Lemalattiepossonoesserestudiateadiversilivelli

epidemiologia

singolipazienti

fisiopatologia

istopatologia

fisiopatologiacellulare

citopatologia

patologiamolecolare

biologiamolecolare

thepresenceorabsenceofa protein or RNA, or anincrease ordecrease in theamount/activity of thesemolecules

rearrangementsoflargeportionsofDNA(translocations) or specific changes ingenestructure(mutations)

✓ Moleculartestscanbeusedtodiagnoseagivendisease:

Molecularpathology

infections by certain viruses(cytomegalovirus and EBV) canbe diagnosed by moleculartesting for the presence of theirspecificRNAsinblood

in the field of cancer pathology,the demonstration of a specificgene mutation or rearrangementcan help confirm the diagnosis ofcertainlymphomasandsarcomas

✓ Moleculartestscanbeusedtosearchthecauseofagivendisease:

✓ Moleculartestscanhelpwiththeprevention….

Molecularpathology

tests that look for inheritedgeneticdiseaseallowforpreventivemeasurestobegiventothetestedpatientsand/or their relatives; (forex.,colorectalcancerpatientscanbetestedforthepresenceofinheritedmutationsingenessuchasAPC)

new generationdrugs can specifically target crucial sitesofmolecules involved in thepathogeneticmechanism(s)ofadisease

…..andtreatmentofadiseaseinseveralways:

● Examplesoftherapeutictreatmenttargetingpathogeneticmechanismsatmolecularlevels:

➢acutelymphoblasticleukemia(ALL)

➢chronicmyeloidleukemia(CML)

➢rheumatoidarthritis(RA)

Molecularpathology

Acutelymphoblasticleukemia(ALL)

• Janus kinases, JAKs, are abundantly expressed inleukemic cells of childrenwith ALL themost commonformofchildhoodcancer

• recentstudieshavecorrelatedJAKactivity inALLcellswithsignalsdown-regulatingapoptosis

• apoptosisdefect contribute to increase thenumberofproliferatingleukemiccell

A,modelof JAK3showingmolecular surfaceofprotein (blue)andcatalytic (ATP-binding)site(yellow).B,ribbonrepresentationofJAK3kinasedomain.Theinhibitor moleculeisshownasaspace-fillingmodelinthecatalyticsiteofJAK3.C,close-upviewofcatalyticsiteofJAK3modelwithdockedinhibitor(green).

Structure-basedDesignof Specific Inhibitorsof JanusKinase3asApoptosis-inducingAntileukemicAgents

IlcromosomaPhiladelphianellaleucemiamieloidecronica

Il gene di fusione ABL-BCR produce una proteina adattività tirosin chinasica che non risponde più ainormalicontrolli(lossofnegativeregulatorydomains)

Molecularpathogenesisofchronicmyeloidleukemia

1960: 22Ph’ identified

1845: CML described

1986: BCR-ABL identfied

1973: t(9:22) identified

1998: BCR-ABL inhibitor tested in CML patients

2001: Gleevec (Imatinib) approved by FDA

Milestones of CML research

TrattamentodellaleucemiamieloidecronicaIlfarmacoagisceinibendol’attivitàtirosinchinasicadi bcr-abl: “congela” la proteina nella suaconformazioneinattivaincapacedilegarel’ATP

● elevataefficacia● bassatossicità

Firsthumantrialin1998:thisdrugrestorednormalbloodcountsinall31treatedpatients

Rheumatoid arthritis (RA) is a systemic, chronicinflammatory autoimmune disease affecting manytissuesbutprincipallyattackingthejoints.

mildform aggressiveform

J. Clin. Invest., 2008

BiologictherapyinRA

Inflammation:keynotes

●Protective response involving various host bloodvessels,cells,proteinsandothermediators

● Intendedtoeliminate the initial causeofcell injury,aswellasthenecroticcellsandtissuesresultingfromtheoriginalinsult,andtoinitiatetheprocessofrepair

●Maintained under strict control to minimize tissuedamage

● The inflammatory reaction and the subsequent repair processcanthemselvescauseconsiderableharm

● Inflammatorydamagemayevenbecomethedominantfeatureifthereactionis:● verystrong(e.g.,whentheinfectionissevere)

