IZSLER Brescia La risposta immunitaria a stressori infettivi e non- infettivi nel suino: distinzione tra la risposta costitutiva in interferon-alfa rispetto a quella virus- indotta Massimo Amadori, Elisabetta Razzuoli IZSLER - Brescia Background Established view A new conceptual framework Open problems
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IZSLER Brescia
La risposta immunitaria a stressori infettivi e non-infettivi nel suino: distinzione tra la risposta
costitutiva in interferon-alfa rispetto a quella virus-indotta
Antibiotico-resistenza di agenti di zoonosi: in aumento in tutto il mondo
E’ necessario rafforzare la sorveglianza
Sviluppare nuovi antibiotici
Sviluppare nuove strategie di controllodella antibiotico-resistenza(http://www.efsa.europa.eu/cs/BlobServer/Scientific_Opinion/1372.pdf?ssbinary=true )
Preoccupanti i livelli di resistenza osservati con Salmonella e Campylobacter spp
Possibile contributo di biocidi (disinfettanti, antisettici e conservanti)
Livelli diversi nei Paesi UE
Difficile adozione di strategia unitaria
Fonti principali di contagio alimentare: macello e processazione di alimenti
Precauzioni per fluorochinoloni e cefalosporine
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Ceppi Salmonella resistenti a Chinolonici
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Resistenze agli antibiotici e sanità pubblica:un’incognita?
The New England Journal of Medicine
342: 1242-1249, 2000
Ceftriaxone-resistent salmonella infectionacquired by a child from cattle
Paul D. Fey et al.
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Fabbisogno di antibiotici: fronti di intervento
Igiene zootecnica e benessere animale
Tempestività degli accertamenti diagnostici
Genetica animale e adattamento ambientale (“forbice” da controllare)
Funzionalità del sistema immunitario innato ed adattativo
Immuno-modulazione mirata in fasi critiche dei cicli zootecnici
Quali basi concettuali ?
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SISTEMA NERVOSO CENTRALE
SISTEMA IMMUNITARIO
sistema neuroendocrino
stimoli antigenici
stimoli psicosensoriali
rispostaimmunitaria
risposte comportamentali
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The stress response
Immune response, stress and inflammation: ancestral set of responses neutralization of noxae perturbing body homeostasis
Cytokines : homeostatic agents
Effector mechanisms: similar in infectious and non-infectious stress
For instance: IL-1 HPA + cerebral NA after infection, electric shock or restraint
Brain IFN-α after infectious and non-infectious stimuli
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La risposta immunitaria
Può essere rivolta verso:
STRESSORI INFETTIVI
STRESSORI NON INFETTIVI
Degenerazione, stress e distruzione tissutaleda cause varie (es. : alterazioni e disfunzioni metaboliche, stress ox, lesioni articolari, disturbi vascolari, ecc.)
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SISTEMA IMMUNITARIO
Sistema deputato al riconoscimento delle componenti estranee all’organismo animale
Fase Caratteristiche Meccanismo
Immediata (<4 ore)
Non specifica
Innata
Non dà memoria
Atività del SRE
Lisozima, complemento, anticorpi naturali, cellule NK
Precoce (4-96 ore)
Non specifica
Inducibile
Non da memoriaInterferon, cellule NK attivate da IFN
Linfociti T Citotossici, interferon, anticorpi specifici
Componenti della “clearance “ degli agenti di infezione
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Attivazione del Sistema Immunitario Innato
1)PAMPs: strutture microbiche tipiche (LPS)
Riconoscimento mediato da: PRP (es. TLR), RIG-like helicases (RIG-1 e MDA-5 per ac. Nucleici virali), NLRs (es. per PG batterico)
2) DAMPS: da cellule danneggiate (es. acido urico) segnalazione del “pericolo”
Quale teoria alla base del modello ?
