DINOGMIUniversità di Genova

“non esiste la medicina alternativa, esiste solo una medicina che funziona e una che non

funziona”

Richard Dawkins, A Devil's Chaplain, United States, Houghton Mifflin, 2003

Medicina convenzionale: Evidence Based Medicine (EBM)

� È il processo della ricerca, della valutazione e dell’uso sistematico dei risultati della ricerca come base per le decisioni cliniche » Sackett D.L. et al.

�deriva da ricerche,prevalentemente da Studi clinicicontrollati e linee-guida di praticaclinica; dati quindi ottenuti medianteuna valutazione critica degli studiesistenti.

Medicine alternative:inefficaci e pericolose

�… non esiste prova dell'efficacia … se sono state

sottoposte a verifica sperimentale ne è stata ravvisata la inefficacia. Enciclopedia Treccani

� la cui efficacia non è stata dimostrata .

Introduzione alla Medicina alternativa di Francesco D'Alpa

�trattamenti che non hanno mostrato alcuna efficacia quando sottoposti a verifica scientificaNational Science Foundation

compresi esiti mortali a causadell'abbandono di terapie convenzionali diprovata efficacia.

N, South M. Adverse events associated with the use of complementary and alternative medicine in children. Arch Dis Child.

2011 Mar;96(3):297-300

Evidence basedmedicineQ

ual

ity

of

evid

en

ce

Controlled Clinical Studies

Randomization

Cross-over

Double blind

Angell M, Kassirer JP (1998). Alternative medicine--the risks of untested and unregulated

remedies. The New England Journal of Medicine 339 (12): 839–41

- Nel 1998 esorta a non abbandonare la sicura medicinaconvenzionale

Angell Marcia (2009), "Drug Companies & Doctors: A Story of Corruption", New York Review of Books, Vol 56, No 1;

15 January 2009

Marcia Angell, M.D.Editor-in-Chief,

New England Journalof Medicine

««««WhatWhatWhatWhat isisisis trulytrulytrulytruly unrealisticunrealisticunrealisticunrealistic isisisis immaginingimmaginingimmaginingimmagining thatthatthatthat wewewewe cancancancan provideprovideprovideprovideuniversaluniversaluniversaluniversal andandandand affordableaffordableaffordableaffordable healthealthealthealt carecarecarecare inininin aaaa marketmarketmarketmarket basedbasedbasedbased systemsystemsystemsystem»»»»

- Nel 2009 si licenzia da capo-editore del New England Journal of Medicine

�Molti farmaci … sono probabilmente poco migliori dei placebo, ma non c'è modo di

saperlo perché i risultati negativi siano nascosti... i risultati sfavorevoli sono omessi.... Il

disegno dello studio è scelto per evidenziare i risultati positivi

�Praticamente è spesso possibile far risultare dai trials clinici, conclusioni molto vicine a

ciò che si desidera

�Non è più possibile credere alla maggior parte degli studi clinici pubblicati

�Mi dispiace giungere a queste conclusioni, alle quali sono arrivata mio malgrado dopo

vent'anni come editore del New England Journal of Medicine

Emilio PeruccaPresident ILAE

Perucca E. What can we learn from clinical trials of anticonvulsant drugs in epilepsy? Eur J Pain.

2002;6 Suppl A:35-44.

- randomized trials can produce misleading conclusions .

- inappropriate titration rates or suboptimal dosages or dosing schedules

(often favouring the sponsor's product over the comparator)

- published drug trials vs unpublished data:benefits of drugs have been much over-rated and the harms under-rated.

- unpublished trial reports:impossible to get access to them

Gøtzsche PC, Jørgensen AW. Opening up data at the European Medicines Agency. BMJ. 2011 May 10;342.

Kirsch I, et al. PLoS Med. 2008 Feb;5(2):e45.-SSRIs efficacy = placebo efficacy

-SSRIs don’t reach the brain in enough amount to be able toinhibit serotonin re-uptake Pinna G, Costa E, Guidotti A.. Curr Opin Pharmacol.

2009 Feb;9(1):24-30

Storia della farmacologia antidepressiva, anni ‘50- Primi farmaci: oppiacei e amfetamina

- Cercando nuovi farmaci contro la Tubercolosi: Isoniazide, casualmente antidepressiva. Altro

antitubercolotico: l’Iproniazide anche se con maggiori effetti collaterali.

Meccansimo azione:

Isoniazide principalmente su diammino-ossidasi, debole sulle monoammino-ossidasi

(serotonina, noradrenalina,..)

Iproniazide: maggiore sulle monoammine-ossidasi. Nel 1961 ritirata per epotossicità

Le diammino-ossidasi sono principalmente nell’intestino per eliminare istamina.

- Cercando nuovi farmaci Anti-istaminici: TCA. Clorpromazina, studiata per essere un

antistaminico, risultò essere un antidepressivo. L’istamina è una diammina.

Studi paralleli sulla reserpina: una delle 50 erbe fondamentali della medicina cinese,

utilizzata da millenni in India:

- Alessandro il grande la utilizzò per curare un suo generale colpito da una freccia

avvelenata,

- veniva utilizzata per curare da morsi di serpenti e

- sembra che anche Gandhi la usasse come tranquillante.

- La sua azione sedativa è confermata dal suo uso per sedare cavalli.

- Ha una marcata azione ipotensiva e viene usata anche per curare le discinesie del

Parkinson.

La reserpina è sintetizzata nelle piante condensando il triptofano con la ciclofosfoamide

• Negli anni ’60 era creduto che l’azione antidepressiva dei TCA fosse dovuta all’inibizione

della ricaptazione della noradrenalina

• Partendo da molecole anti-istaminiche, iniziò una ricerca razionale di molecole con azioni

selettive sui sistemi monoamminergici preferibilmente sulla serotonina.

- Il primo SSRIs fu lo zimelidine, nel 1971, presto ritirato per gli effetti collaterali.

- Stessa sorte per l’indalpina, definita un Blockbuster, record di vendite, ritirata in

quanto provoca neutropenia.

