patogen este
inconjurat de
pseudopode
membranare si apoi
internalizat:
- Fagozom
- Fagolizozom
- Vacuola digestiva
- Corp rezidual
Fagocitoza
Proces activ, initiat de legarea la patogen prin intermediul unor
receptori
Agentul patogen este inconjurat de pseudopode membranare si apoi
internalizat
General scheme of an immune response
Most microorganisms do not cause disease in humans

Pathogens
Disease - causing organisms
Protozoa, Bacteria, Viruses, Fungi, Worms etc

Both humans and pathogens are made of


How does the human
proteins immune system
carbohydrates distinguish
& lipids
pathogens from human tissues and harmless
microorganisms?
Fagocitoza
Caracteristici
Definitie: preluarea unor particule de dimensiune mare (in
principal microorganisme), datorita capacitatii de a distinge
intre tipurile de carbohidrati produsi de mamifere si bacterii
(self - nonself)
o Pathogen-associated molecular patterns (PAMPs): LPS,
peptidoglicani, lipoarabinomanani, dsRNA - substante
prezente relativ constant (fara variabilitate antigenica) la
un grup mare de patogeni
Actin-dependenta, chlatrin-independenta
Rata & eficienta mare de internalizare

Receptori fagocitari - pattern recognition receptors (PRRs) or


molecules (PRMs)
o Externi
FcR (CD16, CD64), CR3 (CD11b), Scavenger receptor
(CD36), Mannose receptor
o Interni
TLRs - Toll like receptors
Fagocitoza
(cont)

opsonin
dependenta, fiind
mediata de:
Ac sau
C3b
non-
non-
self
Recunoastere imuna self

origine antigen / context biologic

Ipoteze

Receptori (self vs. non


on--self microbian):
microbian):
PRR Pattern Recognition Receptor
PAMP Pathogen Associated Molecular Pattern
(Medzhitov & Janeway, 1997)
a) ENDOCITOZA - Endocytic PRRs

Bacterie

glicoproteina proteina bact.


bact. LPS, LTA
man C3b

receptori manoza receptori scavenger receptori opsonine


(lectina C)
C) (CD36, CD68, SRB-
SRB-1) (CR1
CR1))

FAGOCIT
PAMP lipopolizaharid
peptidoglican
acid lipoteichoic
lipoproteine
invariabile manoza
inalt conservate ADN
specifice microbilor ARN dc
(patogeni + non-patogeni) flagelina
comune pentru o clasa de microbi pilina
vitale pentru microorganisme zimozan

Gram-
Gram -negative Gram-
Gram -positive
Receptori fagocitari de suprafata - PRR:
leaga carbohidrati bacterieni

1. Mannose-binding receptor
Recunoaste reziduuri de manoza cu o orientare
spatiala certa/unica microorganismelor
Se gaseste doar pe MQ (nu i pe Mo sau PMN)
2. Glucan Receptor
Prezent pe toate fagocitele
Receptori fagocitari de suprafa - PRR:
leag ali compui bacterieni

3. Scavenger Receptor - CD36


Recunoaste liganzi:
o polianionici (ds-RNA, LPS)
o carbohidrati
o Lp cu densitate mica (LDL) acetilate

Se gasesc pe toate fagocitele


Specific MQ, leaga peretele celulei bacteriene &
LPS
Fagociteaza celule apoptotice
o factor nou MFG-E8 (eliberat din MQ activate se
leaga la celulele apoptotice via fosfatidilserina)
b) TRANSMITERE SEMNAL - Signaling PRRs

TLR
Toll--like receptors
Toll

NOD
nucleotide--binding
nucleotide
oligomerization domain

RIG-1
RIG-
retinoic acid-
acid-inducible gene-
gene-1
Receptorii fagocitari interni
Toll-like (TLRs)

TLRs sunt proteine transmembranare


Toll - molecula identificata ca fiind esentiala pentru patern-ul
embrionic de Drosophila
Conservat pe parcursul evolutiei la insecte & oameni
TLRs la mamifere este omolog cu domeniul citoplasmatic al receptorului
IL-1 si IL-18 (Toll-IL1-R sau TIR domain)
Domeniul extracelular este diferit
Au fost raportati 10 TLRs (1-10)
Exprimati diferentiat pe celulele imune (nivel scazut)
Expresia este modulata (inductibila) ca raspuns la anumiti
stimuli
Exprimati si pe alte tipuri celulare (e.g., celule endoteliale)
Toll-
Toll -like receptors (TLR)

proteine transmembranare tip I


(Drosophila om)
conservate filogenetic (Drosophila
domeniu extracelular bogat in leucina
domeniu intracelular TIR (similar IL-
IL-1R)
TLR mamifere (11-
(11-om, 13-
13-soarece):
recunoastere PAMPs
asociere cu alti TLR sau alte proteine (MD2,CD14)
TLR2
+(TLR1/TLR6) TLR4 TLR5 TLR9
Lipoproteins, lipopeptides LPS Flagellin CpG DNA
Peptidoglycan
Taxol
Zymosan
LPS Leptospira interrogans HSP60
LPS P.gingivalis Fibronectin
GPI (T.cruzi)
(T.cruzi) F Protein (RSV)
Lipoarabinomannan
Phosphatidylinositol dimannoside
(M. tuberculosis)
Functiile imunitatii innascute

PAMP

Stimulare PRR stimuleaza fagocitoza


induce activitate microbicida
induce citokine inflamatorii:
IL-
IL -1, IL-
IL-6, TNF-
TNF- (NF
(NF--kB)
activeaza imunitatea dobindita
expresia molec.
molec. co-stim.
(MHC cls.II, CD80/CD86)
CD80/CD86
Fagocitoza

Mecanism

dependent de oxigen independent de oxigen

O2 - defensine
H2 O2 - cathepsina B
1O - lizozim
2

OCl - lactoferina
OH - enzime proteolitice

eliminare a microorganismelor patogene


Rol
prelucrare Atg pt. prezentare
Mecanismele uciderii bacteriilor - cont.

Explozia oxidativa
Activata in urma fagocitozei
Stimulata de PRRs
Consum crescut de O2
Produce substante care sunt toxice directe pentru
bacterii:
o Produsi derivati ai Oxigenului
o Produsi derivati ai Nitrogenului
NO (monoxidul de azot)
Produs de NO sintetaza inductibila (iNOS)
Enzima este indusa de cytokine, LT, TNF
Explozia oxidativa-speciile reactive ale O2

Speciile reactive ale oxigenului se formeaza prin


Explozia oxidativa, care presupune:
Cresterea consumului de O2
Glicogenoliza - cresterea oxidarii glucozei
Formarea anionului superoxid, apei oxigenate si a acidului
percloric:
o 2O2 + NADPH 2O2- + NADP+ + H+
(NADPH oxidaza)
o O2- + 2H+ H2O2 HO
(SOD superoxid dismutaza) (Fe2+ )
o H2O2 + Cl2 2HOCl
(MPO mieloperoxidaza - granulele azurofile,
doar in PMN nu si in MQ)
Radicalii derivati de O2 sunt detoxifiati de ceruloplasmina,
transferina, superoxid dismutaza (SOD), catalaza & glutation
peroxidaza (H2O2 )
Danger hypothesis

Naive
T cells
Signal 1
Signal 2 (co-
(co-
stimulation)

APC

Danger
signal
- infection
- tissue damage
- stress cells
Damaged Normal
- hypoxia cell cell
- temperature shifts
- hsp
Citokine Macrofag activat

IL-
IL-1 TNF-
TNF- IL-
IL-6 IL-
IL -12

EFECTE LOCALE
- activeaza - activeaza - activeaza Ly - activeaza NK
endoteliul vascular endoteliul vascular - producere Atc. - induce dif. Th1
- activeaza Ly - creste
- acces cel. efect. permeab. vasculara
- acces cel. efect.

EFECTE SISTEMICE
- febra - febra - febra
- producere de IL-6 - mobilizare metaboliti - inducere proteine
- soc faza acuta
Mediatorii specifici

Citokinele
Produsi celulari de natura proteica cu rol de mesaj pentru alte
celule, carora le spun cum sa se comporte
IL-1, TNF- si -, IFN- sunt importante in mod special in inflamatii.
Cresc expresia endoteliala a moleculelor de adeziune, activarea si
agregarea PMNs, etc.
Interferonii
Produsi de celulele infectate cu virusuri, actioneaza ca si mesageri de
scurta durata care protejeaza celulele invecinate de infectia virala.
interferon:
o Inhiba replicarea virala, creste numarul NK si induce antigenele MHC-I
interferon:
o Inhiba replicarea virala, creste numarul NK si induce antigenele MHC-I
interferon:
o Activeaza macrofagele si induce antigenele MHC-II
o Apararea imuna impotriva infectiilor si proliferarilor maligne.
Interferonii
Proprietati IFN- IFN- IFN-

NOMENCLATURA Type I, Type I, Type II, Immune


Leukocyte Fibroblast
Inductori MAJORI Virus Virus, LPS Antigens,
mitogens, TNF-
+ IL-12

Nr de GENE 26 1 1

SURSA CELULARA T cells, B cells Fibroblasts, T cells, natural


Macrophages epithelial cells killer cells

Dupa expunere corespunzatoare cele mai multe celule sunt apte sa produca cel putin un tip de IFN I.
Tipul I de IFN poate fi indus de asemenea de LPS (endotoxina bacteriana), IL-1 si TNF.
Sinteza IFN- este inalt reglata numai in anumite tipuri de celule si este indusa de stimuli specifici
IFN- factorul major de activare macrofagica; rol crucial intre mecanismele de aparare ne-
specifica a gazdei impotriva a numerosi patogeni.
Functii efectorii: Rolul interferonului
interferonului

Celula infectata viral Celula neinfectata


IFN receptor
IFN-
IFN-/

2-5 (A) PKR


Sintetaza
degradare mRNA translatie mRNA

Inhibare sinteza proteica


Pathogens Epithelial barrier Activated epithelial cells

Epithelial cytokines Permeabilised endothelium Cell & fluid migration

Interaction with
other cells of the
innate and adaptive
Opsonisation
immune systems
Phagocytosis
Timing of innate immunity after infection

Barriers - Epithelial activation - Complement -


Seconds Minutes Minutes Cytokines/chemokines -
Minutes to days

Neutrophils - Monocytes/macrophages -
Hours Hours to days NK cells -
Hours to days

Short-
lived
Long-lived & connect with
adaptive immune system
Eosinophil Eosinophil Basophil

Neutrophil Eosinophil

Neutrophil

Lymphocyt
e
Basophil
Monocyte
Eosinophils attacking a schistosome larva in the presence of serum (IgE)
from an infected patient.
Mast Cells

Resting Mast cell Degranulated mast cell


Mediators released include:
Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor,
Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-

Causing:
Vasodilation, increased vasopermeability, contraction of smooth muscle,
bronchoconstriction, increase neutrophil chemotaxis, increase eosinophil,
neutrophil and monocyte chemotaxis, anticoagulation, increased
fibroblast proliferation & platelet activation
Natural Killer (NK) cells

Non-T, non-B cells


No classical antigen receptors
Part of the innate immune system
Recognise and kill abnormal cells such
as tumour cells
Directly induce apoptosis in virus
infected cells by pumping proteases
through pores that they make in
target cells
Similar cytolytic mechanisms to
cytotoxic T lymphocytes (CTL)
Involved in antibody-dependent
cellular cytotoxicity (ADCC)
Missing self

MHC
NKR cls I
Celula Celula Absenta
NK tinta citotoxicitatii
NCR Ligand
activator

NKR
Celula Celula
tinta
Citotoxicitate
NK
NCR Ligand
activator
Interactions between phagocytes and other innate
immune components: Natural Killer cells

IFN
NK

Cytokines TNF, IL-12

Growth factors, angiogenic


factors, proteinases -
REPAIR & REMODELLING

Activated macrophage
Interferon
(IFN) receptor
Sistemul proteinelor plasmatice -
raspunsul imun nespecific umoral
Secreted Pattern Recognition
Molecules (sPRM)
Produse mai ales de ficat, dar si de fagocite, cu rol in:
Activarea Complementului
Opsonizarea celulelor microbiene

Proteine de faza acuta


Cascada coagularii
Complement System

Nonspecific defense system


The combination of antibodies with antigens does not cause destruction of
the antigens or pathogen.

Antibodies serve to identify the targets for immunological attack. Antibody-


induced activation of the complement.

The complement proteins are designated C-1 to C-9.


These proteins are in an inactive state. Become activated by the
attachment of antibodies to antigens.

Complement proteins can be subdivided into 3 components:


C1q, r, s: recognization.
C4, C2, C3: activation.
C5-C9: attack (complement fixation).

Activated via classic (C1) or alternative (C3) pathways to generate MAC (C5
C9) that punch holes in microbe membranes
In acute inflammation
o Vasodilation, vascular permeability, mast cell degranulation (C3a, C5a)
o Leukocyte chemotaxin, increases integrin avidity (C5a)
o As an opsonin, increases phagocytosis (C3b, C3bi)
Activation of C System - cont.