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An innate sense of danger(Matzinger P., 2002, Ann. N.Y. Acad. Sci. 961, 341-342)
Il paradigma “self – not self” è insufficiente
Presenza fisiologica di cellule T e B auto-reattive ma innocue, proteine del latte, Agalimentari, batteri commensali, feti, sperma: fenomeni non spiegati dal paradigma
Driving force: riconoscimento del “pericolo”
Danger: stress e/o distruzione tissutale: rilascio di segnali (alarmine o DAMPs)
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Il ruolo delle “alarmine”
DAMPs o “alarmine”: prodotto di cellule necrotiche, stressate e degenerate
Alarmine: HMGB1 (e istoni in generale): lega TLR2 e 4, RAGE
ROS ciclofillina A, proteine S100 (A8, A9): legame a TLR-4
Trasduzione di potenti segnali in senso pro-flogistico: SAPK cJun NH2, p38 MAPK
HSP72: antagonista naturale di tali risposte (vedi bovine da latte !)
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Alarmine: secrezione di istoni in cellule BHK-21 infettate da FMDV O1 Losanna
30’ k30 120’ k120’ 240’ k240’ 360 k360’
1 2 3 4 5 6 7 8
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Infiammazione mortale, no infezione.Il mistero SIRS
(Systemic Inflammatory Response Syndrome)
Esempio: pazienti con gamba rotta, dopo 2 giorni febbre altissima, shock, resp. artificiale
Ipotesi trad. Ridotto flusso ematico all’intestino, aumento permeab. Intestinale, batteriemia
In realtà: quadro plasmatico sterile in vena porta
Plasma: alto contenuto di DNA, MITOCONDRI !
Per SII: mitocondri = Batteri = risposta flogistica
HBS (Hemorragic Bowel Syndrome) nelle bovine da latte ??
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Organizzazione della risposta flogistica: il ruolo dell’inflammosoma
Indotto da segnali di PAMPs e DAMPs
Complesso di oligomeri NLRs/adattatore (ASC) caspasi 1 IL-1β
Discriminare l’ambiente microbico e non microbico per individuare le condizioni di “pericolo” (Danger Theory)
Repertano segnali infiammatori endogeni in assenza di infezioni microbiche (vedi SIRS!)
Sono alla base dell’efficacia di alcuni adiuvanticome Al (OH)3 DC inflammosomaNALP3 IL-1β
Collegamento immunità innata / adattativa
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Come si manifesta la risposta astressor non-infettivi ?
1) Fenomeno flogistico citochine e chemochinecircuiti regolazione omeostatica
2) Risposta linfocitaria allo stress (specie linf. T γδ):
Riconoscimento di “neo-antigeni” e antigeni da stress: T10, T22, CD1c (DC!), IPP, ATPseF1, altri Ag MHC-like(MICA, MICB, H60). Ovvero:
Danno epit. Linf.Tγδ DC risposta T conv.
(inversione del flusso consueto)
Lymphoid Stress-Surveillance Response
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Una nuova frontiera: il concetto di“Behavioral Immunization”
Precedenti blandi stress sono “educativi”
Fenomeni immunitari implicati ?
Topi deficitari di cellule T fronteggiano peggio lo stress mentale
Stress: aumentato traffico di linfociti al cervello
Effetto protettivo verso nuovo stress
Minore ansietà dopo vaccinazione con peptideSNC
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Esistono evidenze in medicina veterinaria?