- Il primo lancio di successo fu nel 1984 con la fluvoxamina, approvata dalla FDA

nel 1993,

- quello più famoso fu quello della paroxetina nel 1987.

Zimeldine Side effects

• Neuropatie centrale o periferica

• Esantema cutaneo

• Artralgia

• Eosinofila

• Ideazione suicidi

SSRIs increase 5-HT in synaptic cleftReserpine: lower 5-HT in vescicules, then less 5-HT released in synaptic cleft

Reserpine irreversibly blocks the vesicular monoamine transporter (VMAT). This normally

transports free norepinephrine, serotonin, and dopamine from the cytoplasm of the

presynaptic nerve terminal into storage vesicles for subsequent release into the synaptic

cleft ("exocytosis"). Unprotected neurotransmitters are metabolized by MAO in the cytoplasm

and consequently never reach the synapse.

Forstner J, Roomi N, Khorasani R, Kuhns W, Forstner G. Effect of reserpine on the

histochemical and biochemical properties of rat intestinal mucin. Exp Mol Pathol. 1991

Apr;54(2):129-43.

SSRIs vs Reserpine

?

Storia della farmacologia antiepilettica. Serendipety

PB: 1904 registrato come sedativo e ipnotico

1912 fu casualmente prescritto come tranquillante ad epilettico

Primo farmaco: KBr per suo effetto collaterale di provocare impotenza, si ritenevano

le crisi dovute ad eccessiva masturbazioneGoodman; Gilman (1970). "chapter 10: Hypnotics and Sedatives".

The Biological Basis of Therapeutics (4th ed.). London: MacMillan. pp. 121–2

DPH: sintetizzata nel 1908, testata casualmente come AED nel 1938

CBZ: Studiata per la nevralgia del trigemino, successivamente scoperto anticonvulsiva

VPA: solvente scelto casualmente per solubilizzare molecole da testare su modelli di

epilessia

Levetiracetam: scoperto casualmente, non ha dato risultati ai test anticonvulsivi, non

agisce sulle correnti ioniche di membrana. Definito «farmaco del futuro»

Valeric acid

Valproic acidSCFAs: Short Chain Fatty Acids

�Il tempo di azione deve essere ben osservato,

cosicché l'essenziale e l'accidentale non siano confusi.

� L'effetto di un farmaco deve essere visto accadere costantemente o in molticasi, se ciò non avviene, deve essere considerato un effetto accidentale.

Avicenna (Ibn Sina). In “Canone della Medicina”, 10 25

�Il farmaco deve essere testato con tipi contrari di malattia, perché

alcune volte un farmaco cura una malattia per le sue qualità

essenziali ed un'altra per le sue qualità accidentali.

�La qualità del farmaco deve corrispondere alla forza della malattia.Per esempio esistono farmaci, il cui calore è minore che la freddezza di

alcune malattie, cosicché essi potrebbero non avere effetti su tali malattie.

Effetto accidentale= effetto placebo: 30% nelle pat.neurologiche, 80% nelle pat. psicosomatiche

Artrite reumatoide- Il 30% risponde al placebo

- nel sottogruppo di pz con pregresse esperienze positive ai farmaci diventa l’83%

L’artrite reumatoide è una patologia psicosomatica???Morison RAH et al. Placebo responses in an arthritis trial. Ann. Rheum. Dis (1961), 20, 179

An elemental diet contains amino acids (no whole proteins), mono/di-saccharides and medium/long-chain triglycerides supplemented with vitamins and trace elements.

It provides complete nutrition and avoids many of the problems associated

with long-term steroid use (especially growth retardation in children).

Although its mechanism of action is not fully understood, it is a safe and effective method of treatment

for some patients with active Crohn’s diseas

- Sclerosi multipla: riduzione delle lesioni del 20% con placebo- Epilessia: riduzione delle crisi >50% anche con placebo

Placebo= condizionamento ambientale

Parkinson’s disease:

The placebo effect was very strong in this study

Oken BS. Placebo effects: clinical aspects and neurobiology. Brain 2008; 31: 2812-2823:

- dolore: effetto placebo (30%) bloccato dal naloxone

-Transplantion of dopamine neurons. Mcrae C et al. Arch Gen Psychiatry 2004; 61:412-420

de la Fuente-Fernández R et al. . Science. 2001 Aug 10;293(5532):1164-6:

- La dopamina rilasciata correla con il miglioramento anche del gruppo in placebo

- relazione dose dipendente tra rilascio ed effetto

- Effetto comparabile con quello con levodopa e apomorfina

Terapia del dolore

dimora somministrazione

Wilhelmsen I. Brian- gut axsis as an example of bio-psyco social model: Gut 2000; (Suppl IV)47:iv5–iv7

=

Brain (2008) Gut (2000)

-Muore di over dose se la dose giornaliera gli è somministrata

nella gabbi di dimora

-Ha crisi di astinenza se non gli viene somministrata dopo

averlo messo nella gabbia dove è solito riceverla

8

“Non c’è nulla di male a chiedere o cercare segni dal cielo – tuttavia spesso,

come per i farisei e sadducei, ciò accade perché non riusciamo a vedere

quelli che ci sono già dati, dal momento che attendiamo segni che

corrispondano all’idea che noi ci siamo fatta di Dio ed al modo con cui

abbiamo ‘pianificato’ il suo modo di agire”.

Matteo, 15,35 -37; 16,1-4; 16,17

La dieta è una delle più antiche e comuni forme di “medicina”. L’epilessia non fa eccezione.

- 460 aC. Ippocrate: ogni paziente richiede un regime dietetico:

- 300 aC. Erasistrato : connessione tra epilessia, intestino e organi digestivi

-“niente può guarire questo ragazzo se non il digiuno e la preghiera”. The Bible, King James Version. St.

Mark 9-29

- II secolo. Galen: dieta e purghe per controllo crisi: Bladin PF, Eadie MJ. England. John Libbey; 2001

- V secolo . Caelius Aurelianus. Riduzione peso su paziente obeso

- XIII secolo . Arnold di Villanova: “la dieta è l’aspetto più importante della cura dell’epilessia”

- XVII secolo. George Cheyne: dieta moderata ai pazienti epilettici.