C4b C3b
+ +
C2b Bb
Imunitatea Innscut - faze
Rspunsul inflamator sistemic (1)
Induction of fever
Caused by many bacterial products (endotoxins
from G(-) bacteria)
Endogenous pyrogens from monocytes and
macrophages (IL-1 and certain interferons)
Increased WBC production / releasing
Increased synthesis of hydrocortisone and
adrenocorticotropic hormone (ACTH)
Production of acute phase proteins C-reactive
protein binds to membranes of certain
microorganisms to activate the complement
system
Rspunsul inflamator sistemic (2)
Rspunsul inflamator sistemic (3)
Febra are efecte pozitive i negative asupra infeciei i
funciei organismului

POSITIVE NEGATIVE
indicate a reaction to extreme heat enzyme
infection denaturation and interruption
stimulate phagocytosis of normal biochemical
slow bacterial growth reactions
increases body temperature > 39 C (103F) is dangerous
beyond the tolerance of some
> 41C (105F) could be fatal and
bacteria
requires medical attention
decreases blood iron levels
Imunitate innascuta
Nu este doar un sistem de aparare simplu,
menit sa tina in loc infectia pina la
interventia imunitatii dobindite
Instruieste sistemul imunitatii dobindite
pentru a raspunde la infectii

Decizia majora de a raspunde


sau nu unui antigen este luata de
imunitatea innascuta, prin
receptorii codificati in genom !
Immune responses

Barriers Skin & Mucous membranes


rapidly regenerating surfaces,
Invasion peristaltic movement, mucociliary
& infection escalator, vomiting, flow of urine/tears,
coughing

Innate immunity Cellular and humoral defences


+ lysosyme, sebaceous/mucous secretions,
stomach acid, commensal
organisms,complement proteins,
phagocytosis, NK cells

+ Inflammation
Cellular and humoral defences
Adaptive immunity Antibodies, cytokines, T helper cells,
cytotoxic T cells
Imunitatea
innscut
ROL i FUNCTII

originea si contextul
Recunoastere Ag

Functii efectoare
prevenire intrare microorganisme
eliminare patogene

Rol instructiv asupra imunitatii specifice

initierea si tipul raspunsului


INNATE ADAPTIVE IMMUNITY
IMMUNITY cellular immune response

PAMP

pathogen

TLR
Endocytic IL-12 Th1
PRR IFN-

CD80/86 CD28
DC
TCR Naive Th1
MHC-II
T cells IFN-

(adapted after Medzhitov R,


Nature Reviews Immunology, 1,
2001, 135-
135-145)
n ANTIGEN
n ANTICORP
Antigene - Anticorpi n REACTIA ANTIGEN-ANTICORP
n Aplicatii ale reactiei Ag-Ac

Dr. Minodora Dobreanu

Antigens Haptens
Antigen (Ag) the molecule an antibody (Ab) binds
to
n Small organic molecules can become antigens
usually a foreign substance if they bind to proteins.
each antigen has different sites that antibodies n Become antigenic determinant sites on the
can bind to, so that one antigen can be bound by proteins.
several different antibodies By binding haptens to proteins in the
laboratory, new antigens are created for
research or diagnostic purposes.

Allergen n hapten: low molecule weight substance that combines


with specific antibodies but is incapable of eliciting an
immune response by itself
examples in the case of allergy could be pollen,
(e.g., sugars, amino acids)
cat dander, or a chemical in soap

Antigens, immunogens, epitopes: Antigene


nantigen: a substance that interacts with either Factorii care determin / influeneaz potenialul imunogen al unui
antigen sunt:
antibodies (immunoglobulins) or antigen-specific T- n Dimensiunea moleculei (>10.000 D)
cell receptors (TCRs) found on T lymphocytes n gradul de diferen structural fa de componentele endogene
nimmunogen: substance that induces an immune n distana filogenetic ntre antigen i gazd
accesibilitatea epitopului pentru recunoatere imun
response (able to trigger) - Not every antigen can
n
n calea de administrare a antigenului (intravenos pentru Ag
trigger an immune response. corpusculare virusuri, bacterii; subcutan pentru Ag solubile
proteine, fosfolipide, acizi nucleici)
molecular size (MW of 10,000+), molecule n doza de antigen: ptrunderea ntr-un organism a unui antigen n
complexity, molecular form important in cantiti excesive, poate s produc paralizia sitemului imun al
gazdei, ducnd la manifestarea unei imunotolerane; tolerana imun
determining immunogenicity apare i dac un antigen este administrat n doze similare pe ci
diferite: enteral, subcutanat sau intravenos.
nonrepeating polymers (e.g., proteins, n frecvena de administrare a antigenului (administrri multiple l fac
carbohydrates) = good immunogens mai imunogen)
imunogenicitatea Ag solubile este crescut prin adugarea
repeating polymers (e.g., lipids, nucleic acids) = n
adjuvanilor (de exemplu la vaccinuri, adugarea srurilor de
poor immunogens aluminiu sau de calciu); nu se cunoate cu precizie mecanismul, dar
se pare c sunt angrenate i macrofagele

1
Antigene Antigene
Criterii de clasificare Criterii de clasificare

Sursa
Capacitatea de a induce un raspuns imun -externa
-antigene complete -interna (normale, modificate de diferiti factori)
-antigene incomplete (haptena)
Modalitate de obtinere Grad de inrudire genetica
-antigene naturale -antigene autologe
-antigene artificiale (antigene naturale modificate) -antigene alogenice
-sintetice (homopolimeri, heteropolimeri liniari sau -antigene xenogenice
ramificati)
Origine Tip de celula implicata in raspunsul imun
-antigene animale -antigene timodependente (antigene T-dependente)
-antigene vegetale limfocit T - limfocit B - sintetiza anticorpi :
-antigene bacteriene majoritatea antigenelor
-antigene virale -antigene timoindependente (antigene T-independente)
-heteroantigene (origini multiple) direct limfocitului B):
antigene cu epitopi repetitivi

How Antibody Binds to Antigen

n ANTIGEN
n ANTICORP
n REACTIA ANTIGEN-ANTICORP
n Aplicatii ale reactiei Ag-Ac
The top part of this figure shows how different shaped antigens
can fit into the binding site of antibodies: left, pocket; center,
groove; right, extended surface. The panels below show space-
filling or computer-generated models indicating where contact
between the peptide antigen and antibody occurs.

Recunoasterea Ag scopul raspunsului imun Imunoglobulinele


o imunoglobulinele
o receptorii pentru Ag de pe celulele sistemului imun Diversitate si heterogenitate

Definitie - Glicoproteine prezente in ser, fluide


tisulare i ca receptori membranari pe limfocitul B
(rol n contactul cu Ag pentru diferenierea Ly B n
plasmocit).

Clase i subclase (Izotopuri si Allotipuri)


(secventa de Aa, mrime, ncrcarea cu carbohidrai)
IgG : IgG1, IgG2, IgG3, IgG4
IgM
IgA : IgA1, IgA2
IgD
Abdul K. Abbas, Celular and Molecular Immunology, 6th Ed, 2007 IgE

2
Bifunctionalitatea moleculei de imunoglobulina Imunoglobuline - structura

Situsuri de legare specifica pentru antigen

Regiunea Fab

C1q
Regiunea Fc

Imunoglobuline - structura Antibody Structure


n 100 million trillion
antibody molecules that
contain 4 polypeptide
chains (Idiotypes).
n 2 long H chains are joined
to 2 shorter L chains.
Fab regions are
variable.
Provide specificity
for a binding to an
antigen.
Fc region of different
antibodies are
constant.
n B cells have antibodies
that serve as receptors
for antigens.

Several Flavours of Abs: Antibody Structure

3
Major antibody classes

Properties of antibody classes

Properties IgM IgD IgG IgE IgA


Heavy m
chain
mean 1 0.03 12 0.0003 2
human
adult
serum level
(mg/ml)
Half-life in 5 3 25 2 6
serum
(days)
Number of 5 1 1 1 1-3
four-chain
monomers
Special Early Found Activates Stimulate Found
properties appearance; mainly on complement; s mast mostly in
& function fixes B-cell crosses cells to secretions
complement; surfaces; placenta; secrete
activates traces in bind to histamine
macrophage serum macrophage s
s s and
granulocytes

Distributia Ig in diverse lichide biologice The Number Dilemma


Concentratiile imunoglobulinelor in secretiile umane externe (mg/l)

Secreie IgA IgG IgM n You have about a trillion different antibodies able to react
with millions of different types of Ag
Ser 2.500 10.047 1.500
Lacrimi 80-400 0-16 0-18 n but you only have about 30,000-60,000 genes which code for
all the proteins you need in your entire body, most of which
are not Ab
Fluid nasal 70-846 8-304 0
Saliv 194-206 42 64 n so there cannot be one gene for one antibody to code for
Fluid Intestinal 166 4 8 these we wouldnt have enough antibodies!
Urin 0,1-1,0 0,06-0,56 -
So how can your body produce Ab to so many antigens,
Fluid Cervical 3-133 1-285 5-118
even those its never seen?
Fluid Vaginal 35 52 -
Tonegawa Theory
Fundamental Immunology (5th Edition)
Lippincott, Williams & Wilkins Chapter 31 (p.971)

4
Since there are multiple types of each gene segment, there
are many thousands of possible V-D-J combinations so that
Antibody Genes each B cell gets a unique combination of segments! Additional
diversity occurs because there are two types of light chains.

Genes for antibodies arent like most other genes -


they come in pieces (gene-lets):
k - crz 2
l - crz 22
, , m, , - crz 14

variable segments (V) many different versions


diversity segments (D) several different versions
joining segments (J) a few different versions
constant segments (C) a few different versions that are
nearly identical

A unique recombination A unique recombination occurs in each B cell


occurs in each B cell

n each B cell combines these


gene segments to make an
Ab chain like shuffling a Unique combination of segments becomes joined by
deck of cards somatic gene rearrangement

- V, D, and J for the


heavy chain, V and J for
the light chain

n since there are multiple


types of each gene each B cell combines these gene segments to make an
segment, there are many Ab chain like shuffling a deck of cards
thousands of possible V-D-
J combinations so that - V, D, and J are joined to C for the heavy chain, V
each B cell gets a unique and J are joined to C for the light chain
combination of segments!

Mecanisme care contribuie la generarea


diversitii anticorpilor
H Lk Ll
Gene din linia
germinativ n ANTIGEN
Gene V 250-1000 250 2
Gene J 4 4 3 n ANTICORP
Gene D 12 0 0

Diversitate
n REACTIA ANTIGEN-ANTICORP
combinatorie
V x J (x D) 10.000-40.000 1000 6
n Aplicatii ale reactiei Ag-Ac
Asocierea lanului H si L
H x Lk 1 - 4 x107
H x Ll 5 - 10 x104

Total potenial
cu diversitate joncional 109 - 1011

5
Question:

What types of immune


reactions are IgG, IgA, IgM,
IgE and IgD involved in?

Functii efectoare Funcii efectoare (1) - Opsonizare

Imunoglobuline de membrana - receptori pentru antigen de pe


limfocitul B
ontogenia B - IgM, IgM+IgD, orice fel de Ig de suprafata
activarea B - legarea incrucisata a Ig de membrana de catre
Ag multivalente

Neutralizarea Ag de catre Ac circulanti


toxine, medicamente, virusuri, bacterii, paraziti se
complexeaza cu Ac si astfel nu se leaga de tinte celulare

Activarea complementului (IgG, IgM)


complexele imune (Ag-Ac) se leaga de C1q - activarea caii
clasice a complementului (IgG-C2, IgM - Cm3)
Ac se leaga la bacterii si maresc capacitatea de fagocitoza
Opsonizarea (IgG) - creste capacitatea fagocitant Cresterea fagocitozei implica intensificarea legarii de suprafata a
fagocitele au FcR si leaga particule opsonizate cu Ac bacteriilor opsonizate si activarea fagocitului (procese mediate de FcR)
IgM creste indirect fagocitoza C3b

Funcii efectoare (2) How an Antibody Works


When an Ab finds its Ag on an invader, it will bind there and
Citotoxicitatea celular mediat de Ac (IgG, IgE, IgA) act as a trash tag, marking it for destruction by killer
Celule care lizeaz celule: NK, neutrofile, eozinofile cells, macrophages or complement
Ac mbrac intele celulare care urmeaz a fi lizate
Receptor for
agregarea FcR de pe NK activare, TNF, IFN constant region The target cell dies
After binding, the
Antibody binds to
eozinofilele au FcR si leaga Ac (IgE, IgA) care au complexat Ag target antigen
of antibody on NK NK cell is signaled by apoptosis and/or
cell recognizes a to kill the target membrane damage
parazitare secretie de enzime bound antibody cell

6
Funcii efectoare (3) Functii efectoare (4)
Hipersensibilitatea imediata (IgE)
Imunitatea de la nivelul mucoaselor
mastocitele si bazofilele au FcR pentru IgE monomeric, agregarea FcR (IgA)
in prezenta alergenului duce la eliberarea de mediatori ai inflamatiei celulele epiteliale au FcR,
capteaz IgA din snge i l trec n
(amine vasoactive, leukotriene, prostaglandine, citokine)
secreiile mucoaselor ca IgAs,
hipersensibilitatea imediata unde neutralizeaz Ag

Imunitatea neonatala la mamifere (IgG


matern)
IgG matern trece prin bariera
placentar n circuitul sanguin
fetal (om)
IgG din lapte e captat de FcR din
intestinul nou-nscutului i intr n
snge (porc, oarece) invers ca
IgA

Inhibarea rspunsului imun (IgG)


LyB au FcR i leag Ac din
complexele imune se inhib
activarea B

Antibody functions Antibody functions

Monoclonal Antibodies n ANTIGEN


n Commercially prepared antibodies are used: n ANTICORP
To provide passive immunity
In research, clinical testing, and treatment of certain n REACIA ANTIGEN-ANTICORP
cancers
n Monoclonal antibodies are pure antibody preparations
n Aplicaii ale reaciei Ag-Ac
Specific for a single antigenic determinant
Produced from descendents of a single cell
n Hybridomas cell hybrids made from a fusion of a tumor cell
and a B cell
Have desirable properties of both parent cells
indefinite proliferation as well as the ability to produce a
single type of antibody

7
Aplicaiile reaciei Antigen-Anticorp
Tehnici de imunodetecie

Western Blotting
Imunoprecipitarea
Imunohistochimia / Imunocitochimia
Citometria in flux
Radioimunoanaliza (RIA)
ELISA - Elispot
Mutiplexare

8
SELF - non SELF

Limfocite T si B

MHC - Procesare / Prezentare


Antigene
Cells of the specific immune
system

T cell B cell

Involved with cell mediated


immunity
Involved with humoral
Two types: immunity
helper T cells (CD4)
Secrete antibodies
cytotoxic T cells (CD8)

Generally eliminate Generally eliminate


intracellular pathogens extracellular pathogens
B cells

Lymphocytes that react directly with


antigens
produced and mature in bone marrow
require stimulation from Helper T Cells
each B cell produces and wears a
unique antibody on its surface
clonal selection - when a B cell
encounters a matching antigen, it
begins to divide rapidly.
- some then become plasma cells that all
produce the same antibody (huge
numbers of antibodies -2000/second),
and then die. Others become memory
B cells.
the specific antibody produced by a
plasma cell is also secreted in soluble
form and circulates in the blood
T cells
T cells display TCR as their antigen recognition protein
When stimulated they become Cytotoxic or Helper T cells
Secrete cytokines that recruit other cells of the Immune
System
Types of T cells:
helper T cells start the immune response
Inflammatory T cells (TH1) - Activate macrophages to kill
intracellular bacteria
Helper T cells (TH2) - Activate B cells to make antibody
cytotoxic T cells (CTLs)
Kill virally infected cells
Kill cells containing cytosolic bacteria
Kill tumor cells
suppressor T cells suppress the activities of other T cells,
helping to end the immune response
TCR Structure The TCR plus CD3 is referred
to as the TCR complex

The TCR is composed


of two protein chain:
or
Crz
Crz

The extracellular part


binds to MHC+peptide.
The intracellular part
signals through the
CD3 complex.