Lymphoid Stress-Surveillance Response: modelli umani e murini
Secrezione di citochine in risposta a stressorsnon-infettivi: evidenziata anche in bovini e suini
Interferon I (specie IFN-α) gioca un ruolo regolatorio centrale
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Interferon: a dogma in immunology
Dogmas / paradigms = serendipitous
Discovery of new components of the immune system
Interferon = no exception to this rule
1957 = Isaacs and Lindenmann discovery of a potent antiviral substance accumulated in embryonated eggs
50 years later: most immunologists share the same view: antiviral activity + “accessoryproperties”
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IFNs as homeostatic agents
Three types of IFNs (I, II and III)
Type I IFN: 10 sub-families (α, β, ω, τ, etc.); many α subtypes
IFN-α : a highly diversified system (13 genes and 5 pseudogenesin humans; at least 14 genes and 2 pseudogenes in pigs) shouldserve diverse functions and effector mechanisms
Low levels of IFN α/β in tissues of healthy subjects: constitutive expression
Steady-state role under health conditions
Most important: frequent involvment in the stress response
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The physiological IFN response
IFN is produced under health conditions: e.g. after meals and drinking
Not obligatorily related to infection status
This is true of the whole type I family
IFN-β : maintenance of memory T cells
IFN-τ : survival of the conceptus in ruminants
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Aims of the physiological IFN response
Strong cellular responses to viruses and cytokines
To control the effects of growth factors
To prevent unnecessary harmful inflammatoryresponses in tissues
Default function of IFN-α
It would complement other establishedbiological activities after infectious and non-infectious IFN-inducing stimuli
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In vitro activity of IFN-α
Very low concentrations (0.05 – 5 U/ml) down-regulate CD14 expression in swinePBMC and PAM, as opposed to higherconcentrations
Signalling by LPS/LPS binding protein isinhibited
Released CD14: potent scavenging system forLPS
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How does the control action take place?
A direct modulation of the inflammatoryresponse in target cells
Release of second messengers forneighbouring cells
Neighbouring cells would be thus instructed todown-regulate the inflammatory response
An in vivo propagation of these signals todistant sites would be possible as well.
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The IFN-α response in farm animals
Low-titered IFN-α response in pigs after earlyweaning and transportation (calves, too)
Early weaning: activation of inflammatorycytokine genes in the small intestine and up-regulation of IFN-γ in PBMC
IFN-α at weaning: (1) A SPF pig model . (2) Aconventional pig model
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The SPF pig study
1 litter of L x Lw SPF piglets (n. 8)
Matched for weight at day 20 of age (4.9±0.9 versus5.4±0.8 Kg in treatment and control groups, respectively) and weaned one day later.
At weaning, 4 piglets: 20 IU freeze-dried human IFN-alpha / Kg b.w. for 10 days in RG lactose per os. 4 piglets: placebo (the same mass of RG lactose) in a separate cage.
Daily clinical inspections. Blood samples at days –1 / +3 / +21 with respect to weaning
Individual weight controls at days –1/ +21 / +30
RT real time PCR for inflammatory cytokine genes in PBMC. Interferon-α and TNF-α bio-assays on serumsamples (MDBK and WEHI 164 cells).
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Results obtained on SPF piglets
No clinical difference between treated and control animals
Daily weight gain greater in IFN alpha-treated groups between days -1 and +21 and also between days 21 and 30; the effect of the treatment betweendays –1 and + 30 was almost significant (p<0.06).
Evidence of a low-titered serum IFN-alpha response in control animals, only
No evidence of serum TNF-α response but in 1 control pig at day +21
In RT real time PCR, IFN-γ was significantly up-regulated at day +3; expression significantly lower at days +3 and +21 in treated piglets.
TNF-α expression significantly different between sampling times.
IL-1β significantly increased in IFN-treated pigs at days +3 and +21
IL-6 at day +3, only.
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N-fold gene expression values by the 2-ΔΔCt method (basis: mean value at day -1)
Housekeeping gene: TATA box-binding protein gene (TBP)
IFNA
b
a
IL6
α
β
IFNG IL1B TNF
-5.0
-4.0
-3.0
-2.0
-1.0
0.0
1.0
2.0 -h
IFN
-alp
ha
+hI
FN-a
lpha
-hIF
N-a
lpha
+hI
FN-a
lpha
-hIF
N-a
lpha
+hI
FN-a
lpha
-hIF
N-a
lpha
+hI
FN-a
lpha
-hIF
N-a
lpha
+hI
FN-a
lpha
-hIF
N-a
lpha
+hI
FN-a
lpha
-hIF
N-a
lpha
+hI
FN-a
lpha
-hIF
N-a
lpha
+hI
FN-a
lpha
-hIF
N-a
lpha
+hI
FN-a
lpha
-hIF
N-a
lpha
+hI
FN-a
lpha
3d 21d 3d 21d 3d 21d 3d 21d 3d 21d
log2
(n-fo
ld)
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The conventional pig model
Aims:
1) To check the oral, IFN-α treatment under practical, large-scale conditions (in drinkingwater)
2) To evaluate the endogenous IFN-αresponse
3) To assess the influence of the weaning age
4) To characterize such a stress-inducedresponse
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The field trial: role of the endogenous IFN-α response
39, LxLW piglets, of a healthy, farrow-to-finishherd, randomly allocated to:
Group 1: weaned at 28 days (19 pigs, 2 litters)
Group 2: weaned at 22 days (20 pigs, 2 litters)
Group 3: weaned at 22 days and orally treatedwith hIFN-α in drinking water (1 IU / Kg b.w. over 10 days) (20 piglets, 2 litters).