- 1866. Radcliffe: dieta ricca di grassi povera di carboidrati

- 1921. Geyelin HR. Fasting as a method to treating epilepsy. Med Record. 1921; 99: 1037-9.

- 1922. Conklin HW. Cause and treatment of epilepsy. J Am Osteopathic Assoc. 1922; 26: 11-14.

con digiuno risultati sul 90% dei ragazzi, 50% degli adulti

-1930. Talbot: 50% del “Treatment of epilepsy” riguarda la dieta chetogena: Talbot FB.

Treatment of epilepsy. Ney York Mc Millan, 1930.

-1960. Lennox: Epilepsy and related disorders: solo un capitolo è sulla KD. Lennox WG, Lennox

MA. Epilepsy and related disorders. Vol 2. Boston.; Little Brown, 1960.

Starvation and epilepsy

From the role of intestine in metabolic syndrome to neurological diseases.

A think of the past.

Dr. Conklin, 1922

The explanation of the improvement is first that thepatient is kept in bed and secondly that thefermentative and putrefactive intestinal processes, sooften the exciting causes of convulsions even in a nonepileptic child, are reduced for the time being to aminimum.

So far as children are concerned, one often sees childrenwho have been considered epileptics in whom theepilepsy is found to be purely of intestinal origin andwho are therefore permanently cured by the applicationof strict dietetic measures. There can be no doubt alsothat such children, if they were regarded as epilepticswould be permanently cured by the starvationtreatment.

Alton Goldbloom. Some Observations on the Starvation Treatment of Epilepsy. Can Med AssocJ. 1922 August; 12(8): 539–540

18

Dieta ipoglicemica: una dieta chetogena “liberalizzata”Pfeifer HH, Thiele EA. Low-glycemic-index treatment: a liberalized ketogenic diet for treatment of intractable epilepsy.Neurology. 2005 Dec 13;65(11):1810-2.

Kang HC, Kim HD. Diet therapy in refractory pediatric epilepsy: increased efficacy and tolerability. Epileptic Disord. 2006Dec;8(4):309-16.

Pfeifer HH, Lyczkowski DA, Thiele EA. Low glycemic index treatment: implementation and new insights into efficacy.Epilepsia. 2008 Nov;49 Suppl 8:42-5.

Muzykewicz DA, Lyczkowski DA, Memon N, Conant KD, Pfeifer HH, Thiele EA. Efficacy, safety, and tolerability of the low glycemic index treatment in pediatric epilepsy. Epilepsia. 2009 May;50(5):1118-26.

Coppola G, D'Aniello A, Messana T, Di Pasquale F, Della Corte R, Pascotto A, Verrotti A. Low glycemic index diet in children and young adults with refractory epilepsy: First Italian experience. Seizure. 2011 Apr 12

19282006

-La restrizione calorica conferisce un controllo delle crisi, senza aumentare il livello di corpi chetogeni cerebrali:

-Yudkoff M, Daikhin Y, Nissim I, Horyn O, Luhovyy B, Lazarow A, Nissim I. Short-term fasting, seizure control and brain amino acid metabolism.

Neurochem Int. 2006 May-Jun;48(6-7):650-6.

- Lennox WC, Cobb S. Studies in epilepsy. VIII. The clinical effect of fasting. Arch Neurol Psychiat 1928; 20: 771-779

Osnato M. The pathogenesis of epilepsy. A critical review with a new interpretation of the available data.

Read at meeting of the Clinical Society, Oct. 6, 1922 and of the New York Neurological Society, Nov. 14, 1922

-Read A.L.

- pone le cause delle origini delle crisi convulsive nell’intestino. 1916

- riporta controllo delle crisi dopo interventi chirurgici intestinali in pz epilettici

Read AL Diagnostic Methods and Pathologic Constants in Idiopathic Epilepsy J.A.M.A: 66:336 (Jan.29) 1916; Constipation and

Intestinal Infection in Epileptics, ibid.67:1157 (Oct 14) 1916

Read AL. The bacillus epilepticus. Third report. J. Am Med Assoc. 1916; LXVI (21): 1607-1611

G. Akhan, F. Andermann, M. J. Gotman, Ulcerative colitis, status epilepticus and intractable temporal seizures Epileptic Disorders. Volume 4, Number 2, 135-7, June 2002

-Gerolamo Cuneo (1865): studioso delle malattie del ricambio: gotta, diabete,

leucemia, epilessia, malattie mentali

- Albuminose nel sangue di pz epilettici che se iniettate in animale inducono crisi

- sangue, feci e urine acide dopo le crisi: in urine total organic acidity 80% vs 5% in controls

Riporta nell’epilessiaUNA ELEVATA PERMEABILITA’ INTESTINALE =

INFIAMMAZIONE INTESTINALE

1922: evidenza del ruolo dell’intestino nei meccanismi patogenetici delle crisi

Efficacia della KD: Quanto?

Keene D. A systematic review of the use of ketogenic diet in childhood epilepsy. Pediatr Neurol 2006 ; 35: 1-5

Lefevre F Arosnosn N. Ketogenic diet for the treatment of refractoary epilepsy in children: A systematic review of efficacy. Pediatrics 105 (4) 1-7. 2000

12

Samuel T. Henderson, Ingrid Van der Auwera, Stefaan Wera and Fred Van Leuven. A ketogenic diet reduces amyloid beta 40 and 42 in a mousemodel of Alzheimer’s disease. Alzheimer's and Dementia , Volume 2, Issue 3, Supplement 1 , July 2006 , Pages S637-S638

Freeman JM, Vining EP, Pillas DJ, Pyzik PL, Casey JC, Kelly LM. The efficacy of the ketogenic diet-1998: a

prospective evaluation of intervention in 150 children. Pediatrics. 1998 Dec;102(6):1358-63

Neal EG, Chaffe H, Schwartz RH, Lawson MS, Edwards N, Fitzsimmons G, Whitney A, Cross JH. The ketogenic diet for the treatment of childhood epilepsy: a

randomised controlled trial. Lancet Neurol. 2008 Jun;7(6):500-6.