The TCR is always associated


with CD3 () and 2
(zeta) chain. These molecules
are involved in signaling the T
cell when it engages antigen
TCR Gene Rearrangement

Similar to Ig gene rearrangement:


Heavy () chain has V+D+J(+C) segments.
Light () chain has V+J(+C) segments.

TCRs can be either or .


The mechanisms are the same as in B cells.
Creates a highly diverse repertoire of TCRs.
T cells

there are millions of different T cells the difference is


in their receptors (surface markers)

each T cell has a unique receptor that will recognize a


different foreign substance: V , VCDR1, CDR2,
CDR3

mature in the thymus, where they learn to tell the


difference between self and non-self
- critical, because if they did attack self,
autoimmune disease could result
MHC & T cells
T cells have a requirement to recognise both the ANTIGEN and the
MHC molecule
This is because the molecular structure of the MHC-Antigen complex is
arranged so that some of the polymorphic amino acids of the MHC
molecule are in direct contact with the TCR
Therefore T cell recognition of antigen is said to be MHC
restricted.

Helper T cells (Th)


recognize peptide
associated with MHC
class II molecules

Cytotoxic T cells (Tc)


recognize peptide
associated with MHC
class I molecules
TCR Structure accessory molecules

Accessory molecules
which also bind
MHC:

CD4 on helper
T cells, binds
MHC class-II.

CD8 on killer T
cells, binds MHC
class-I.
CD4 and CD8 Molecules

Transmembrane
molecules
Referred to as co-
receptor or
accessory
molecules
Member of the Ig
superfamily
T cells are either
CD4 CD4(+)
CD8(-)
CD8 CD4(-)
CD8(+)
HLA, MHC Clasa I si Clasa II

Clasa I Clasa II
Distribuie tisular Distribuie tisular
o Toate celulele o Celule prezentatoare
nucleate de Antigen (APC)
o Hematiile la unele Macrofage
specii Celule dendritice
Limfocite B
Celule stromale
timice
Functie Functie
o Prezinta antigene o Prezinta antigene
Ly Tc (CD8+) Ly Th (CD4+)
Proteinele MHC Clasa I si Clasa II

Clasa I Clasa II

Lanul Alfa Lanul Alfa


o 3 domenii externe o 2 domenii externe
o 1 transmembranar o 1 transmembranar
o 1 rest C citoplasmatic o 1 rest C citoplasmatic
o Codificat in MHC o Codificat in MHC
(crz 6p) (crz 6p)
Beta-2 Microglobulina Lanul Beta
o 1 domeniu extern o 2 domenii externe
o nu e ancorat in o 1 transmembranar
membrana o 1 rest C citoplasmatic
o fara rest citoplasmatic o Codificat in MHC
o codificat in crz 15 (crz 6p)
Proteinele MHC I si MHC II
MHC = CMH - Expresie

MHC I - pe toate MHC II - limitat, pe


celulele nucleate din imunocite: limfocitele B si
organism monocite (APC)
* Exceptii (He, celule ** Exceptii :
germinale) limfocitele T activate
Alctuit din 2 lanuri: Celule epiteliale / timus
ancorat - alfa cu 3 Alctuit din 2 lanuri:
regiuni ancorat - alfa cu 2
liber - beta 2 (2-mg) regiuni
Leag strns oligopeptide ancorat - beta cu 2
mici (aproximativ 9 Aa) regiuni
Sunt extrem de polimorfe Accept oligopeptide cu cel
puin 15 Aa
Polimorfism limitat
Distributia tisulara a exprimarii MHC

Expresia moleculelor MHC


difera de la tesut la tesut.
Moleculele MHC clasa I sunt
exprimate pe toate celulele
nucleate, foarte bine
exprimate pe celulele
hematopoietice.
Moleculele MHC clasa II
sunt exprimate nativ doar
pe un subset de celule
hematopoietice si de
celulele timice stromale,
alte celule exprimndu-le
doar in urma expunerii la
citokine inflamatorii
(interferon-).
MHC & Antigenele

MHC Clasa I MHC Clasa II-a


prezinta peptide prezinta antigene
endogene exogene care au fost
originea peptidelor: self fagocitate si procesate
sau de origine virala (de ex. bacterii) sau
deoarece MHC Clasa I Ag care nu necesit
este prezent pe toate procesare
celulele din organism, procesul este realizat
orice celula poate de celule
interactiona cu limfocite profesionale (de ex.
T, daca este infectata Macrofage, Celule
cu virus dendritice, LyB)
Structura moleculara a MHC clasa I

Doua structuri in helix


ale zonelor 1 si 2 ale
lantului formeaza
peptide-binding groove al
moleculei

TCRs se leaga la vrful


moleculei, care contine
cele mai polimorfe reziduuri.
MHC clasa I

2-microglobulina
Structura molecular a MHC clasa a II-a

Cele doua domenii


polimorfe (1, 1)
formeaz peptide-
binding groove al
moleculei

TCRs se leaga la
vrful moleculei,
care conine cele mai
polimorfe reziduuri.
MHC clasa a II-a

DR chain
Polimorfismul MHC clasa I

Variatia allelica survine


la nivelul unor situsuri
specifice ale moleculei
MHC. Aceste situsuri
corespund aminoacizilor
care vin in contact cu
antigenul.

Variabilitate similara
se observa in cazul
moleculelor MHC clasa
II.
Polimorfismul MHC clasa I

Structura cristalina
(clarificata trziu - 80) a
dovedit existenta unui sant
alcatuit din foi -pliate anti-
paralele (la baza santului) si
-helixuri (pe laturile
santului).
Regiunile 1 si 2 ale MHC
clasa I si 1 / 1 ale MHC
clasa II formeaza imaginile
in oglinda cu rolul de a
alcatui santul de legare al
peptidului antigenic.
Aminoacizii pozitionati de-a
lungul santului interactioneaza
(prin legaturi de hidrogen si
atractii de tip ionic) cu amino
acizii peptidului, stabilizindu-l
Expression of HLA is co-dominant

Father Mother

Kids
Polymorphism and polygeny of MHC

Two different properties make it difficult


for pathogens to evade immune responses

Definitions:

Alleles: different forms of one


gene
Allotypes: different forms of one
protein (isoforms)

Polymorphic: alternative forms of


one gene = Many alleles
Oligomorphic: a few forms of one
gene = Few alleles
Monomorphic: no polymorphism

Homozygous: same allele on both


inherited chromosomes
Heterozygous: different allele on
both inherited chromosomes
The MHC gene region

http://www.ncbi.nlm.nih.gov/mhc/MHC.fcgi?cmd=init&user_id=0&probe_id=0&source_id=0&locus_id=0&locus_group=0&proto_id=0&banner=1&kit_id=0&graphview=0
Genele MHC

Determina capacitatea unui organism de a


interactiona cu antigene
Sansa unei persoane de aceeasi etnie sa aiba gene
MHC compatibile este de aprox 1:1000
Genele MHC sunt denumite HLA de la human
leukocyte antigen
La alte specii au denumiri diferite (la soarece
genele sunt denumite H-2)
Genele MHC I si MHC II sunt niruite pe acelai
cromozom 6p
Genele MHC sunt poligenice i polimorfe
Genele MHC

Genele MHC utilizeaza loci diferiti pe cromozomul 6p


Genele MHC sunt mostenite de la ambii parinti, combinatia
genelor numindu-se haplotip

Expresia genelor MHC este co-dominanta


Indivizii pot exprima 2x3 subclase de gene I si 2x3(4)
subclase de gene II

Alte gene ale sistemului imun nnscut sunt localizate ntre


cele dou regiuni (I i II) n acelai ciorchine (cluster)
- C2, C4, factorul properdinic B, TNF
Major Histocompatibility Complex
Human MHC (HLA) encoded on cromosome 6
Genele MHC

Gradul de polimorfism variaza in cele doua clase


(Polimorfism = numarul de allotipuri sau izoforme posibile)

Genele MHC Clasa I sunt denumite: HLA-A (414 allele), HLA-B


(728), HLA-C (210), HLA-E (6), HLA-F (1), HLA-G (15)

Genele MHC Clasa II-a sunt denumite: HLA-DP(23 allele),


HLA-DP(120 allele), HLA-DQ(32 allele), HLA-DQ(68
allele), HLA-DR(3 allele), HLA-DR(503 allele), HLA-DM
(10), HLA-DX, HLA-DO (16), HLA-DZ

In consecin, moleculele MHC sunt limitate n capacitatea lor


de a lega peptide
Aceasta se numete restricie MHC
Figure 5-13
Human Leukocyte antigen (HLA=MHC
in humans) polymorphism - alleles

http://www.anthonynolan.com/HIG/index.html
Figure 3-24 part 1 of 2


Heavy Chain

Heavy Chains
Diversitatea imunologic generat de
locusurile MHC
HLA Diversity - 2 x 3 HLAs per person

http://www.stanford.edu/dept/HPS/transplant/html/tt_1.html
Celulele prezentatoare
de Ag - APC

Macrofage/monocite
Celule Dendritice
(Langerhans cells)
Ly B
Proprietatile
APC
RELATIONSHIP BETWEEN PATHOGEN AND MHC
COMPARTMENTS: A TWO-PRONGED APPROACH
TO ANTIGEN PRESENTATION

Two places for pathogens to be found within eukaryotic cells, cytosol/endogenous


and vesicles/exogenous.
Two pathways for antigen presentation for peptides (MHC class I and II).
One place for antigens to end up in order for the TCR to recognize foreign antigen-
on the surface of the cell in the context of MHC.
Demarcation
between MHC
class I and
class II
Processing
Pathways
MHC Class I pathway

Figure by Eric A.J. Reits


Endogenous Antigen Processing - Generation of
Peptide-class I MHC Complexes

Calnexin
Tapasin +
Calreticulin

TAP =
Transporter
associated
with Ag
Processing
MHC class II pathway

Figure by Eric A.J. Reits


Exogenous Antigen Processing
APC engulf antigens (bacteria, etc.)
MQ, dendritic cells eat anything
B cells only endocytose Ags bound by their mIg
receptors
Ag peptides generated in endocytic vesicles
Demarcation between MHC class I and
class II Processing Pathways
MHC Proteins

Ambele tipuri de proteine MHC sunt


importante pentru activarea Ly T:
Class I proteine MHC
o Sunt recunoscute de Ly T CD8
o Prezenta peptide (antigene) endogene

Class II proteine MHC


o Sunt recunoscute de Ly T CD4
o Prezenta peptide (antigene) exogene
Co-receptors on Helper and Killer T cells
Prezentare Ag
Toxins that act as superantigens:

superantigen binds to
regions outside the
normal binding sites on
TCR, MCH II

result is massive T
cell activation, cytokine
release, systemic
inflammation

Enterotoxin A - Staphylococcus aureus


Sistemul limfatic

Organe si tesuturi limfoide


Cells of the specific immune
system
T cell B cell

Involved with cell mediated


immunity Involved with humoral
Two types: immunity

helper T cells (CD4) Secrete antibodies


cytotoxic T cells (CD8)
Generally eliminate
Generally eliminate extracellular pathogens
intracellular pathogens
Lymphatic System: Overview

Function:

Drain fluid from


around cells
Absorb fat from
intestines
Circulate lymph
Filter lymph
Immunity
ANATOMY OF THE IMMUNE SYSTEM
The immune
system is localized
in several parts of
the body
immune cells
develop in the
primary organs
- bone marrow
and thymus
(yellow)
immune
responses occur
in the
secondary
organs (blue)
The bursa of Fabricius in birds
Originea celulelor implicate n
rspunsul imun
Celula stem
Hematopoietica Progenitor mieloid
Monocit
GM CSF M CSF

IL1,3,6 G CSF

IL 3
Progenitor
Basofil Macrofag
limfoid IL 5
Celula
dendritica
IL7
B Neutrofil

NK Timus Mastocit

Eosinofil
CD8 CD4
T T

Plasmocit
Lymphocyte Maturation
ANATOMY OF THE IMMUNE SYSTEM

Bone marrow blood-producing tissue located inside certain


bones (5% BW)
blood stem cells give rise to all of the different types of
blood cells

Thymus glandular organ near the heart where T cells learn


their jobs

Spleen serves as a filter for the blood


removes old and damaged red blood cells
removes infectious agents and uses them to activate cells
called lymphocytes

Lymph nodes small organs that filter out dead cells,


antigens, and other stuff to present to lymphocytes

Lymphatic vessels collect fluid (lymph) that has leaked out


from the blood into the tissues and returns it to circulation
B-CELL DEVELOPMENT
B-CELL DEVELOPMENT
Thymus
A bilobed organ in mediastinum
above the heart
The size of the thymus varies
with age
In infants, it is found in the
inferior neck and extends
into the mediastinum where
it partially overlies the heart
It increases in size and is
most active during childhood
It stops growing during
adolescence and then
gradually atrophies
T-CELL DEVELOPMENT
T-CELL DEVELOPMENT
Steps in T cell development

Step 1. Positive selection


occurs in the thymic cortex

T cells (CD4+CD8+) that


recognise foreign antigen
presented in the form of
antigen/MHC complexes by
antigen-presenting cells
within the thymus are
allowed to live MHC self-
recognition
molecules

This is called positive


selection
Steps in T cell development
Step 2. Negative selection
occurs in the thymic medulla.