Clinical checks and blood samplings at days -1 / +6 / +12 with respect to weaning
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Clinical findings
All piglets remained healthy during the trial
No clinical difference between early and late weaninggroups. DMWG: greater in the late weaning group(28 days) and in IFN-treated animals (both: +1.5 Kg on average at 48 days of age) (p<0,001)
Correlated to lower serum TNF-α levels in the 3rd week after weaning
Blood samples were investigated for viral infections: all samples tested negative for Influenza A, PRRS, PCV-2, Pseudorabies viruses in PCR and Abassays
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Groups Day -1 Day + 6 Day + 12
1 (weaned at 28 days) 1/19 7/19* 0/19 2 (weaned at 22 days) 1/20 20/20 1/20
3 (weaned at 22 days and IFN alpha-treated)
0/20 18/20 0/20
*Significantly different, p<0.01
InterferonInterferon alphaalpha--positivepositive pigspigs out of the totalout of the total
The IFN-α response in serum samples
Assay: VSV cpe inhibition assay on MDBK cells, calibrated with Porcine rec. IFN-α1
Low titres: 1-27 IU/ml
Activity neutralized by anti-Porcine IFN-α1 mAb
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GelGel--filtrationfiltration on a on a SephadexSephadex GG--75 column of IFN75 column of IFN--αα in in samplessamplesfromfrom groupgroup 2 2 pigspigs at day + 6at day + 6
2727--30 30 kDa*kDa*
1717--18 18 kDa*kDa*
< 14 < 14 kDakDa
R2 = 0,9909
00,10,20,30,40,50,60,70,8
4,1 4,2 4,3 4,4 4,5 4,6 4,7 4,8 4,9
PM log
Kav
y = -0,5959x+3,1898
SerumSerum
Fractions checked by a bioassayon MDBK cells
Log MW
PBMC NP-40 lysates
Also: 58 kDa (dimer)
and 41 kDa
Fractions checked by ELISA with mAbs F17 and K9
* Also shown by rec. IFN-α1
(Apparent MW)
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FlowFlow CytometryCytometry: : analysis of intracellular IFN-α in fixed and permeabilized swine PBMC at day +6
IFN-α positive samples out of the total:
• Group 1: 4 / 19 (0.6 – 3.9% positive cells)
• Group 2: 10 / 20 (0.6 – 8.8% positive cells)
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Anti-IFN α mAbControl
Flow cytometry assay for intracellular porcine IFN-αin fixed and permeabilized swine PBMC at day +6
2% IFN α−positive cells on average
(0.1 – 0.3% in 2-3 month-old pigs)
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RT RT realreal time PCR time PCR forfor IFNIFN--αα genesgenes ((groupgroup 2 2 pigspigs))Day Day --11
Day +12Day +12
Day +6: no Day +6: no expressionexpression evidencedevidenced
Assays carried out according to Cheng G. et al., (2006), Gene 382, 28-38
(No valid results for IFN α 10 gene)
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Day -1
Day +6
Day +6
Day -1
Day +12
Day +12
RT real time PCR for IFN-α 5/6
The test was carried out on PBMC of the same pig at days –1 / +6 / +12 with respect to weaning
IFN-α gene expression at days -1, +6, +12
Day +12
Day -1
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Constitutive and virus-induced expression
Previous data on PK-15 cells in vitro:
Virus-induced expression: IFN-α 2/3/4/8/9/10/13
Constitutive expression: IFN-α 1/2/5/6/7/11/12
Truncated Porcine IFN-α genes: IFN-α 1/2/3/11
Genes with N-glycosylation site: IFN-α 7/9/10/11
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Issues raised by the field trial and in vitro tests
Is this pattern of response also applied to infectious (viral) stressors ?