Helen Cross: 2008 Studio clinico “controllato” (Arruolamento casuale):

Mainardi P, Albano C. Is the antiepileptic effect of the ketogenic diet due to ketones? Med Hypotheses. 2008;70(3):536-9.

- sono i chetoni i responsabili dell’azione anticonvulsiva della KD?

62% vs 137%Mean % of baselineseizures

Szot P, et al. Brain Res Dev Brain Res. 2001 Aug 23;129(2):211-4.

Norepinephrine

norephineprine

0

50

100

150

200

250

0 10 20 30

day of starvation

plas

mat

ic le

vels

(n

mol

/l)

Ahern TH, et al. Neuropsychopharmacology. 2006 Apr;31(4):730-8.

• Norepinephrine has both anticonvulsant and antidepr essive effects

Martillotti J, et al. Epilepsy Res. 2006 Mar;68(3):207-11.

Fischer W, Müller M. Biomed Biochim Acta. 1988;47(7):631-45.

- increases anticonvulsant activity of drugs.

• Norepinephrine is needed for the anticonvulsant eff ect of KD

• Starvation and KD increase Norepinephrine

- increases anticonvulsant effect of KD

• Augmentation of norepinephrine:

Curcumin in neurological diseases:

- Epilepsy

- Alzheimer’s

- Parkinson

- Major Depression

- Stroke

- Schizophrenia

- Neurodegenerative disorders

- Neuropsychatric disorders

- Neuroproctetive actions

It’s scarcely absorbed by intestine

Curcumin in oncology:

Curcumin in intestine:

- Antinflammatory

Bereswill S, et al. Anti-inflammatory effects of resveratrol, curcumin and simvastatin in acute small intestinal inflammation.

PLoS One. 2010 Dec 3;5(12):e15099

Alfa-lattoalbumina: prebiotico intestinale

-40% delle proteine del ns colostro

- responsabile attivazione processi assorbitivi difensivi intestinali

-Modula lo sviluppo cerebrale del neonato

ALAC is the best intestinal prebiotic for human

26

HELSINKI

2006

FOOD SUPPLEMENT OF ALAC

7th European Congress

On Epileptology

27

Single oral administration

0

3

6

9

0 1 2 3 4 6 12 24 48

Time (hours) after last ALAC administration

Sei

zure

sco

re vehicle2505007501000

N = 5 animals per dose and time group

0

3

6

9

0 1 2 3 4 6 12 24 48Time (hours) after last ALAC

administration

Seiz

ure

scor

e

vehicle2505007501000

5 days chronic administration

0

3

6

9

0 1 2 3 4 6 12 24 48Time (hours) after last ALAC

administration

Sei

zure

sco

re

vehicle2505007501000

12 days chronic administration

Study on more than 400 animals:

� GEPR-9 rats

� Pilocarpine

� MES test

� WAG Rij rats

GEPR-9 rats

28

Animal Group

Dose (mg/kg)

Pilocarpine (mg/kg)250 270 300

Control 10/10 10/10 10/10

CBZ 20 10/10 10/10 10/10

CBZ 40 10/10 10/10 10/10

CBZ 80 10/10 10/10 10/10

ALAC 125 4/10 6/10 9/10

ALAC 250 0/10 3/10 9/10

ALAC 500 0/10 8/10 10/10

ALAC on seizures induced by Pilocarpine

010203040506070

0 200 400 600

n E

EG

ALAC dose (mg/Kg)

ALAC 15 Days

010203040506070

0 10 20 30 40 50

n E

EG

CBZ dose (mg/Kg)

CBZ

Acute SeizuresChronic Seizures

After these results ALAC entered in the new drug screening

program of NIH (US)29

Chronic Administration of ALAC Increases Trp/LNAAs plasmatic ratio

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0 5 10 15

R

Days of ALAC (250 mg/Kg) Chronic Administration

R Trp/LNAAsn= 7

Citraro et al.

The initial hypothesis has been confirmed:

An increase in Trp/LNAAs ratio permits to improve the seizure control

ALAC on Experimental Models

Choi S, DiSilvio B, Fernstrom MH, Fernstrom JD. Effect of chronic protein ingestion on tyrosine and tryptophan levels and catecholamine and

serotonin synthesis in rat brain. Nutr Neurosci. 2011 Nov;14(6):260-7.

ALAC ORALLY ADMINISTERED FOR 9 DAYS

and hypothalamic serotonin synthesis rate

30

Unified Myoclonus Rating Scale,section 4 (action myoclonus)

Unified Myoclonus Rating Scale,section 5 (functional performance)

Beck Depression Inventory Pittsburgh Sleep Quality Index

Mov Disord. 2011 Dec;26(14):2573-5

Striano P, D'Amato E, Pezzella M, Mainardi P, Zara F, Striano S. Sudden death in Unverricht-Lundborg patients: is serotonin the key? Neurol Sci. 2010 Feb;31

Chew NK, Mir P, Edwards MJ, Cordivari C, Martino D, et al. 2008. The natural history of Unverricht-Lundborg disease: A

report of eight genetically proven cases. Movement Disorders 23:107-13

Unverricht-Lundborg disease would begin to progress early and lack of effective treatment

meant a quick progression. In many cases the patient would require a wheelchair for mobility,

and would die at a young age.

31

WAG/RiJ mice Pilocarpine

PTZ NMDAAfter 3 weeks of treatmentaudiogenic mice continue

to be protected by seizuresfor at least 1 month

ALAC increasesepiletogenic threshold

32

Diet and Epilepsy: Ketogenic Diet

NE Intestinal Stimuli:

� Starvation….... (Biblical times)

� Fats…….. (Ketogenic diet)

� Medium Chain Triglycerids… (MCT diet)

� Tryptophan and or Serotonin…. (SSRIs, AEDs, ALAC)

Brain Synthesis:� Leptin� NPY “endogenous anticonvulsant”� Norepinephrine

All of them are reported to be anticonvulsant when injected in the brain

APR: Acute Phase ReactionStig Bengmark, Acute and ‘‘chronic’’ phase reaction - a mother of disease. Clinical Nutrition (2004) 23, 1256–1266

� La scienza, che per secoli ha cercato di scomporre il mondo in parti semplici,ormai si è convinta che capire le parti è cosa ben diversa dal capire il tutto.