T cells are presented with self


antigen/MHC complexes by
antigen-presenting cells within
the thymus
If T cells bind and recognise
these self antigens they are
destroyed by apoptosis
The immune system destroys T
cells specific for self-antigen

This is called negative


selection
T-CELL DEVELOPMENT

The result is a
T cell
repertoire that
recognises
foreign antigen
and is tolerant
towards self
antigen
Know your Flow!
Spleen
Largest lymphoid
organ
In upper left
quadrant of
abdomen
Has a hilum and a
capsule
Sinuses contain
blood instead of
lymph

Filters blood
Worn out RBC
Bacteria
Lymphocytes
Monocytes
Structure of the Spleen
Surrounded by a fibrous
capsule, it has trabeculae that
extend inward and contains
lymphocytes, macrophages,
and huge numbers of
erythrocytes and platelets
Two distinct areas of the
spleen are:
White pulp area
containing mostly
lymphocytes suspended on
reticular fibers and
involved in immune
functions
Red pulp remaining
splenic tissue (MQ)
concerned with disposing
of worn-out RBCs and
bloodborne pathogens
Additional Spleen Functions

Stores breakdown products of RBCs for


later reuse
Spleen macrophages salvage and store
iron for later use by bone marrow
Site of fetal erythrocyte production
(normally ceases after birth)
Stores blood platelets
Lymph

Fluid of the lymphatic system


Similar to blood plasma and interstitial fluid

Lymphatic Vessels

Transport lymph
Lymph is returned to the circulatory system at either the
right or left subclavian veins

Lymph Nodes

500-600, 1-10 mm
Filter lymph, Microorganisms, Cancer cells, Lymphocytes
Monocytes
Lymph Nodes
Lymph is filtered through
lymph nodes
Found in clusters
Vary in size
Principal groupings in deep
thoracic, abdomen and
cervical, axillary, inguinal
regions.
Provide biological filtration
Site of cancer growth and
metastasis
Lymph Node

Capsule, cortex and medulla:


Cortex contains lymph
nodules
Follicular dendritic cells
Germinal centers B cells
proliferate
Lymphatic vessels enter
node on convex side
Lymph passes through
irregular channels called
sinuses
Leaves node through one
or two efferent vessels at
the hilum or hilus
Flow of lymph
MALT
MALT mucosa-associated
lymphatic tissue is
composed of:
Peyers patches, tonsils,
and the appendix
(digestive tract)
Lymphoid nodules in the
walls of the bronchi
(respiratory tract)
MALT protects the
digestive and respiratory
systems from foreign
matter
Tonsils

Lymphoid tissue of tonsils contains


follicles with germinal centers
Tonsil masses are not fully encapsulated
Epithelial tissue overlying tonsil masses
invaginates, forming blind-ended crypts
Crypts trap and destroy bacteria and
particulate matter
Tonsils

Simplest lymphoid organs;


form a ring of lymphatic tissue
around the pharynx
Location of the tonsils
Palatine tonsils either
side of the posterior end
of the oral cavity
Lingual tonsils lie at the
base of the tongue
Pharyngeal tonsil
posterior wall of the
nasopharynx
Tubal tonsils surround
the openings of the
auditory tubes into the
pharynx
Aggregates of Lymphoid Follicles

Peyers patches isolated clusters of lymphoid


tissue, similar to tonsils
Found in the wall of the distal portion of the
small intestine
Similar structures are found in the appendix
Peyers patches and the appendix:
Destroy bacteria, preventing them from
breaching the intestinal wall
Generate memory lymphocytes for long-
term immunity
Gut associated lymphoid tissue (GALT) - tonsils,
adenoids, Peyers patches, appendix
Gut associated lymphoid tissue (GALT) - tonsils,
adenoids, Peyers patches, appendix
Lymphatic vessels

Resemble veins (same 3 layers)


Found throughout body except:
Avascular tissues
Central nervous system
Splenic pulp
Bone marrow
Lymphatic vessels join to form
lymphatic trunks

Lymphatic trunks
join to form :
Thoracic duct
(3/4 of body)
Right lymphatic
duct (drains right
arm, and right side
of head, neck and
upper torso)
These empty into
subclavian veins at
junction with
internal jugular
vein.
Fluid Movement

Formation of lymph:

Fluid leaves capillaries by


diffusion and filtration
Escaped proteins

If lymph flow blocked =


tissue swelling or edema

Specialized lymphatic
capillaries in vili of small
intestine transport lipids -
they are called lacteals, and
the fluid is called chyle.
Edema
Accumulation of
interstitial fluid
Causes of Edema
Blockage of lymphatic
system
Increased pressure in
veins
Lack of albumin
Decreases fluid
returning to blood
capillaries by osmosis
Inflammation
Overview of the immune response:

Antibody
mediated
Cell (humoral)
mediated
(CMI)

(Fig. 22.6, p. 762, Madigan et al.)


Antigen Recognition

Provides the key for the immune system


to recognize the presence of
intracellular microorganisms
MHC proteins are ignored by T cells if
they are complexed with self protein
fragments
Antigen Recognition

If MHC proteins are complexed with


endogenous or exogenous antigenic
peptides, they:
Indicate the presence of intracellular
infectious microorganisms
Act as antigen holders
Form the self part of the self-antiself
complexes recognized by T cells
T Cell Activation: Step One
Antigen Binding

TC cells are activated by antigen fragments


complexed with class I MHC proteins
APCs produce co-stimulatory molecules that are
required for TC activation
TCR that acts to recognize the self-antiself
complex is linked to multiple intracellular signaling
pathways
Other T cell surface proteins are involved in
antigen binding (e.g., CD4 and CD8 help maintain
coupling during antigen recognition)
T Cell Activation: Step One
Antigen Binding

Figure 21.16
T Cell Activation: Step Two
Co-stimulation
Before a T cell can undergo
clonal expansion, it must
recognize one or more co-
stimulatory signals
This recognition may require
binding to other surface
receptors on an APC
Macrophages produce
surface B7 proteins
when nonspecific
defenses are mobilized
B7 binding with the CD28
receptor on the surface
of T cells is a crucial co-
stimulatory signal
Other co-stimulatory signals
include cytokines and
interleukin 1 and 2
T Cell Activation: Step Two
Co-stimulation

Depending on receptor type, co-stimulators can


cause T cells to complete their activation or
abort activation
Without co-stimulation, T cells:
Become tolerant to that antigen
Are unable to divide
Do not secrete cytokines
T cells that are activated:
Enlarge, proliferate, and form clones
Differentiate and perform functions
according to their T cell class
Cytokines

Mediators involved in cellular immunity, including


hormonelike glycoproteins released by activated T
cells and macrophages
Some are co-stimulators of T cells and T cell
proliferation
Interleukin 1 (IL-1) released by macrophages co-
stimulates bound T cells to:
Release interleukin 2 (IL-2)
Synthesize more IL-2 receptors
Cytokines

IL-2 is a key growth factor, which sets up


a positive feedback cycle that encourages
activated T cells to divide
It is used therapeutically to enhance the
bodys defenses against cancer
Other cytokines amplify and regulate
immune and nonspecific responses
Helper T Cells (TH)
Helper T Cell
TH cells interact directly
with B cells that have
antigen fragments on
their surfaces bound to
MHC II receptors
TH cells stimulate B cells
to divide more rapidly and
begin antibody formation
B cells may be activated
without TH cells by
binding to T cell
independent antigens
Most antigens, however,
require TH co-stimulation
to activate B cells
Cytokines released by TH
amplify nonspecific
defenses
Cytotoxic T Cell (Tc)
TC cells, or killer T cells, are the only T cells
that can directly attack and kill other cells
They circulate throughout the body in search
of body cells that display the antigen to which
they have been sensitized
Their targets include:
Virus-infected cells
Cells with intracellular bacteria or parasites
Cancer cells
Foreign cells from blood transfusions or
transplants
T-CELL FUNCTIONS
T-CELL FUNCTIONS
Mechanisms of Tc Action

Figure 21.18a, b
A Cytotoxic T Cell Attacking and Killing a Virus-
Infected Target Cell

CELLS alive!

Here, the smaller cytotoxic T cell or Tc (arrow) is attacking and killing a


much larger virus-infected cell. The T cell will survive while the infected
cell is destroyed.
Other T Cells

Suppressor T cells (TS) regulatory


cells that release cytokines, which
suppress the activity of both T cells
and B cells
Gamma delta T cells (Tgd) 5 10% of
all T cells found in the intestines that
are triggered by binding to MICA
receptors
Selection of B cells by antigen (clonal selection)

Different types of B cells have


different receptor molecules.


When a pathogen (germ) locks
on to a receptor, that type of B
cell is selected.


The selected B cell divides
rapidly to make lots of copies
of itself. The copies make lots
of antibodies against the
pathogen.
Plasma cells secrete antibody at a high rate but can no longer respond to
antigen or helper T cells.
Clonal Selection Theory (continued)

Some of the cells


become plasma
cells that secrete
primary response.
Others become
memory cells that
secrete
antibodies during
the secondary
response.
Antigens select
lymphocytes that
are already able
to make
antibodies.
Memory & specificity key features
of the adaptive immunity
Cinetica rspunsului imun la o infecie
viral tipic
Raspunsul imun in tumori
Factori cancerigeni

o de origine extern, fizic (radiaii ionizante),


chimic (benzen, tetraclorur de carbon, etc) i
biologic (mai ales viral),
Exist virusuri oncogene:
de tip ADN: EBV (Epstein-Barr)- atac limfocitele B
determinnd limfomul Burkitt, Hodgkin sau carcinomul nazo-
faringean; HPV (Papiloma virus) determin carcinomul cervical
uterin; HBV (virusul Hepatitei B), poate cauza carcinom
hepatocelular
de tip ARN: HTLV (human T lymphotropic virus): poate
determina Leucemia, respectiv limfomul cu limfocite T; HSV 8
(Herpes simplex virus 8) poate determina sarcom Kaposhi
o sau de origine intern (oncogene)
Gene supresoare tumorale
Inhib sau frneaz creterea Gena supresoare p53 a
celular i ciclul celular, prevenind
dezvoltrii tumorilor crz
dezvoltarea tumorilor
17p13.1, este inactiv sau
Mutaii ale acestor gene lipsete la peste 50% din
determin ignorarea din partea celulele canceroase
celulei a unuia sau mai multor
componeni ai reelei de semnale
inhibitorii, nlturnd frnele
ciclului celular, ceea ce conduce la
o cretere celular necontrolat.
p53 = Proteina citopl. Supres. a
creterii tu, monitorizeaz
integritatea ADN n timpul
diviziunii celulare (poate opri /
ncetini ciclul celular n faza S,
pentru a permite repararea ADN) -
in caz de leziuni ireparabile,
declaneaz apoptoza celulei
Antigene tumorale
TSA-Tissue
TSA Specific Ag - Antigene care aparin doar celulelor
tumorale
TAA
TAA-Tissue Associated Ag - Antigene care aparin i celulelor
normale

Dei nu toate celulele neoplazice dispun de markeri deosebii de cei


fiziologici, la majoritatea pot fi identificate structuri antigenice
diferite, numite neoantigene
neoantigene. Aceste structuri pot fi nirate n
urmtoarele 2 grupe:

Antigenele retrogenetice
retrogenetice: n condiii fiziologice ele sunt
exprimate exclusiv pe suprafaa celulelor embrionare, dar nu i a
celor de dup natere. Din aceast cauz se numesc i antigene
oncofetale. n aceast grup sunt nirate: -fetoproteina (AFP)
i antigenul carcinoembrionar (CEA), care pot reapare dup
natere
Antigenele de difereniere:
difereniere apar numai ntr-o faz determinat
a maturaiei unor serii celulare, ca n cazul elementelor imature
din cadrul unor leucemii (limfatice sau mielocitare).
Antigenele de difereniere
Pentru leucemii acute:
LLA: CD10 (CALLA)-forma comun; CD19, TdT(n) - forma null
LLA cu preB: CD19, lanuri libere intracitoplasmatic
LLA cu T: CD3 (citoplasmatic), CD7, TdT(n)
LMA: CD13, CD33, mieloperoxidaza
Terminal deoxynucleotidyl transferase (nuclease)

Pentru leucemii cronice:


LLC cu B: CD19, CD21, CD5, CD23
Sindromul Sezary: CD3, CD4
LLC cu T: CD3, CD8

TdT-CD34-HLA DR-CD19-CD10-CD79b-cy Ig-FMC7-CD5-CD23-CD22-sIg-CD38


Markeri tumorali - definiie
Markerul tumoral este substana produs de ctre un esut tumoral,
care rmne n / pe celula productoare (markeri
markeri celulari, de ex.
crz Ph, p53, receptori hormonali)) sau este secretat n umorile
nconjurtoare (markeri
markeri umorali / solubili n lichide biologice) i a
crei identificare / dozare este util in diagnosticul sau management-
ul clinic al afeciunilor canceroase.
Natural Killer (NK) Cells
Lymphocytes that are
related to, but distinct
from T cells.
Provide first line of cell-
mediated defense.
NK cells destroy
tumors in a
nonspecific fashion.
NK cells attach to
cells that lack class-
1 MHC antigens.
Release
perforins
and
granzymes.
Do not require prior
exposure for
sensitization to the
tumor antigens.
Stimulated by
interferon.
Markeri tumorali - utilitate
screening populaie la risc (Calcitonina in familii cu
afeciuni neoplazice endocrine multiple tip 1, AFP la
pacienii cu ciroze hepatice alcoolice - risc nalt de carcinom
hepatocelular)

diagnosticul tumorilor, pentru completarea


simptomatologiei clinice (5 HIAA urinar n sindr.
carcinoide)
stabilirea prognozei (n unele cazuri, concentraia
markerului este n strns relaie cu masa esutului
tumoral)

monitorizarea rspunsului la tratament


chirurgical, chimioterapic sau radioterapic
stabilirea recurenei / recderii / metastazrii
Markeri tumorali clasificare
utilitate n practica clinic

A. Antigene oncofetale CEA i AFP


B. Carbohidrai CA 15-3, CA 27.29, CA 19-9, CA
125, CA 72-4, TPA, PSA
C. Hormoni hCG, Calcitonina

D. Proteine non-oncofetale Tireoglobulina,


Feritina, 2-mg, S-100, Bence Jones
E. Enzime NSE, TK
F. Markeri genetici p53
Caracteristicile unui marker
tumoral ideal
1.sensibilitate 100% - capacitatea de a identifica toi
pacienii cu tumor, n stadiu ct mai precoce;
2.specificitate 100% - capacitatea de a discrimina
ntre indivizii sntoi i cei cu neoplazii;
3.specificitate de organ capacitatea de a localiza
tumora;
4.corelaie ntre concentraia seric a markerului i
stadiul tumorii;
5.capacitatea de a indica modificrile induse de
tratament;
6.capacitate prognostic n funcie de concentraie.
Valoarea cut off: este valoarea concentraiei la care
sensibilitatea i specificitatea metodei sunt optime.
De obicei corespunde concentraiei la care
specificitatea este de minimum 80%
Antigene oncofetale CEA
n viaa embrionar se produce n pancreas i n tractul gastro-
intestinal.
Este o glicoprotein complex 180 kD cu 45-60% carbohidrai,
fixat n membrana celulei neoplazice (de unde se elibereaz n snge)
S-au descris cel puin 6 epitopi (heterogenitatea se datoreaz
coninutului variabil n glucide)

Valori normale serice: 0-4,6 ng/ml, Fumtori : < 10 ng/ml


Timpul de njumtire n ser : 1-2 sptmni

Valori crescute se ntlnesc n carcinoame ale tractului gastro-


intestinal: colo-rectal, pancreatic, gastric i pulmonar, mamar, cervical,
ovarian.
- n urin, concentraii de peste 35 ng/ml (dac infecia urinar
este exclus) poate indica un carcinom al vezicii urinare
valori crescute n lcr, apar n metastaze cerebrale
Antigene oncofetale CEA
Sensibilitatea n tu. primare i secundare

Tumori primare Se (%) Locul metastazei Se (%)


Carcinom pulmonar cu celule mici 60-70 Multiple 87
Carcinoame pulmonare altele 75-85 Ficat 71
dect cele cu celule mici
Oase 57
Cancer mamar preoperator (T3) 40-95
Cancer mamar postoperator 75-85 Plmni 56
Ficat 30-40 Peritoneu 33
Pancreas 30-40 Sistem limfatic 11
Carcinom colo-rectal preoper. 10-85
Carcinom colo-rectal postoper. 65-75
Concentraia plasmatic a markerului
Vezic urinar <10 se coreleaz destul de slab cu masa
Prostat <10 tumoral

Ovar 20-30 Se/Sp sunt modeste nu se poate


utiliza n scop de screening
Tiroid <10
Persistena valorilor patologice
Cap, gt 60-80 postoperator indic o tumor rezidual
Antigene oncofetale AFP
Este o glicoprotein de origine oncofetal, cu masa molecular 70 kD
(conine 4% carbohidrai), sinteza ei fiind suprimat la adult.
n cursul vieii intrauterine se formeaz n sacul vitelin, iar mai trziu
n ficat i tract gastro-intestinal (sinteza max - n spt 14-20), fiind o
protein plasmatic; n serul mamei concentraia max este atins n spt
35-38).

Valori normale: < 5 ng/ml (< 3,5 U/ml) la adult


2-5 mg/ml la ft, < 500 ng/ml la femeia gravid
Raportul normal al concentraiei de AFP n :
serul fetal : lichidul amniotic : serul matern = 10.000 : 100 :1

Timpul de njumtire: 5 zile

Valori crescute (peste 500 ng/ml): carcinom hepatocelular (poate fi


utilizat i pentru screening la persoane AgHBs+), testicular,
moderat crescute: n neoplazii testiculare, ovariene, retroperitoneale,
mediastinale i n boli non-maligne: hepatita alcoolic, ciroza hepatic,
hepatita acut viral, hepatita cronic activ
Sensibilitatea AFP n diverse afeciuni
tumorale i non-tumorale

AFP ng/ml <10 10-215 215-7500 >7500

Persoane sntoase ~100%

Ciroza hepatic/hepatita ~80% ~15% ~5%

Carcinoame extrahepatice ~65% ~30% ~5%


cu metastaze hepatice

Carcinoame primare ~5% ~10% ~35% ~50%


hepatocelulare
CA 15-3
Este o mucin (GP) polimorf epitelial Sensibilitatea CA 15-3 i CEA n
(300 - 450 kD) diverse stadii ale cc mamar

A fost identificat cu 2 Ac Mo
diferii: CA15-3 >
Stadiul CEA >
115 D8 (obinut prin imunizare cu 25
bolii 5 U/ml(%)
lipide din laptele uman) i U/ml(%)
DF3 (prin imunizare cu membrana
celulelor din cc mamar). T1 25 9
T2 32 11
Valori normale: 0-22 U/ml T3+T4 35 14
Timpul de njumtire: 4-6 zile Media: 30 11
Peste 40 U/ml probabilitatea Meta
neoplaziei este de 80%. Locale 46 12
Osoase 79 44
Are o sensibilitate semnificativ mai Hepatice 79 75
mare n comparaie cu CEA n
detectarea cc mamar aflat n Media: 69 41
diferite stadii
CA 15-3
CA 15-3 este folosit pentru monitorizare
postoperatorie, pentru c indic recidiva cu cteva
luni nainte ca diagnosticul clinic s fie posibil.

Lead time este perioada de timp care trece de la


prima cretere a concentraiei markerului i pn
cnd detectarea clinic a recderii devine posibil.

Valoarea CA 15-3 n monitorizarea postoperatorie


Cut-off Valori crescute nainte Valori n momentul Lead time
(U/ml) de diagnostic clinic (%) diagnosticului (%) maxim (luni)
25 70 76 9
40 63 76 7
Specificitatea CA 15-3
Concentraia CA15-3 n alte boli benigne i maligne
U/ml >22 >25 >30 >35 >40 X +/- DS
Persoane 9,4 5,5 1,3 0,5 0,09 13,3+/-6
sntoase%
Hepatita acut % 63 50 38 25 0 24,7+/-10
Hepatita cronic % 57 43 29 29 14 25,4+/-13
Ciroz % 39 39 31 27 23 26,8+/-19
Boli maligne
Stomac % 14 7 7 7 7 20,5+/-30
Ficat % 36 28 21 14 14 44,2+/-124
Pancreas % 80 80 70 70 50 51,2+/-53
Prostat % 100 50 30 28 20 26,2+/-11
Plamni % 71 71 60 53 40 63,6+/-76
Ovare % 66 64 55 39 34 59+/-64
Recomandri pentru utilizarea Markerilor
pentru dg. Cancerului de sn

CA 15-3 nu se utilizeaz ca i marker de screening de


mas (Se )
Determinarea receptorilor pentru Estrogen /
Progesteron este recomandat pentru a aprecia raspunsul
la terapie hormonal
Determinarea CD 15-3 i CA 27.29 sunt utile pentru
detecia precoce a recurenei la pacientele clinic
asimptomatice, dup tratament n std II i III
Determinarea CA 27.29 este utilizat doar in
monitorizarea pacientelor aflate n faz avansat de
boal
Descreterea CD 15-3 = succes terapeutic
CEA dg precoce al metastazelor
Antigenul specific prostatic
(Prostate-Specific Antigen)- PSA

Este o glicoprotein cu masa molecular de 36 kD (Ablin, 1970),


sintetizat de celulele epiteliale prostatice i secretat n lichidul
seminal.
Valori normale: 0-2,5 ng/ml, iar cut-off: 3 ng/ml
Timp de njumtire: 1-2 zile.
Valori crescute apar n boli benigne (prostatit, hipertrofie de
prostat) i n carcinom de prostat.
In intervalul 4-10 ng/ml, pentru excluderea dg de hipertrofie
benign, se recomand dozarea free PSA
Este un marker specific de organ, care poate fi folosit i pentru
screening-ul grupelor de risc (brbai cu vrste cuprinse ntre 50-70
ani), mai ales n asociere cu tueul rectal
PSA poate fi folosit i pentru monitorizarea terapiei i detectarea
recidivelor.
Recomandri pentru utilizarea PSA
pentru dg. Cancerului de prostat

PSA se utilizeaz n asociere cu tueul rectal ! (cut-off: 3 ng/ml)

Recoltarea sngelui pentru analiz, se face nainte de orice manipulare /manevre


asupra prostatei

La valori < 4 ng/ml, nu se recomand biopsia

n intervalul 4-10 ng/ml, pentru excluderea dg de hipertrofie benign, se recomand


dozarea f PSA (dac < 25% din PSA, risc )

Standard PSA Probabilitate % free PSA Probabilitate


cancer cancer
0-2 ng/mL 1% 0-10% 56%
2-4 ng/mL 15% 10-15% 28%
4-10 ng/mL 25% 15-20% 20%
>10 ng/mL >50% 20-25% 16%
>25% 8%
Recomandri pentru utilizarea CEA pentru
dg. Cancerului colo-rectal

CEA nu este recomandat pentru screening (Se ~),


este utilizat pentru completarea stadializrii

Dozarea CEA imediat post-operator nu este


recomandabil, datorit eliberrii unei mari cantiti
intra-operator

Poate fi utilizat pentru monitorizarea rspunsului


terapeutic i progresiei bolii
Recomandri pentru utilizarea Markerilor
pentru Cancerul pulmonar (NSE, CEA,
CYFRA 21-1, SCC)
Sensibilitateaa markerilor NSE (SCLC) CEA (NSCLC) n cancerul pulmonar

Tipul bolii N NSE (> 13 ng/ml) CEA (> 5 ng/ml)


Carcinom cu celule mici 81 74% 47%

Alte carcinoame 108 15% 46%

Boli pulmonare benigne 79 5.1% 13.9%

Persoane sntoase 92 0% ( 7.1 +/- 2.0) 0% ( 1.8 +/- 0.8)

CYFRA 21-1 fragmente solubile de Citokeratina 19 (n NSCLC)


Valori normale: < 3,3 ng/ml
Valori crescute: carcinom bronic non-parvicelular specificitate 95%.
Antigenul celulelor carcinomatoase scuamoase (SCC)
Valori normale: < 3 ng/ml
Se recomanda dozarea tuturor markerilor pt a putea depista Leading
marker-ul, cu care se va monitoriza tratamentul !!!
(cel mai adesea biopsia este imposibil !)
Recomandri pentru utilizarea
Markerilor pentru Cancerul gastric
Sensibilitatea i specificitatea comparativ a CA72-4, CA19-9, CEA
CA72-4 > 8 ng/ml CA19-9 > 37 U/ml CEA > 5 ng/ml

Carcinom 78% 60% 51%

Boli benigne 1% 7% 9%

Corelaia ntre valorile crescute ale CA72-4, CA19-9, CEA i stadiul tumorii

Stadiul CA 72-4 > 8ng/ml CA 19-9 > 37 U/ml CEA> 5ng/ml

I 14% 4% 1%
II 28,5% 12,5% 12,6%
III 64% 46% 32%
IV 85% 70% 65%
Recomandri pentru utilizarea
Markerilor pentru Cancerul ovarian

Nu se recomand utilizarea CA 125 pentru


screening-ul populaiei asimptomatice (Se )

Istoric familial de cancer n sfera genital se


recomand dozarea la 6 luni interval + Ultra-
sonografie transvaginal

CD 125 poate fi utilizat ca marker prognostic n


terapia primar
Markeri tumorali de prim
linie recomandai n neoplazii
Tumori Markeri tumorali
Hepatic primar AFP, CEA
Germinale AFP + hCG
Prostat PSA, fPSA
Gastric CA 72-4, CEA, TPA

Pulmonar cu celule mici NSE, TPA, CEA

Pancreatic CA 19-9, TPA


Ovarian CA 125, TPA,, CA 72-4

Vezica urinar TPA


Metoda Limita deteciei
(fmol) Colo-rectal CEA, TPA, CA 19-9
EIA 1-0,1
Neuroblastom NSE
FIA 0,01
Tiroidian Tireoglobulina, Calcitonina
LIA 0,01-0,00001
Combinarea markerilor
tumorali n practica clinic