Are inflammatory cytokines released at weaning implied in the modulation of type I IFN system ?
This is the reason why we investigated IFN genes and proteins in a relevant model of in vitro cultured swine PBMC from healthy, 70 to 80-day old animals, far away from the weaning stress.
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In vitro tests
IFN proteins (α nd γ) and IFN-α gene expressionin PBMC cultures of 70/80-day old pigs
Time 0
Unstimulated control
Priming (100 IU/ml IFN-a)
Priming + Paramixovirus (NDV) stimulation
NDV, only
Supernatant collection / RNA extraction after 18 hours of culture
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In vitro results
IFN-α1, α2, α4, α5/6 and α7/11 genes were shown to be involved in constitutive expression in uncultured PBMC (Time 0 samples).
Further IFN-α genes were then expressed in culture.
Interestingly, expression was often shown in unstimulatedcultures, as well.
As a result, NDV stimulation often caused only increased expression compared with unstimulated cultures
In January 2002 a herd of 1100 sows wasaffected by reproductive PRRS:» Enzootic abortion
Oral administration of IFN-α was carried out on all pregnant sows in the ad libitum food ration for 15 days in a row
The treatment was repeated 15 days later
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Results
010203040506070
June
Augu
stO
ctobe
rDe
cem
ber
Febr
uary
April
June
Augu
stO
ctobe
rDe
cem
ber
Abortions
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Dose/response curve of IFN-α
Foundation in the dose/response curve
Immune effector functions: bell-shaped curve
Positive/decrease/even reversion (e.g. Ab responseto sheep RBC in mice)
Concept: low dose priming / high dose suppression
Timing and concentration: crucial roles (subtypes?)
At odds with antiviral / anti-proliferative activities: it increases with increasing IFN-α concentrations
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Long-distance journeys of calves:
Serious sensory dullness
Dehydration (HCT, serum protein, albumin, Hb)
Homeostatic role of IFN-α: truck transportation of calves
Adrenal response (glucose, urea)
PO4 increase e Fe decrease
Detection of IFN-α in serum
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05
10152025303540
T-2 T-1 T0 T+4 T+15
tempo
conc
entraz
ione
IL-6 (ng/ml)
TNF alfa (ng/ml)
Aptoglobina (mg/dl)
Risposta al trasporto: Risposta al trasporto: citochinecitochine e risposta APP+e risposta APP+
in vitelli trasportati dalla Poloniain vitelli trasportati dalla Polonia
Elevata risposta in IL-6 e TNF-alfa (carattere compensativo) prima e dopo il trasporto
Risposta APP+
Elevata pressione infettante in allevamento di origine
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01234567
T-2 T-1 T0 T+4 T+15
Tempo
IFN-alfa
Negativi
Positivi
IFNIFN--alfa e risposta alfa e risposta adattativaadattativa in vitelli trasportati in vitelli trasportati
dalla Polonia dalla Polonia
Risposta in IFN-alfa in tutti gli animali nel centro di raccolta e dopo il trasporto
Diminuzione a 4 giorni dall’arrivo in Italia
Conferma del suo ruolo adattativo di tale citochina
picco di citochine infiammatorie risposta IFN-alfa
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La filiera ANAFI:il trasporto dei bovini di breve durata
5 trasporti controllati di torelli Frisoni di 6-12 mesi al Centro Genetico ANAFI di Cremona (totale 26)
Dicembre 2008, Gennaio 2009, Aprile 2009, Giugno 2009, Luglio 2009
200 – 300 Km, 3-6 ore*, Torino-Cuneo/Cremona
Sopralluoghi a T-4 (esame clinico ed etologico), T0 (carico-scarico), T+4, T+15, T+30
Prelievi ematici nelle stesse date
Riprese filmate da T-4 a T+4
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E’ importante la componente climatica nei trasporti animali ? Suddivisione dei trasporti di