� Un sistema complesso non è una banale giustapposizione di parti semplici.

� È strutturato dalle loro relazioni reciproche, che originano proprietà nuove,collettive, irriducibili a quelle dei costituenti.

FISICA DEI SISTEMI COMPLESSI

35

Neurotrasmettitori

Ammino acidi

La tirosina è sintetizzata dalla

fenilalanina

L’istidina è essenziale nei bambini

Asse intestino-cervello. Quali evidenze?

Apporto nutrizionale

�High urinary skatol levels in epilepticsMori A et al. Clinica Chimica Acta, 84 (1978) 63-68

Marja-Leena Koskiniemi, Journal of the Neurological Sciences, Volume 47, Issue 1, July 1980, Pages 1-6,

� Trp oral loads carry out an high urinary inidican levels in epileptics

Ammino acidi essenziali come precursori: - Triptofano: Serotonina- Tirosina: dopamina,

noradrenalina, adrenalina

- Istidina: Istamina

Triptofano Indolo Scatolo

Istidina Istamina

Tirosina Tiramina

�Low Trp/LNAAs plasma values in epileptics (in depression, in multiple sclerosis,…, too )

� Intestinal dysbiosis: excessive decarboxylation of proteic amino acids:

� Lunardi G, Mainardi P, et al.. Tryptophan and epilepsy. Adv Exp Med Biol. 1996;398

M. S. Wallin and A. M. Rissanen. Food and mood: relationship between food, serotonin and affective disorders.

Acta Psychiatrica Scandinavica (89) s377; 36-40. 1994

Serotonina e umore

- Sintesi cerebrale di Serotonina F(quantità di trp captata dal cervello)

- La captazione cerebrale di trp F( rapporto plasmatico trp/LNAAs)

LNAAs (Tyr, Val, Met, Ile, Leu, Phe, Trp)

Chugani by PET

- Bassi rapporti trp/LNAAs in depressione, epilessia, Sclerosi multipla, ...

1/3 reduction in brain influx rate

� Marion JL, Bigot JC, Goas JY . Presse Med 1985;14(12):681–3

� Ko FJ, Chiang CH, et al.. Ko Hsiung I Hsueh Ko Hsueh Tsa Chih 1993;9(3):131–42

� Lunardi G, Mainardi P, et al. Tryptophan and epilepsy. In: Recent Advances in Tryptophan Research (Eds G. Allegri Filippini & V. L.

Costa) NY & London, Plenum Press, 1995: pp. 101–102

� Lunardi G, Mainardi P, et al.. Tryptophan and epilepsy. Adv Exp Med Biol. 1996;398

Arnold E, Fineberg N, Hannah P, Glover V, Pitt B, Sandler M. Is the tyramine test for depressive illness useful in elderly

patients? J Affect Disord. 1992 Sep;26(1):1-5

- Risposta insulinica dopo assunzione di carboidrati

- L’insulina sposta nei tessuti i LNAAs

- Il Trp è l’unico LNAA legato alle proteine

- L’insulina agirà meno sul Trp, quindi

La risposta insulinica porta ad:- un aumento del rapporto plasmatico Trp/LNAAs, - quindi ad un aumento della sua captazione cerebrale e - ad un corrispondente aumento di sintesi cerebrale di serotonina

Risposta serotoninergica all’assunzione di carboidrati

La fame nervosa è un disperato tentativo di aumentarela serotonina cerebrale

Asse intestino-cervello. Quali evidenze?

1) Traumatic brain injure on intestinal membrane

before after

Lactulose/ mannitol permeability

Hang Ch et al, Intestinal mucosa structure after TBI. World J Gastroenterol 2003,9 (12) 2776-2781

Hung CR, Cheng JT, Shih CS. Gastric mucosal damage induced by arecoline seizure in rats. Life Sci. 2000 May 5; 66 (24): 2337-49

2) Gastric mucosal damage induced by arecoline i.c.v. in rats

�Riazi K, Honar H, Homayoun H, Demehri S, Bahadori M, Dehpour AR. Intestinal inflammation alters thesusceptibility to pentylenetetrazole-induced seizure in mice. J Gastroenterol Hepatol. 2004 Mar;19(3):270-7.

3) To produce intestinal inflammation by croton oil decreases epileptogenic threshold.

Ruolo dell’infiammazione

Neuroinflammation plays a key role in the progression or resolution of pathological

conditions.

Inflammatory responses in the brain parenchyma have been associated with the

etiopathogenesis of different neurological disorders, including central nervous system (CNS)

infection, brain ischemia, multiple sclerosis, Alzheimer’s disease, and Parkinson’s disease

Lima IV, Bastos LF, Limborço-Filho M, Fiebich BL, de Oliveira AC. Role of prostaglandins in neuroinflammatory and

neurodegenerative diseases. Mediators Inflamm. 2012;2012:946813

Vulnerabilità

“il lubrico sacco oe si tramuta in merda quel che si trangugia”.

Small intestine or bowel length

• Duodenum (adult 25 cm length)• Jejunum (adult 1.4 m length)• Ileum (adult 3.5 m length)• Large intestine or bowel• Cecum (caecum)

• Vermiform appendix ("appendix", adult 2 to 20 cm length)

• Colon• Ascending colon (adult 25 cm length)• Transverse colon• Descending colon• Sigmoid colon Intestinal Functions

Small Intestine• absorption of nutrients and minerals found in food• Duodenum -principal site for iron absorptionCecum• connects the ileum with the ascending colon• separated by the ileocecal valve (ICV, Bauhin's valve)• connected to the vermiform appendix ("appendix")Colon• absorbs fluid, water and salts, from solid wastes• site of commensal bacteria (flora) fermentation of

unabsorbed material

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Alessio Fasano et al Zonulin, a newly discovered modulator of intestinal permeability, and its

expression in coeliac disease, The Lancet, Volume 355, Issue 9214, 29 April 2000, Pages 1518-1519,

BRAININTESTINE

49

Nel cervello immaturo scarsa inibizione da parte del GABA.