ROC (Receiver
Operating
Characteristics)
markeri tumorali n
carcinomul mamar

CEA: 42%
hCG: 21%
CA 15-3: 30%
p53: 10%
C-erB-2: 20-30% MCA = mucinous carcinoma associated
antigen (cut off: 11-14 U/l)
Combinarea
markerilor
tumorali n
practica clinic

Carcinoamele
pulmonare:
Calcitonina: 54%

CEA: 88%

NSE: 90%

Carcinomul cu celule
n boabe de ovz:
C-k-ras2: 20%
C-myc: 45%
Imunoterapia in tumori
Imunoterapia cu anticorpi antitumorali
Doar anticorpii specifici antiTSA pot fi utilizati n scop
diagnostic sau terapeutic. Actualmente exist puini
anticorpi specifici reali antitumorali:
Receptorii B (n LLC); idiotipul anticorpilor care este
receptorul limfocitar B poate dezvolta antiidiotip (de ex.
anticorpi anti CD21).
O form mutant a receptorului factorului de cretere
epidermic (cu o del a domeniului extracelular) este antigenic
i poate fi administrat sub forma unui vaccin (cuplat cu
celulele tu) de baz pentru terapia cu anticorpi a tumorilor.
Terapia antitumoral cu anticorpi izolai: produce citoliz
mediat de complement sau prin fagocitoz (ADCC):
Anticorpi antiHER2/neu (Herceptin) -n cancerul de sn
Anticorpi antiCD20 (Rituxan) -n LLC
Anticorpii 17-1A reacioneaz cu un antigen asociat
carcinomului colorectal
Necesitatea obinerii unor anticorpi monoclonali umani sau
umanizai este obligatorie pentru a putea fi utilizai timp
ndelungat.
Imunoterapia in tumori
Terapia antitumoral cu anticorpi
cuplai cu toxine sau radioizotopi:
Anticorpii cuplai cu toxine (de ex. Ricina),
inhib molecule / procese intracelulare
eseniale.
Anticorpii cuplai cu radioizotopi mediaz
citoliza prin denaturarea ADN.
Acest tip de terapie poate ucide ns i celule
nvecinate normale (inocent bystander)
Terapia antitumoral cu anticorpi
bispecifici:
la TAA
la o molecul trigger de pe T (de ex. CD3
declaneaz eliberarea / formarea de
perforine)
Este terapia antitumoral de viitor,
deoarece utilizeaz nalta specificitate a
anticorpilor monoclonali i capacitatea
citolitic a sistemului imun celular specific.
Imunitate de transplant
Transplant
Definitii
Autogrefa: transfer de tesuturi de la la
acelasi organism (piele)
Allogrefa: transplant intre indivizi diferiti ai
aceleiasi specii
Isogrefa (singenic): transplant intre gemeni
identici (univitelini)
Xenogrefa: transplant de la o specie la alta
Compatibilitate de Transplant
Pentru a creste sansa de supravietuire a
transplantului:
Cel mai important: compatibilitate ABO
Absenta Ac citotoxici preformati impotriva
Ag HLA ale donatorului
Compatibilitate HLA, in particular pentru
locii D
VIROLOGICAL ASSESSMENT

Both donor and recipient are tested for: HBV, HDV, HCV,
HIV 1/2, CMV, EBV, HSV 1 and 2, VZV, HTLV 1/2 ,
rubella virus, toxoplasma gondii and chlamydia.

Methods
Indirect diagnostic tests (serological) - Antibodies
Direct diagnostic tests, molecular biology tests (PCR, RT-
PCR) DNA- RNA-virus
HLA
IMMUNOGENETICS
1. Cross- match
- CDC
- ELISA

2. HLA Typing by molecular biology methods PCR

SSOP- sequence-specific oligonucleotide probe hybridization


(medium resolution )
SSP sequence-specific primers (high resolution)
SBT Sequence Based Typing allele SEQR (the highest available
resolution)

3. Anti-HLA antibody detection and identification


- AHG CDC
- ELISA
Major Histocompatibility Complex

Lymphocyte crossmatch
Used to screen recipient serum for anti-HLA
antibodies
Recipients serum, complement and donor B
lymphocytes are mixed together in a test tube. Lysis
of donor lymphocytes is indicative of cytotoxic
antibodies in the recipients serum directed against
donor lymphocytes
The identity of these antibodies must then be
determined in order to find a suitable donor who is
negative for the corresponding HLA antigen(s).
Sample of cells or tissue

Combine DNA with sequence-


specific primer fix for each
allele

Amplify by
PCR
DNA
80ng for Class I
40 ng for Class II
Importance of DNA Quality

100 ng Genomic DNA 1% Agarose Gel


Assign-SBT Resolves Ambiguities
Sequences are arranged in layers
Master sequence
Types of Transplants
Corneal
Best graft survival rate since the cornea is
avascular and the lymphatic drainage from the
eye is not as well developed as in other tissues
Associated with transmission of prions-
Creukfeld-Jacob disease-Transmissible
spongiform encephalopathy; also has been
associated with amoebic transmission
(granulomatous amebic encephalitis)
Types of Transplants
Renal
Between living donors with a 2 haplotype match = 90-
95%, 5 year survival
With a 1 haplotype match = 80%, 5 year survival
Cadaver transplants between unrelated donors is the
most common type of transplantation. Similar statistics
to 1 haplotype match when the recipient receives
multiple blood transfusions prior to the surgery
(induces tolerance to the allograft) and is placed on
immunosuppressive therapy
Types of Transplants
Liver
In adults with chronic active hepatitis or
cirrhosis
In children with biliary atresia
1 year survival rate is slightly greater than 90%
Types of Transplants
Cardiac transplantation
In adults, used in patients with chronic ischemic
heart disease and congestive cardiomyopathy
In children, endocardial fibroelastosis is the
usual indication
Endomyocardial biopsies are the best means of
diagnosing allograft rejection
Approximately 80% of transplants survive 1
year
Types of Transplants
Bone marrow transplants
Used in the treatment of aplastic anemia,
leukemia and immunodeficiencies
Goal is to infuse donor marrow containing
pluripotential hematopoietic stem cells that will
eventually repopulate the lymphoid, erythroid,
myeloid, and megakaryocytic series in the
recipient.
GVH occurs in almost 2/3rds of cases
Increased incidence of CMV pneumonitis
Transplant Rejection

The chance of a sibling in a family having another


sibling with 0, 1, or 2 haplotype match is:
25% - 0 haplotype match
25% - 2 haplotype match
50% - 1 haplotype match
However, a 2 haplotype match is rarely
achieved due to crossovers between the
individual loci during meiosis when
homologous chromosomes line up close to each
other
Transplant Rejection
Three types of transplant rejections
Hyperacute rejection
Acute rejection
Chronic rejection
Transplant Rejection
Hyperacute rejection:
occurs within minutes of attaching the allograft to the
recipients blood supply
Due to the presence of an ABO mismatch or preformed
cytotoxic antibodies in the host against foreign HLA
antigens in the donor tissue (example; a blood group A
recipient would have anti-B IgM antibodies and would
react against a group B donor heart)
Hyperacute rejection is rare because ABO and
anti-HLA cytotoxic antibody screening is
performed prior to the surgery
Transplant Rejection

Acute rejection
Most common type of rejection encountered
Usually occurs within the first 3 months of the
transplantation
Involves cell-mediated and antibody-mediated reactions:
Cell-mediated has the greatest role in rejection
The type II antibody-mediated hypersensitivity produces
a necrotizing vasculitis with subsequent vessel damage
and intravascular thrombosis
Transplant Rejection
Acute rejection
Vessel events can occur over a period of time
leading to fibrosis and vessel lumen obliteration
The cell-mediated component involves
cytotoxic T cells producing extensive interstitial
infiltrate in the graft with edema and damage to
the tissue (Type IV hypersensitivity)
Can be reversible with immunosuppressive
drugs such as cyclosporin A, corticosteroids,
and OKT3.
Transplant Rejection

Chronic rejection
Irreversible
Occur over a period of months to years
Extensive fibrosis and loss of organ structure
characterize the histologic findings in the transplant
Activated macrophages release growth factors that
stimulate fibroblasts to deposit collagen
There is also chronic ischemia secondary to antibody-
mediated damage to the vessels
Transplant Rejection
Cyclosporin A inhibits CD4 helper T cell release
of interleukin-2 (blocks calcineurin) which
stimulates the proliferation of cytotoxic and helper
T cells
Corticosteroids inhibit macrophage production of
interleukin-1 and tumor necrosis factor and are
cytotoxic to immature cortical derived thymocytes
OKT3 is a monoclonal antibody preparation that
attaches to the CD3 antigen receptor of T cells,
blocking their reaction with the graft
ID/CC A 45 year old male with refractory acute myeloid
leukemia is brought to the emergency room with fever, a
generalized rash, jaundice, right upper quadrant pain,
severe diarrhea, and dyspnea; two months ago, he
underwent an apparently uncomplicated bone marrow
transplantation.
HPI Prior to the transplant, he received radiotherapy and
chemotherapy as well as broad-spectrum antibiotics
PE VS: normal blood pressure. PE: cachexia; moderate
dehydration; 2+ jaundice; violaceous and erythematous
macules as well as papules and bullae with scale
formation over extremities
Labs Elevated IgE level. CBC/PBS: falling blood counts;
relative eosinophilia. Elevated direct serum bilirubin
and transaminases, no infectious agents on stool
exam
Graft versus Host Reactions

Potential complication in bone marrow and liver


transplants and in blood transfusions administered to
patients with T cell immunodeficiency
Donor lymphocytes produce interleukin-2
-->activation of NK cells (primary effector cells in acute
GVH reactions)-->lymphokine-activated NK cells are
called LAKs and produce extensive epithelial cell
necrosis in the biliary tract (jaundice), skin
(maculopapular rash), and GI tract (diarrhea)
Graft versus Host Reactions
May progress into chronic GVH which is
marked by the presence of extensive
fibrosis
To lessen the risk of GVH, donor tissue is
pretreated with anti-thymocyte globulin to
remove donor T cells.
Cyclosporin A is used also
Transplant complications
Immunosuppressive therapy has increased
the incidence of:
Cervical cancer
Malignant lymphomas (immunoblastic)
Basal and squamous cell carcinomas of the skin
Squamous cell CA is the most common overall
malignancy
Other complications include infection and
bone marrow suppression
Diagnostic Immunopathology.

Immunodeficiency Disorders
What is Immunodeficiency?
Under-reaction to antigen

A failing of one or more of the bodys defensive


mechanisms resulting in morbidity or mortality.
Any part of the immune system can be deficient
cells, proteins, signalling mechanisms.
The body is susceptible to infection by organisms that
meet with little or no resistance.
Or, in certain cases, other homeostatic systems in the
body will be disrupted by the defect.
Severity is variable.
Immunodeficiency may be Primary or Secondary.
Learning Points

Be aware of the range and classification of


immunodeficiency disorders
Understand the nature of disease,
diagnosis and therapy options for primary
immunodeficiencies involving:
Phagocytes (Chronic Granulomatous Disease)
The Complement cascade
(HereditaryAngioedema)
Lymphocytes (X-Linked Agammaglobulinaemia)
Primary Immunodeficiency:

Arises without a pre-existing


causative disorder.

Usually has a genetic basis.


Secondary Immunodeficiency:

Arises because of another pre-existing


pathology or intervention, e.g.
Infection
Renal failure, or protein losing enteropathy
Leukaemia or Lymphoma
Myeloma
Extremes of age
Certain Drug Therapies
Immunodeficiency Disorders
Most immunodeficiency disorders are
acquired (most common acquired disorder
is AIDS)
Approximately
50% are B cell disorders;
40% T cell disorders; and
5% each for phagocytic and complement
problems
Most cases are in children less than 15
years of age (80% male)
Under-reaction to antigen
Immunodeficiencies

occur when some part of the immune system


is defective or missing
T cells, B cells, phagocytes, or complement

these deficiencies are grouped as:


primary: inherited or congenital
severe combined immunodeficiency
(SCID)
secondary: acquired
HIV infection David Vetter 1972-1984,
the original bubble boy
Phagocyte deficiencies:
Cyclic neutropenia, oscillation of neutrophil numbers.
Chronic Granulomatous disease,
Specific granule defects, defective killing.
Adhesion molecule defects, affects chemotaxis.

Antibody deficiencies:
X-linked agammaglobulinaemia
Selective Ig deficiency, differentiation failure leading to
decrease in one or more of the IgG subclasses or IgA
subclasses.
Common variable immunodeficiency (CVID), a common but poorly
defined collection of syndromes with reduced IgG, and
IgA/IgM.
T cell deficiencies:
DiGeorges Syndrome, gene defects affecting thymus
development and hence T cells. Now known as CATCH-22
syndromes.
Wiskott-Aldrich, progressive reduction in T cells, also low
platelets and low IgM.

Complement deficiencies:
Deficiency of individual components, e.g. C3, C5 C9, or
inhibitors Factor H, Factor I.
Hereditary Angioedema (C1-INH deficiency).
Primary Immunodeficiency:

Phagocyte-specific Disorder
?
Chronic Granulomatous
Disease (CGD) ?
What is CGD?
A group of diseases in which there is a defect in a
major killing mechanism of phagocytes.
The diseases are rare, inherited and result from

? genetic abnormalities in key enzymes of the


oxidative burst.
CGD is the most significant of the phagocyte
defects.
Patients are vulnerable to specific bacterial and
fungal infections.
?
Clinical findings
Recurrent severe bacterial and fungal infections
occurring at sites acting as environmental barriers:
Skin -Lymph nodes
Liver or spleen -Lungs
Gastrointestinal tract
Infecting organisms are specifically catalase
positive which means the faulty phagocyte cannot
use the organisms endogenously produced hydrogen
peroxide to kill them.
Patients are no more susceptible to viral infection
than anyone else.
Organisms include: Staph. Aureus, Salmonella,
Pseudomonas, Aspergillus, Candida..and unusual
organisms such as Serratia marcescens

Patients are usually children, although milder forms


may present in adulthood

Symptoms may include boils, diarrhoea, gum disease,


fever, abscesses, lymphadenopathy, pneumonia, GI or
urinary obstruction.