breve durata su base stagionale
Si possono distinguere:
2 trasporti invernali (range: 5,7-13,7 °C; 2,6-9,4 °C ). Trasporti 1 e 2
1 trasporto in stagione termo-neutrale (range11,3-16,1 °C) Trasporto 3
2 trasporti estivi (range 23-33 °C, THI 46-73,5). Trasporti 4-5. Temp. > superiori a quelle prospettate nel doc. Commissione UE
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Lesioni muscolari, enzima CK plasmatico
CK
0
100
200
300
400
500
600
700
800
900
T-4 T0 T+4 T+15 T+30
giorni dal trasporto
Gruppo 1Gruppo2Gruppo3 Gruppo4Gruppo 5
Andamento differente nel trasporto 1 a causa di forti interazioni agoniste di 2 torelli, mai tenuti in gruppo prima. Elevata prevalenza di risposte IFN-alfa sino a T+15 !!
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Risposta negativa (APP-) e positiva di fase acutaAlbumina
0
5
10
15
20
25
30
35
40
45
T-4 T0 T+4 T+15 T+30
giorni dal trasporto
Gruppo 1Gruppo2Gruppo3 Gruppo4Gruppo 5
La risposta APP+ nel gruppo 5 è accompagnata da risposta APP-(ipo-albuminemia) a T+15, come evidenziato nelle bovine da latte nel periparto. Forte correlazione con la risposta in IFN-alfa !!
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Citochina IL-6 e risposta di fase acuta
Andamento atteso per i trasporti 2 e 5: come atteso, in assenza di lesioni, RFA dipende da incremento ad alto titolo di IL-6 plasmatica.
IL 6
1
10
100
1000
10000
T-4 T0 T+4 T+15 T+30
giorni dal trasporto
Gruppo 1Gruppo2Gruppo3 Gruppo4Gruppo 5
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Trasporto breve: conclusioniNon è possibile considerare l’impatto del trasporto avulso dal contesto ambientale (origine + destinazione): strategia adattativa globale.
L’esito finale in termini di sanità e benessere animale dipende dal complesso di condizioni pre e post trasporto, oltre che dal trasporto stesso.
2 strategie di adattamento:
A basso impatto infiammatorio (gr. 3): incremento e/o allungamento delle alterazioni da stress acuto (cortisolo, NEFA, glucosio, lattato)
Ad alto impatto infiammatorio (gr. 1): da traumi nel trasporto (CK, APP+, IFN-alfa, NEFA)
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The “hinge” role of IFN-α
A “hinge” role of IFN-α between innate and adaptive immunity is widely recognized
PDC IFN-α type and extent of the adaptive immune response
The concept should now be widened
IFN-α would also promote a balance betweendanger and response
A homeostatic control action: the “spring”mechanism
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The “spring” mechanism of IFN-α
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Flow chart of actions
Start of innate immunity: IFN-α released at moderate/high concentrations after TLR recognition of microbes
Later on clearance of microbes decreaseof IFN-α release
Shift to an inflammatory control action basedon the transcriptional control of inflammatorycytokine genes and PAMP receptors (CD14)
Major tissue damage is avoided
The system gets at a “stand-by” status
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Tuning of the IFN-α response
Tuning of the response of paramountimportance
Detrimental effects because of high therapydosage, overexpression in transgenic mice, genetic defects of regulation even withoutinfection
This confirms the induction of IFN-α under health conditions and the need for a strictcontrol of this response
Excessive concentrations of IFN-α checked byAb responses in the host (self /not self ??)
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A note of caution
Several features need to be investigated
4 issues:
which effector cell populations ?
how are they activated ?
how can they sense differentconcentrations of IFN-α ?