Formazione dei canali del Cl e TJs intestinali

� La microflora rappresenta la prima linea difensiva.

� Lo sviluppo del microbiota avviene in seguito a complessi processi inflenzatidall’interazione tra il microbico e l’ospite e da agenti esterni.

� Gli agenti esterni sono la composzione microbiotica materna, la dieta, il tipo diparto e le medicazioni

51

� Durante la gestazione e alla nascita la completapermeabilità intestinale permette il passaggio dianticorpi e fattori di crescita.

� Il colostro sostituisce il cordone ombelicale nelladuplice funzione di proteggere e nutrire

� La maturazione intestinale rappresenta ilgraduale aumento di un’autonomia protettiva daparte del neonato.

� In questa fase sempre più funzioni vitali sonotrasmesse al microbiota

Xu RJ. Development of the newborn GI tract and its relation to colostrum/milk intake: a review. Reprod Fertil Dev. 1996; 8 (1) :35-48.

These changes are apparently related to the onset of colostrum ingestion, becausestarved or water-fed newborns showed little changes in the GI tract.

52

Profondi cambiamenti nel periodo postnatale: modificazioni morfologiche ematutazione funzionale:

• L’esofago aumenta la produzione di muco

• Lo stomaco aumenta la secrezione di acidi

• L’intestino perde la capacità di assorbire macrmolecole, ammino acidi liberi e disintetizzare enzimi digestivi

“developmental programming”

Nel periodo postnatale fattori ambientali hanno un elevato impatto sullo sviluppo futurodell’organismo, sulle sue strutture e funzioni

“an environmental factor acting during a sensitive or vulnerable developmental

period exerts effects that impact on structure and function of organs that, in some

cases, will persist throughout life” Seckl JR, Meaney MJ (2004) Glucocorticoid programming. Ann N Y

Acad Sci 1032:63–84

One such environmental factor is the gut microbiota

that it has been shown to contribute to developmental programming of

� epithelial barrier function,

� gut homeostasis, and angiogenesis,

� the innate and host adaptive immune function.

53

Il microbiota intestinale controlla lo sviluppo degli altri organi in tutto il corpo

exposure to microbial pathogens during similar developmental periods result inbehavioral abnormalities,

including anxiety-like behavior and impaired cognitive function in rodents

Desbonnet L, Garrett L, Clarke G, Bienenstock J, Dinan TG (2008) The probiotic Bifidobacteria infantis: An

assessment of potential antidepressant properties in the rat. J Psychiatr Res 43:164–174.

“developmental programming”

54

Studi epidemiologici hanno indicato un'associazione tra comuni disturbi dello svilupponeurologico, quali

autismo e schizofrenia

e le infezioni dei patogeni microbici durante il periodo perinatale

Finegold SM, et al. (2002) Gastrointestinal microflora studies in late-onset autism. Clin Infect Dis 35

(Suppl 1):S6–S16

Mittal VA, Ellman LM, Cannon TD (2008) Gene-environment interaction and covariation in

schizophrenia: The role of obstetric complications. Schizophr Bull 34:1083–1094

the role of the gut flora in health and disease, and its modification as therapy

o 1012 viable bacteria/ g

o 400-500 different species

o Number of genes is 50-100 times than in our genoma

normal gut structure and function is the end-point of a complex set of interactions betweenthe host and microorganisms colonizing the gut.

Io

ospito

lui!!

Schultz D, Wolynes PG, Ben Jacob E, Onuchic JN. Deciding fate in adverse times: sporulation and competence in Bacillus subtilis. Proc Natl Acad Sci U S A.

2009 Dec 15;106(50):21027-34. 55

“Our results suggest that excess caloric consumption is not only a result of undisciplined

eating but that intestinal bacteria contribute to changes in appetite and metabolism”

Are belly bacteria making us overeat?

Andrew Gewirtz, associate professor of pathology and laboratory medicine at Emory University School of Medicine.

Toll-like receptorsrecognise molecules of pathogens, i.e. TLR-5 recognizes flagellin

TLR5 plays a prominent rolein controlling bacteria in the intestine.

1) were about 20 percent heavier than regular mice andhad:

- elevated triglycerides, cholesterol, blood pressure,- mildly elevated blood sugar- increased production of insulin.

2) consumed about 10 % more food than their regular relatives.

The TLR5-deficient mice:

3) exhibit “metabolic syndrome,”

4) develop colitis, an inflammatory bowel disease.

56

By inducing the production of pro-inflammatory cytokines and cell adhesion molecules in

immune cells, TLRs may indirectly damage neurons in conditions such as ischemic stroke

and multiple sclerosis.

neurons also express a subset of TLRs and that their activation promotes neuronal

degeneration in experimental models of stroke and Alzheimer’s disease.

TLRs may also play roles in regulating the processes of neurogenesis and neurite

outgrowth, suggesting roles in neuronal plasticity.

57

58

Peroxisome Proliferator-ActivateReceptors

Citraro R et al. Antiepileptic action of N-palmitoylethanolamine through CB1 and PPAR-

α receptor activation in a genetic model of absence epilepsy. Neuropharmacology. 2013

Jun;69:115-26.

Peroxisomes function to rid the cell of toxic substances, in

particular, hydrogen peroxide -- a common byproduct of cellular

metabolism.

These organelles contain enzymes that convert the hydrogen

peroxide to water, rendering the potentially toxic substance safe

for release back into the cell.

PPARs are nuclear hormone receptors that bind peroxisome

proliferators and control the size and number of peroxisomes produced

by cells.

PPARs mediate a variety of biological processes, and may be involved

in the development of several chronic diseases, including diabetes,

obesity, atherosclerosis, and cancer

PEA plays a key role in the regulation of the inflammatory response and pain through,

among others, activation of nuclear peroxisome proliferator-activated receptors (PPAR-α).