● prolonged(e.g.,whentheelicitingagentresistseradication)

● inappropriate(e.g.,whenitisdirectedagainstself-antigens,suchasinautoimmunediseases)

● excessive (e.g., inallergicdisordersagainstusuallyharmlessenvironmentalantigens)

Inflammation:keynotes

Inflammation:adouble-edgedsword

Examplesofhumanpathologiesassociatedwithinflammation

● Infections(bacterial,viral,fungal,parasitic)

● Traumaandtissuedamagebyphysicalandchemicalagents

● Immunereactions

● Tissuenecrosis

● Hemodynamicdisorders(atherosclerosis,thrombosis)

● Neoplasia

Inflammation: the «backbone» of pathology (Lord Howard Florey, 1945 Nobel Prize for Medicine)

• These two basic formsof inflammationmay coexist, andmany variables modify their course and histologicappearance

• Chronic inflammation can have serious pathologicconsequences

Inflammation:keynotes

Thecomponentsofacuteandchronicinflammatoryresponsesandtheirprincipalfunctions:anintegratedview

Inflammation:keynotes

Acuteinflammation

Recognition of the INFLAMMATORY STIMULUS

RELEASE OF MEDIATORS

vascular changes

Protein and cell recruitment into tissues

Main events in acute inflammation

formation of the inflammatory exudate

Recognition of the INFLAMMATORY STIMULUS

Main events in acute inflammation

● Vertebrates evolved two different systems to recognize andeliminatepathogens:theinnate(natural)andadaptive(acquired)immunity

● Theinnateimmunesystemisthefirstonetobeactivatedandcansense a wide range of pathogenic microbes through a limitednumberofreceptors,calledpattern-recognitionreceptors(PRRs)

● PRRs are expressed by many cell types (mainly macrophages,monocytes,neutrophils,dendriticcells),allowingthedetectionofpathogenstotakeplacedirectlyatthesiteofinfection

● Inaddition to recognize infectiouspathogens,PRRs aredesignedtosensethepresenceofsubstancesreleasedfromdeadcells

Phase1:recognitionofmicrobesandnecroticcells

Phase1:recognitionofmicrobesandnecroticcells

● Microbes and necrotic cells elicit “danger signals” thatdistinguish them from normal tissues and mobilize the hostresponse

● PRRsrecognizestructures(i.e.,molecularpatterns)commonto:

➢manymicrobes(Pathogen-AssociatedMolecularPatterns,PAMPs)

➢deadcells(Damage-AssociatedMolecularPatterns,DAMPs)

● OnceactivatedbyPRRs–PAMPs interaction, thecellsof theinnate immunesysteminitiatestheinflammatoryresponsebysecretingcytokinesandchemokines

● This leads to the expression of adhesion and co-stimulatorymolecules able to (1) recruit immune cells and (2) stimulatetheadaptiveimmuneresponse

● Because of the need todistinguish between pathogenic andnon-pathogenic (or commensal) microbes, it has beenproposed that the innate immune system is activated by therecognition of an antigen, but only in presence of dangersignalsreleasedbycells(DAMPs).

Phase1:recognitionofmicrobesandnecroticcells

Damage-AssociatedMolecularPatterns

●DAMPs:[allarmins]

● intracellularproteins,suchasheat-shockproteins(HSP)orHMGB1(high-mobilitygroupbox1),Ca++bindingproteins

● non-proteinDAMPs:ATP,uricacid,DNA,RNA,alteredphospholipids

● proteinsderivedfromtheextracellularmatrixgeneratedupontissueinjury,suchashyaluronanfragments

Summaryofmolecularinteractionsinvolvedintherecognitionofpathogensand/orsubstancesreleasedbynecroticcells

Patternrecognitionreceptors(PRRs)

Damage-associatedmolecularpatterns(DAMPs;allarmins)

Pathogen-associatedmolecularpatterns

(PAMPs)

Toll-likereceptors(TLRs)

NOD-likereceptors(NLRs)

PRRsrecognizepathogen-associatedmolecularpatterns(PAMPs)fromdifferentclassesofpathogens

Viruses, bacteria, fungi, and protozoa display several different PAMPs,someofwhicharesharedbydifferentclassesofpathogens.MajorPAMPsare nucleic acids, surface glycoproteins (GP), lipoproteins (LP), andmembranecomponents[peptidoglycans(PG),lipoteichoicacid(LTA),LPS].PAMPsarerecognizedbydifferentfamiliesofPRRs.