Chronic inflammation at various sites e.g. gingivitis,


lymphadenopathy, granuloma.

Delayed growth and malabsorption in children.


Laboratory Diagnosis
Patients often
have an elevated Nitroblue tetrazolium test
white cell count (NBT) where NBT (colourless)
(neutrophil is reduced to formazan (blue)
by the phox enzymes.
leucocytosis).
Slide or tube assay observing the
presence of blue inclusions in
phagocytes.
Neutrophils are
morphologically
normal. Dihydro-rhodamine (DHR)
reduction assay fluorescent
rhodamine (reduced DHR) is
Chemotaxis and detected by flow cytometry.
endocytosis are Flourescence in cells is measured and
normal. flourescing cells are counted.
When to suspect CGD?

In a patient (particularly a male child):


with recurrent infection with catalase
positive organisms
Infection with rare organisms
Infection in unusual sites e.g. St.
aureus liver abscesses
Therapy and Management
Prompt aggressive treatment of
infection with antibiotics.
Antibiotic prophylaxis.
Trials of Gamma IFN may reduce
number and severity of episodes.
Steroids and NSAIDS sometimes
used to treat granulomas.
Tissue biopsy/drainage may need
hospitalisation.
Individual susceptibility varies but
1 serious infection can be expected
every 3-4 years in X-CGD cases
even with prophylaxis.
Primary Immunodeficiency:

Disorder of the Complement


System

Hereditary Angioedema (HAE)


Complement Disorders

C1 esterase inhibitor deficiency (hereditary angioedema)


Autosomal dominant disease resulting in excessive
release of C2-derived kinins that increase vessel
permeability resulting in edema
See involvement of the face and oropharynx
sometimes with obstruction of the airway; also GI
Laboratory tests show a decreased level of C4
(best screen) and C2. A low level of C1 esterase
inhibitor is the confirmatory test
Treatment with synthetic androgens which increase
the synthesis of the inhibitor
? What happens in HAE? ?
A deficiency of C1INH due to genetic mutations
of the C1INH gene on chromosome 11p11-q21.

The loss of control of the complement and


contact systems results in severe subcutaneous
and submucosal oedema due to fluid leakage into
the extravascular space.

Patients are also susceptible to immune complex


disease and SLE (failure to remove ICs).
Secondary or Acquired forms (AAE) occur in

?
some LPDs or in association with C1INH
autoantibodies.
?
Different variants exist:
Variant I Variant II

80-85% of cases 10-15% of cases


Low levels of normal Normal or increased levels of
C1INH are found (10- abnormal C1INH are found
30% of normal) Autosomal dominant, genetic
C1INH present is mutations are usually single
produced by the normal point mutations
allele
Autosomal dominant, Variant III
genetic mutations often Not associated with C1INH
missense mutations deficiency (X-linked)

No one with homozygous HAE has ever been observed.


Clinical findings

Occurs in childhood or adolescence.

Extreme swelling of tissues oedema.

Often in hands, face, feet, airways.

Pain and swelling of abdomen, nausea and vomiting often


mistaken for acute abdomen.

Swelling of larynx may cause fatal asphyxiation.

Tingling or tightness may precede attacks.

Oedema lasts usually 24-72 hours.

Episodes may be as frequent as weekly or as rarely as once


a year or less.
Triggers
Minor trauma
Surgery
Anxiety/stress
Mild illness colds or flu
Dental procedures
Hormones pregnancy or OCP
Physical activity typing, writing, gardening
etc.
Laboratory Diagnosis

Firstly - measure C4 levels.


If low:
Measure C1INH level (single
radial-immunodiffusion)
and function (ELISA).
Low C1INH with normal C1, C3, C1q (Type I HAE)
Normal or raised levels of dysfunctional C1INH with
normal C1, C3, C1q (Type 2 HAE)
Primary Immunodeficiency:
Lymphocyte-specific disorder
affecting antibody production.

X-Linked Agammaglobulinaemia
- XLA (Brutons Disease)
Clinical findings
LITTLE BOYS WITH BIG INFECTIONS!

Symptoms appear at 6-9 months of age (after loss of


maternal Ig) or as late as 3-5 years of age.

Sites of infection: mucous membranes, ear (otitis


media), lungs (bronchitis/pneumonia), blood (sepsis), gut
(Giardia, or enterovirus), skin, eyes, meningitis.

Also seen: joint problems, kidney


problems, neutropenia, malignancy in
older patients.
Typical organisms involved include: Haemophilus
Influenzae, Streptococcus Pneumoniae.

Affected boys have little or no tonsils or palpable


lymph nodes.

Growth retardation may be present.

There may be no family history of XLA.

If diagnosis has been delayed then permanent lung


damage (bronchiectasis), or hearing loss due to
repeated infection may be present.
Significant Laboratory Findings

Serum protein electrophoresis


Microbiological evidence of
early-onset, severe, recurrent
infection.

Profound reduction in
circulating B lymphocytes.

Hypogammaglobulinaemia
(decreased immunoglobulin
levels) absent in severe
cases.

For confirmed diagnosis


mutation in the Btk gene.
Counting lymphocytes by
Flow Cytometry
488 nm

CD19-PE

CD3-FITC

CD45-Per CP
Lymphocyte subsets by Flow
Cytometry
Therapy Options & Management
Intravenous
Immunoglobulin (IvIg).

Prompt treatment of any


infection and use of
prophylactic antibiotics.

Avoidance of live
vaccines (Polio), including
siblings.
Education of patient and
family.

Genetic counselling.
Prenatal testing.
Prognosis

No treatment:
Death at an early age.

Treatment but with chronic lung disease:


May have a shortened life span.

Early diagnosis and treatment:


Normal active life.
AIDS

Acquired Immunodeficiency caused by the human immunodeficiency


syndrome is the most virus (HIV - 1984) and is spread by
common acquired contact with body fluids
immunodeficiency in the E.U.,
U.S., first identified in 1981 infects CD4+ (helper) T cells, which
HIV 1 is the most common decrease in number
isolate in the E.U., U.S.,
Canada; HIV 2 is primarily decreased numbers of CD4+ T cells lead
isolated in West and Central
Africa-transmitted to increased susceptibility to
heterosexually opportunistic infections.
AIDS is the most common
cause of death in men and treatments include drugs that inhibit
women between 25-44 years RT-ase, the activity of HIV proteins,
of age
thereby preventing production of the
virus
AIDS
Modes of Transmission
Subsets of individuals that become HIV positive in decreasing
order of frequency
Homosexual or bisexual men
IV drug abusers
Heterosexual contacts
Individuals receiving blood or blood products (includes
hemophiliacs)
Men account for approximately 88% of all people who are HIV
positive (women-IV drug abusers- represent the remainder)
Adults:
Intimate sexual contact is the most common mode of transmission,
usually by anal intercourse between homosexuals or by vaginal
intercourse in heterosexuals
Inoculation of the virus through blood or blood products is the second
most common mode of transmission with the majority of cases related
to the sharing of needles between intravenous drug abusers and less
commonly by receiving a blood transfusion (1:40.000 to 1:200.000 risk)
Health care workers most commonly contract the disease from
accidental needle sticks where the risk for becoming HIV positive
is 0.3%
AIDS
Modes of Transmission
Children
Aids is contracted in greater than 90% of cases from an infected mother
who is usually an intravenous drug user
Transplacental (vertical) transmission accounts for approximately 30-50%
of cases, while perinatal factors associated with blood contamination
during delivery and breast feeding (most common perinatal factor)
represent the remainder of cases
Pregnant women who have CD4 helper T cell counts less than 400
cells/uL or who are positive for the viral p24 antigen (a marker of
disease activity) have an increased risk for transmitting HIV to their
baby.
Treatment of asymptomatic pregnant women with AZT has reduced the
rate of infants developing AIDS from 13-40% to less than 10%
All newborns of HIV positive women are HIV Ab positive due to
transplacental passage of IgG antibodies; therefore, documentation
of HIV infection in infants is often difficult during the first 2
months
Tests in this setting that are useful include the isolation of the
virus in peripheral blood monocytes, the detection of viral RNA
using PCR, and assays for the p24 antigen in the serum
AIDS
AIDS virus attacks and destroys
CD4 T helper cells by first
attaching to the CD4 molecule on
the surface membrane of the
lymphocyte with the gp120
envelope protein

A co-receptor must also be


present with the CD4 molecule for
fusion and entry of T cell tropic
strains of HIV-1.

The co-receptor is CXCR4 on T


cells and chemokine receptor
CCR5 on macrophages

Macrophages are the primary


reservoir for the virus and are
the vehicle for carrying the
virus into the CNS
AIDS
Following binding, fusion with
the host cell membrane occurs
via the gp41 molecule.

The HIV genomic RNA is


uncoated and internalized.
Reverse transcriptase catalyzes
the reverse transcription of
viral RNA into double-stranded
DNA.

The virally encoded enzyme,


integrase, causes the viral DNA
to be integrated into the host
cell DNA
AIDS
Cells with CD4 molecules on their
surface in which the virus can
replicate without killing the cell
are monocytes, tissue
macrophages, dendritic cells in
the skin and lymph nodes, and
microglial cells (macrophages) in
the CNS

Host cell activation is CRITICAL


to the pathogenesis of HIV,
because viral DNA cannot
integrate efficiently into the
host cells genome unless
activation has occurred

Activation of the host cell is also


required for initiation of
transcription of the integrated
proviral DNA into either genomic
RNA or mRNA.
HIV Life Cycle
HIV & Immune System
AIDS
Clinical Findings

Divided into acute, latent and late phases


Acute phase
Develops within 3-6 weeks of contracting the
virus
Initial infection is characterized by fatigue,
sore throat, and lymphadenopathy
CD4 counts are usually normal, but the p24
antigen is increased
AIDS Acute phase

p24
High RNA
levels
CD4+ cell
counts - normal
AIDS: Early Symptomatic
Disease
Generalized lymphadenopathy
The presence of enlarged lymph nodes (> 1 cm)
in two or more extrainguinal sites for more than
3 months without an obvious cause.
This finding is not associated with an increased
likelihood of developing AIDS; however, a loss
in lymphadenopathy or a decrease in lymph node
size may be a prognostic markers of disease
progression
AIDS: Early Symptomatic
Disease
Other non-specific findings:
Fever
Weight loss
Diarrhea
Malaise
A distinct group of patients with early
AIDS develop immune thrombocytopenic
purpura (ITP) due to the presence of
anti-platelet antibodies.
AIDS
Laboratory Findings
Antibodies against gp120
and gp160 (ELISA test)
are not usually present
for approximately 4-8
weeks (window period);
screening tests in
blood banks may be
negative during this
phase
Positive ELISA screens
are confirmed by the
Western blot analysis
which detects p24 and
gp41 antibodies in order
to increase overall
specificity Testul Western Blot. Linia 1: CTR pozitiv, 2: CTR negativ, 3-4: teste
pozitive HIV-1, 5-6: teste pozitive HIV-2, 7-10: teste pozitive seriate la
un pacient infectat HIV-1
AIDS
Asymptomatic latent phase
Virus is actively
proliferating and is
present in follicular
dendritic cells
(macrophages) in
lymph nodes
The rate of disease
progression
correlates with HIV
RNA levels
The CD4+ cell counts
fall progressively
during this
asymptomatic period
at the rate of
approximately 50
cells/microliter/year
AIDS
Symptomatic phase
Clinical Findings
Follows an average span of 10 years
CD4 count drops to below 400 cells/uL
p24 antigen appears again
Patients develop minor opportunistic infections,
but are not severe enough to be indicative of a
defective cell-mediated immune response
Oral lesions: Thrush, hairy leukoplakia and
aphthous ulcers are very common during this
stage. The first two are associated with declining
immunologic function (< 300 CD4+ T cells)
AIDS
Advanced AIDS
Diagnosis of AIDS is based upon an opportunistic
infection (usually Pneumocystis carinii pneumonia)
and/or a CD4 count of 200 cells/uL in an HIV
positive individual
Most common cause of death today is bacterial
pneumonia (most common pathogen is Mycobacterium
avium intracellulare MAIC- causing bacterial
pneumonias), disseminated CMV infections, and
Streptococcus pneumoniae
Due to the use of trimethoprim and aerosolized
pentamidine, Pneumocystis carinii is no longer the most
common cause of death
Average life span from beginning of infections to death
is 10 years
Organ Pathology in AIDS

Lungs are the most frequently involved


organ in AIDS
Gastrointestinal tract
Kaposhis sarcoma is the most common cancer in
AIDS and the most common cutaneous lesion;
associated with HHV-8
Organ Pathology in AIDS

Other malignancies include:


Cervical cancer (HPV)
Anal squamous carcinoma (HPV)
Non-Hodgkins lymphomas
Primary CNS lymphomas (HIV)
Organ Pathology in AIDS