Wahli W, Michalik L. PPARs at the crossroads of lipid signaling and inflammation. Trends

Endocrinol Metab. 2012 Jul;23(7):351-63

Critical nutrients, oxygen and othermolecules are constantly taken upacross these mucosal barriers

another important function of the mucous is to keep invadingpathogens out

Daily these mucous membranes are bombarded by outside elements and it is up

to the unique immune system of the mucous to determine what is potentiallyharmful and what is beneficial

Barriera Mucosale

Thight Junctions, Zot, and Zonulin

A century ago, TJs were conceptualized as a secreted extracellular cement forming an

absolute and unregulated barrier within the paracellular space.

Recent studies had shown TJs are dynamic structures subjected to structural changes that

dictate their functional status under a variety of developmental scenarios.

TJs must be capable of rapid and coordinated responses.

Presence of a complex regulatory system that orchestrates the state of assembly of the TJ

multiprotein network

zonula occludens toxin (Zot), an enterotoxin elaborated by Vibrio cholerae that reversibly

opens TJ and its eukaryotic counterpart zonulin appears to be involved in several

functions, including TJ regulation responsible for the movement of fluid, macro-

molecules, and leukocytes between the bloodstream and the intestinal lumen and vice

versa.

Another possible physiological role of intestinal zonulin is the protection against

microorganism colonization of the proximal intestine (innate immunity)

A common denominator in autoimmune diseases is the presence of several pre-existing conditions that lead to an autoimmune process.

• The first of these conditions is the genetic susceptibility of the host immune

system to recognize, and potentially misinterpret, an environmental antigen

presented within the gastrointestinal tract

• The second is that the host must be exposed to the antigen

• Finally, the antigen must be presented to the gastrointestinal mucosal immune

system following its paracellular passage from the intestinal lumen to the gut

submucosa

• this process is normally prevented by competent TJ

• In many cases, increased intestinal permeability seems to precede diseaseand causes an abnormality in antigen delivery that triggers the multiorganprocess leading to the autoimmune response.

• Autoimmune diseases involve a miscommunication between

innate and adaptive immunity

• Molecular mimicry or bystander effects alone might not explain

entirely the complex events involved in the pathogenesis of

autoimmune diseases.

• The continuous stimulation by nonself-antigens (environmental

triggers) seems to be necessary to perpetuate the process

• Contrary to general belief, this concept implies that the

autoimmune response can theoretically be stopped and

perhaps reversed if the interplay between genes predisposing

individuals to the development of autoimmunity and

environmental triggers is prevented or eliminated

• In addition to genetic predisposition and exposure to triggeringnonself-antigens, the loss of the protective function of mucosalbarriers that interact with the environment (mainly the

gastrointestinal and mucosa) is necessary for autoimmunity to

develop

zonulin is overexpressed in tissues and sera of subjects affected by autoimmune diseases:- Celiac diseases- type 1 Diabetes- Asthma- Ankylosing Spondylitis- Inflammatory Bowel Diseases- Multiple Sclerosis: 29% of MS patients have 2-fold zonulin plasma levels

than controls.

- This mean 71% of MS patients have normal zonulin plasma levels

Alessio Fasano reports:

It is generally accepted that MS is an immune-mediated disease

MS remains largely a clinical diagnosis, not easy because of the heterogeneity of signsand symptoms, many of which are not specific to MS. (Giesser BS. Diagnosis of multiple sclerosis. Neurol

Clin. 2011 May;29(2):381-8)

Demyelination is considered the main neuropathological hallmark of MS

It is a possible epiphenomenon on the basis of the observed disagreement betweenneurological deficit and pathological changes, and on the evidence that the distributionof plaques and the pathological changes don’t always correlate with the clinicalsymptomatology. (Sandyk R. Demyelination as an epiphenomenon in multiple sclerosis. Int J Neurosci. 1993 Oct;72(3-4):141-8)

MULTIPLE SCLEROSIS

Demyelinating plaques are reported also in other neurologic diseases, quite differentfrom MS, but, more surprisingly,

they were discovered unexpectedly at post mortem evaluation of patients who neverexperienced any neurological deficit,!!!!!!!!!

(George W. Multiple sclerosis. Anatomopathological findings of multiple sclerosis in diseases not clinically diagnosed. Schweiz Med

Wochenschr. 1961 May 20;91:605-7; Phadke JG, Best PV. Atypical and clinically silent multiple sclerosis: a report of 12 cases discovered

unexpectedly at necropsy. J Neurol Neurosurg Psychiatry. 1983 May;46(5):414-20)

Silent MS!!!

(Silent is the brain of some researchers!!!!!)

Demyelination is surely the main hallmark of the experimental model mostly used inpre-clinical studies on MS drug-discovering, the Experimental AutoimmuneEncephalomyelitis (EAE) model, since it is induced by sensitization against myelin basicprotein

But it is controversial if it is correct to use EAE model as MS model, since EAE resemblesacute disseminated encephalomyelitis rather than MS (Chaudhuri A, Behan PO. Multiple sclerosis: looking

beyond autoimmunity. J R Soc Med. 2005 Jul;98(7):303-6; Behan PO, Chaudhuri A, Roep BO. The patogenesis of multiple sclerosis

revisited. J R Coll Physicians Edinb. 2002; 32: 244-65.)

Few agents have translated from efficacy in EAE to the treatment of human disease, puttingin evidence as

“everything stops EAE (phosphate buffer saline, too), nothing cure MS”.

(Baker D, Gerritsen W, Rundle J, Amor S. Critical appraisal of animal models of multiple sclerosis. Mult Scler. 2011

Jun;17(6):647-57)

The actually MS therapies are mainly based on the autoimmunity aspect.

(Wingerchuk DM, Noseworthy JH. Randomized controlled trials to assess therapies for multiple sclerosis. Neurology. 2002 Apr

23;58(8 Suppl 4):S40-8.)

Even if IFNββββ is reported to be able to reduce the conversion rate to MS in the first 2-3years of the treatment, after 5 years, no gain in terms of disability has been found,.he IFNB Multiple Sclerosis Study Group. Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. I. Clinical results of a

multicenter, randomized, double-blind, placebo-controlled trial. Neurology 1993;43:655–61.