PRRs:PatternRecognitionReceptors➢Toll-like receptors (TLRs): (ten types)microbial sensorslocated in plasma membranes and endosomes; detectextracellularandingestedmicrobes

➢TLRs recognize products of different types of microbes;providedefenseagainstessentiallyallclassesofpathogens

➢Recognition of microbes by these receptors activatestranscription factors that stimulate the production of anumber of secreted and membrane proteins such as:mediators of inflammation, antiviral cytokines (interferons),andproteinsthatpromotelymphocyteactivation

HumanToll-LikeReceptors(TLRs)

(Toll/IL-1R)

TenhumanTLRsandtheirpathways

SignalingpathwaysactivatedbyTLRsmightbeMyD88dependentorindependent.

MyD88(myeloiddifferentiationfactor88)isanadaptormoleculethatrecruits and activates other proteins that partake to the signalingcascade; resulting in the activation ofNF-κB and other transcriptionfactors(CREBandAP1).

• The activation of both the MyD88-dependent and MyD88-independent pathway finally leads to synthesis of inflammatorycytokinesoranti-viraltypeIinterferons(α-,β-IFNs).

• AllTLRs,exceptTLR3,canactivateMyD88.

http://www.intechopen.com/books/major-topics-in-type-1-diabetes/the-innate-immune-system-via-toll-like-receptors-tlrs-in-type-1-diabetes-mechanistic-insights

LEGENDADELLASLIDEPRECEDENTE

• Afterrecognizingamoleculeonthemicrobe,theTLRsbeginaseries of chemical reactions that activate the innate immunecellandallowittofunctioninthekillingofmicrobes

• If oneormoreof these signalingmoleculesaredefective, theinnateimmunecellcannotkillthemicrobe

• In recent years several deficiencies of TLRs have beenidentifiedinpatientswhopresentwithrecurrentinfections

• The knowledgeof the functionof these TLRshasprovidedanimportant basis for the diagnosis and treatment of thesedisorders

Toll-like Receptors

Examples of TLRs defects will be treated later in the section«Immunodeficiencies»

HumanToll-LikeReceptors(TLRs)

(Toll/IL-1R)

MyD88deficiency

IRAK-4deficiency

PRRsrecognizepathogen-associatedmolecularpatterns(PAMPs)fromdifferentclassesofpathogens

Viruses, bacteria, fungi, and protozoa display several different PAMPs,someofwhichare sharedbetweendifferent classesofpathogens.MajorPAMPsarenucleicacids,surfaceglycoproteins(GP), lipoproteins(LP),andmembranecomponents[peptidoglycans(PG),lipoteichoicacid(LTA),LPS].PAMPsarerecognizedbydifferentfamiliesofPRRs.

NLRs=NOD-likereceptorsnucleotide-bindingoligomerizationdomain-likereceptors

NOD-likereceptors(NLRs):Nucleotide-bindingoligomerizationdomain-likeRs

●The NOD-like receptors (NLRs) are a family of PRRsmostlyexpressed inthecytosolandthereforeabletodetectsignsofintracellularinvaders

●SomeoftheNLRscanalsosensenon-microbialdangersignals and form large cytoplasmic complexes calledinflammasomes

●The inflammasome: multi-protein cytoplasmic complex thatrecognizesproductsofdeadcells,suchasuricacidandextracellularATP,aswellaspathologiccrystals(urateandcholesterolcrystals)andsomemicrobialproducts

● Triggering of the inflammasome results inactivation of an enzymecalledcaspase-1,whichcleavesprecursorformsoftheinflammatorycytokineIL-1βintoitsbiologicallyactiveform

● In the inflammatory response, IL-1β mediates the activation ofendothelial cell and the recruitment of leukocytes whichphagocytoseanddestroymicrobesanddeadcells

PRRs:PatternRecognitionReceptors

Somepeculiaraspectsoftheinflammasomewillbetreatedlaterinthesection«Inflammatoryboweldiseases»