Endocrine disease
Adrenal insufficiency is very common
HIV induced abnormalities in steroid
synthesis, adrenalitis from CMV,
ketoconazole therapy
Clinical diagnosis of adrenal insufficiency
should be suggested by hypotension with
electrolyte disturbances
Organ Pathology in AIDS
CNS and PNS disease
Dementia
Meningitis
Peripheral neuropathies
Spinal cord lesions
Opportunistic infections
Toxoplasmosis is the most frequent cause of encephalitis
and a space occupying lesion in the CNS
CMV chorioretinitis is the leading cause of blindness in
AIDS
Cryptococcus is the most common cause of meningitis in
AIDS
Organ Pathology in AIDS
Renal Disease
Focal segmental glomerulosclerosis (HIV-
associated nephropathy) presents with
nephrotic syndrome - primary renal disease
Dermatologic Disease
Seborrheic dermatitis
Seen in up to 50% of patients with HIV
Caused by Malassezia furfur
Hematologic problems
Cytopenias (neutropenia, lymphopenia,
thrombocytopenia)
Autoantibodies against platelets
Anemia of chronic disease
Autoimmune hemolytic anemia
AIDS
Laboratory Abnormalities

To detect Antibodies against gp120 and


gp160
Detected by EIA (enzyme-linked immunoassays)
within 4-12 weeks after contracting the virus)
False negatives in patients who are
immunocompromised or who have serum
collected before the antibody has developed
False positive in patients with autoimune
diseases, hepatitis, receiving recent vaccins,
with anti MHC II Ab
AIDS
Laboratory Abnormalities
AIDS antibodies Other laboratory
Duplicate negative tests
are reported as negative abnormalities
Second test turning
positive must be Reversal of the CD4/CD8
verified by obtaining suppressor T cell ratio
positive results in 2 out
of 3 repeated tests Absolute lymphopenia
All positive or
indeterminate EIA tests Hypergammaglobulinemia
are confirmed with a secondary to polyclonal B
Western blot assay
which measures p24 and cell stimulation by EB
gp41 antibodies to virus and CMV
improve the overall
specificity of the test Decreased mitogen
A positive EIA in
combination with a positive blastogenesis
Western blot has a positive Anergy to skin testing
predictive value of 99.5%
Western Blot Results
AIDS
Laboratory Abnormalities
Other laboratory
abnormalities
Decreased production
of cytokines
NK cells are not
activated since gamma
interferon from CD4
cells is decreased
Increased p24 core
protein indicates
active disease and has
two peaks; one during
the acute infection
and the other at the
onset of AIDS
AIDS
Treatment and Prevention

Initial treatment: Azidothymidine (AZT)


Blocks reverse transcription
HIV reverse transcriptase is not accurate and produces
frequent transcription errors
This high mutation rate causes resistance to drugs
Treatments include:
Reverse transcriptase inhibitors
Protease inhibitors (saquinavir and ritonavir)
New drugs currently being developed that block HIVs entry to
helper T cells
Other drugs include
Gamma interferon
Granulocyte-macrophage colony stimulating factor
Granulocyte colony stimulating factor
AIDS
Treatment and Prevention
Prevention in order of decreasing
effectiveness
Abstinence
Monogamous relationship with a confirmed
seronegative partner
Manual sex
Kissing
Intercourse with latex condoms using
nonoxynol-9-a spermicide with some anti-viral
activity
Bazele celulare i moleculare ale
reactiilor imune patologice
IMUNITATEA PATOLOGICA

exces

(hipersensibilitate,

autoimunitate)

deficit (genetic,

dobndit)
Hipersensibilitate - Hiperergie

= Rspuns Imun care cauzeaz leziuni tisulare

Se disting diferite tipuri de hipersensibilitate dup:


Evoluia n timp
Tipul de protagoniti imuni implicai (Ac sau Tc)

Alergiile mediate de Ac sunt de tip imediat/acut i subacut


Cea mai important reacie de hipersensibilizare de tip
ntrziat este cea mediat celular
Clasificarea Gell i Coombs (dup mecanismul
de producere) a tipurilor de Hipersensibilitate

Tipul I (Alergia)
Ag solubile, IgE, Mastocite / Bazofile, Eozinofile
Atopia: Tendina exagerat de a dezvolta un rspuns prin
Anticorpi


IgE (forme clinice de prezentare a RIP tip I: astmul, rinita
alergic, urticaria, alergia alimentar, ocul anafilactic)
Tipul II
Ag asociate celulelor, IgG, M, fagocite (Mo/MQ), limfocite
NK (ADCC)
Tipul III
Complexe imune Ag-Ac, sistemul C, PMN
Celule

Tipul IV
Mediat prin limfocite Tc
Type I hypersensitivity sensitization to an
inhaled allergen or bee sting

cytokines

Mast
cell

Antigens (red dots) from inhaled pollen are ingested and presented by
macrophages to T cells. Activated T cells produce cytokines leading
to the production of IgE, which binds to receptors on mast cells and
causes the release of histamine, which is responsible for allergy
symptoms. Onset is usually within minutes of contact with antigen.
Type II hypersensitivity immune-mediated
destruction of red blood cells

Drug (p=penicillin)
modified red blood cells
induce the production
of antibodies, because
the bound drug makes
them look foreign to
the immune system.
When these antibodies
are bound to them, the
red blood cells are
more susceptible to
lysis or phagocytosis.
Onset is dependent on
the presence of
specific antibodies.
Type III hypersensitivity immune complex
formation and deposition

Immune complexes
Immune complexes activate complement
of antigen (red dots) Inflammation and
(green dots- C3a, C4a,
and antibody form in edema occur, and
and C5a), and mast cells
target organ organ is damaged
(yellow cell) degranulate.

In sensitized individuals, allergen (antigen) combined with antibody leads to


the formation of immune complexes, which activate complement and the
inflammatory response. The location of the inflammation depends on the
location of the antigen - inhaled, under skin, systemic. Onset is usually
within 2-6 hours.
Type IV hypersensitivity delayed-type or contact

T cells (blue cells)


that recognize
Antigen (red dots) antigen are Inflammatory
are processed by activated and response causes
local APCs release cytokines tissue injury.

Antigen is presented by APCs to antigen-specific memory T cells that


become activated and produce chemicals that cause inflammatory
cells to move into the area, leading to tissue injury. Inflammation by
2-6 hours; peaks by 24-48 hours.
Nomenclatura OMS a unor alergeni

INHALATORI
polen de mesteacn (Betula verucosa): Bet v1
acarian (Dermatophagoydes pteronissinus): Der p 1-6
gndac buctrie (Blatella germanica): Bla g 1-2
peri pisica (Felix domesticus): Fel d 1
peri cal (Equus cabalus): Equ c 1-3
Aspergillus fumigatus: Asp f 1

ALIMENTARI
ou (Gallus domesticus): Gal d 1-3
lapte vac: caseina, lactalbumina,
-lactoglobulina

VENIN HIMENOPTERE
fosfolipaza A2

MEDICAMENTE
insulina, asparaginaza, latex (Hev b 1)
Hipersensibilitatea imediat tipul I
Contactul iniial cu alergenul este asimptomatic, dar sensibilizeaz sistemul
imun:
mecanismul implic IL-4 secretate de limfocitele Th2
IL-4 stimuleaz B s produc IgE

La contactul ulterior
cu alergenul, are loc:
Legarea IgE de
receptorii specifici
din membrana Ma
Eliberarea de amine
vasomotorii si
producerea de
agenti inflamatorii ->
rspuns inflamator
Simptomatologia
poate fi localizat
sau sistemic
Mastocitul - bazofilul tisular

1. Ubiquitous in connective
tissues:
Long lived >40 days
3x104 IgE receptors
High histamine content
Heparin & high tryptase

2. Mucosal - Gut & lung:


T cell dependent
Short lived <40 days
25x105 IgE receptors
Lower histamine content
Chondroitin sulphate
Lower tryptase

Proteoglicani, Proteaza neutra,


Calea cicloxigenazei:Prostaglandine
-Glucozaminidaza, IL-3, -4, -5, -6,
Tromboxan
GM-CSF, TNF, PAF
Calea lipoxigenazei: SRS-A
(LTC4,D4,E4), LTB4, Lipoxine
Mediatori specifici eliberai

Amine vasoactive:
Histamina: eliberata de Ma, Ba, Pl ca raspuns la injurie (traume, temperaturi
extreme), reactii imune (IgE-FcRI Ma), anafilatoxine (fragmente C3a, C5a),
citokine (IL-1, IL-8), neuropeptide, leukocyte-derived histamine-releasing
peptide, produce vasodilatatie si contractia celulelor endoteliale venulare, cu
largirea jonctiunilor
Serotonina: prezenta in granulele dense Pl; agregarea plachetara declanseaza
eliberarea; efect vasodilatator similar cu al histaminei
Kinine plasmatice:
Formarea bradikininei (BK) prin clivarea HMWK, declansata de factori asociati
cu coagularea (F XII)
Vasodilatatie, permeabilitate vasculara (edem)
Posibil efect chemotactic Leu (declanseaza expresia ECAM pe endoteliul
vascular)
Contractia musculaturii netede non-vasculare (bronsice)
Durere
Inactivate rapid (kininaze)
Actioneaza mai lent decat histamina.
Anafilaxia localizat i ocul anafilactic
Caracteristicile rspunsului imun IgE

component normal a rspunsului imun secundar

durat scurt

inductibil cu doze foarte mici de antigen

modulat de citokine: ntrit de IL-4, suprimat de


IFN

n principal, produs ca reacie imun la interfaa


organism-mediu

relativ uor de ntrerupt: doze foarte mari de


antigen, administrri repetate (imunoterapie
specific)
Tratament
Influentarea raportului Th1/Th2 : desensibilizare
Hipersensibilitatea subacut
Cauzat de IgM i IgG, poate fi transferat via plasm
sau ser
Debut lent (13 ore) dup expunerea antigenului
De durat (1015 ore)

Reacia citotoxic (tip II)

Ac se leag la Ag fixate in
membrana celular,
stimulnd reacia
citotoxic dependent de
Ac i citoliza mediat prin
complement
Exemple: reaciile
patologice din
transfuzia de snge cu
incompatibilitate n
sistemul ABO i Rh
R TSH LATS
(hipertiroidie)
RAC - Miastenia gravis
RPL hiposecreia
lactat
RSTH nanism
Diagnosticul fenomenelor de tip II

Demonstreaza fixarea anticorpilor si/sau a


factorilor de complement la suprafata
celulelor purtatoare de Ag
Tehnica: imunofluorescenta
(Anticorpi anti-Ig umane cuplate cu
o molecula fluorescenta)
Teste functionale:

o Crossmatch (verifica existenta


Ac preformati la primitor,
prevenind rejetul superacut in
transplant)
o test Coombs direct sau indirect:
Direct Cauta Ig sau C3 pe hematiile
pacientului
Indirect - Cauta Ig anti-hematii in sreul
pacientului
Hipersensibilitatea subacut
Reacia imun patologic prin
complexe imune (tip III):
Antigenele sunt rspndite n snge
sau n esuturi (adesea autoAg)
Se datoreaz:
Persistenei infeciei / Administrrii
repetate a Ag (local sau general)
Sintezei continue, ineficiente de Ac
IgM/IgG formeaz CIC:
La un raport corect al concentraiei
componentelor, sunt rapid epurate de
MQ
Complexele mici/solubile (Ag >>> Ac)
sunt greu epurate -> Inflamaia intens,
lezarea vascular, renal, tegumentar
inocent bystander (prin activarea
sistemului complementului i a
proteazelor Leu)
Reacie inflamatorie local (fenomen
Arthus) sau general (boala serului)

Caracterizeaz bolile autoimune


generalizate - de sistem (LED, PAR,
DM)
Relativ comune (mai ales la vrstnici)
Fenomene de tip III localizate

Fenomenul Arthus

Implicatii clinice:
afectiuni pulmonare in care
alergenul este inhalat in mod
repetat (plamanul de fermier,
alveolite alergice ,...)
Boala Serului

Simptome
Febra

Eruptii

Adenopatie

Artrita

Glomerulonefrita
Fenomene de
tip III

Postinfectios
Glomerulonefrita postinfectioasa
(streptococica)
hepatite
mononucleoza
malarie
...
Medicamentos
Reactiile medicamentoase se pot
manifesta clinic prin mai multe
tipuri de reactii imune patologice
Autoimun
LED
o complexe: anticorpi anti-DNA si
DNA depozitati in articulatii,
piele, rinichi, etc.
Hipersensibilitatea ntrziat (Tip IV)

Debut lent (13 zile)


Mediat prin mecanisme
implicnd limfocite Th (pentru
Ag solubile) i Tc (pentru Ag
asociate celulelor)
Citokinele din Tc activate sunt
mediatori
Activeaz Tc (cu Ag procesate
de MQ si prezentate prin MHC
II) rejectia grefelor (HVGR)
Activeaz TH1 (cu Ag procesate
de MQ si prezentate prin MHC I
sau II) cu eliberarea de citokine
(IFN)
TB test cutanat
Dermatitele de contact
(Triggers: chimicale, bijuterii
ieftine (Ni), antibiotice)
B.autoimune (DZ I,Tiroidite)
Patogeneza RIP Tip IV
RIP tip IV mediat de Th1
Alergen injectat
Infiltrat celular!!

Exemplu: testul la tuberculin


RIP tip IV mediat de Th1
Alergen absorbit

Exemplu: Dermatitele de Contact


Nickel RIP tip IV contact cu metale grele

Heavy metals
bind to
proteins and
haptenylate
them.
Stimulates an
immune
response

Transport to lymph
node and T cell
stimulation to
hapten:protein
RIP tip IV DZ I

Insulita beta
Funcii i disfuncii ale Th
Ts

TH1 TH2

SNTATE
(imunoprotecie: rspuns inflamator adecvat;
toleran - auto-, allo-, xeno-)

TH2 TH1

TH2
TH1
Alergie (toleran xeno
Boli autoimune (tolerana self sczut) sczut)
Rspuns excesiv inflamator Rspuns excesiv inflamator