Kinkel RP et al.IM interferon beta-1a delays definite multiple sclerosis 5 years after a first demyelinating event.Neurology. 2006 Mar

14;66(5):678-84.

Filippini G, Munari L, Incorvaia B, Ebers GC, Polman C, D'Amico R, Rice GP. Interferons in relapsing remitting multiple sclerosis: a

systematic review. Lancet. 2003 Feb 15;361(9357):545-52.

A systemic review on Glatiramer acetate reports it does not alter relapse rate orprogression in MS. Moreover, these therapies are ineffective in the secondaryprogressive phase of the disease.Munari LM, Filippini G. Lack of evidence for use of glatiramer acetate in multiple sclerosis. Lancet Neurol. 2004 Nov;3(11):641.

Panitch H, Miller A, Paty D, Weinshenker B. Interferon beta-1b in secondary progressive MS: results from a 3-year controlled study.

Neurology. 2004 Nov 23;63(10):1788-95.

In the meanwhile, MS incidence is reported to increase and until now, the death isattributable to MS in two-thirds of cases, with a median time to death around 30 yearsfrom disease onset. Bronnum-Hansen H, Koch-Henriksen N, Stenager E. Trends in survival and cause of death in Danish

patients with multiple sclerosis. Brain 2004; 127: 844–50.

The severity of MS is unclear, because there is disagreement between the results reportedby clinical studies and those reported in observational studies.

The firsts report MS does not affect life expectancy, while observations from registrystudies reveal an higher death rate in patients with MS compared with the generalpopulation, indicating that MS is an important public health issue.Benson K, Arthur HJ. A comparison of observational studies and randomized controlled trials. N Engl J Med 2000;342:1878–86. Nicholas R,

Rashid W. Multiple sclerosis. Clin Evid (Online). 2012 Feb 10;2012. pii: 1202. Hurwitz BJ. Analysis of current multiple sclerosis registries.

Neurology. 2011 Jan 4;76(1 Suppl 1):S7-13

CCSVIs e SM: metodo Zamboni

Comi, presidente dell’AISM, DIFFIDA

i neurologi ad inviare pazienti MS ad effettuare intervento di angioplastica.

Il Ministero lo vieta!!!!

Zhu Y, Liu HN, Zhang CD, Liang HY. Meta-analysis on relationship between hyperhomocysteinemia and

multiple sclerosis. Zhonghua Yi Xue Za Zhi. 2009 Nov 24;89(43):3055-7.

1) Iper omocistiniuria in pazienti SM

2) Iper omocistiniura responsabile stenosi vascolari

Milosević-Tosić M, Borota J. Hyperhomocysteinemia--a risk factor for development of occlusive vascular

diseases. Med Pregl. 2002 Sep-Oct;55(9-10):385-91.

• Low rTRP plasmatic values in MS patients had already reported in 1979 by Monaco et al.

• On the basis of the low value of rTRP, a low Trp brain uptake could be evaluated and, then, a low brain serotonin synthesis in MS patients could be suggested.

• Confirming the brain serotonin deficit, low HVA and 5-HIAA levels have been reported in the cerebrospinal fluid (CSF) of MS patients,, as well in other neurodegenerative diseases.

Lunardi G, Mainardi P, Rubino V et al. Tryptophan and epilepsy. Adv Exp Med Biol. 1996;398:101-2.

Sonninen V, Riekkinen P, Rinne UK. Acid monoamine metabolites in cerebrospinal fluid in multiple sclerosis. Neurology 1973: 23; 760-763

Andersen O, Johansson BB, Svennerholm L. Monoamine metabolites in successive samples of spinal fluid. A comparison between healthy

volunteers and patients with multiple sclerosis. Acta Neurol Scand. 1981 Apr;63(4):247-54.

Ichikawa N. Study on monoamine metabolite contents of cerebrospinal fluid in patients with neurodegenerative diseases. Tohoku J Exp

Med. 1986 Dec; 150(4):435-46.

Serotonina= serum + tonina: controlla tono del vaso sanguigno

• Most of the brain serotonin is synthesized from Trp by pineal gland, where part of itis converted in melatonin, mainly in the night,

• then a low plasmatic Trp corresponds to a low brain serotonin availability and to ascarce melatonin synthesis.

• low melatonin levels have been reported in MS patients, especially in MS depressedpatients.

• Melatonin is well known to control the sleep and wake cycles day night, but it isrelated with fatigue, too.

• Low plasmatic Trp levels led to low melatonin synthesis, then to sleep disorders andfatigue, symptoms that are often reported by MS patients,.

Lovenberg W, Jequier E, Sjoerdsma A. Tryptophan hydroxylation: measurement in pineal gland, brainstem, and carcinoid tumor.

Science. 1967 Jan 13;155(759):217-9.

Sandyk R, Awerbuch GI. Nocturnal plasma melatonin and alpha-melanocyte stimulating hormone levels during exacerbation of

multiple sclerosis. Int J Neurosci. 1992 Nov-Dec;67(1-4):173-86.

Akpinar Z, Tokgöz S, Gökbel H, Okudan N, Uğuz F, Yilmaz G. The association of nocturnal serum melatonin levels with major depression

in patients with acute multiple sclerosis. Psychiatry Res. 2008 Nov 30;161(2):253-7

Sandyk R, Awerbuch GI. Nocturnal plasma melatonin and alpha-melanocyte stimulating hormone levels during exacerbation of

multiple sclerosis. Int J Neurosci. 1992 Nov-Dec;67(1-4):173-86.

Kaminska M, Kimoff RJ, Schwartzman K, Trojan DA. Sleep disorders and fatigue in multiple sclerosis: Evidence for association and

interaction. J Neurol Sci. 2011 Jan 15.

Melamud L, Golan D, Luboshitzky R, Lavi I, Miller A. Melatonin dysregulation, sleep disturbances and fatigue in multiple sclerosis. J

Neurol Sci. 2012 Mar 15;314(1-2):37-40.

Since melatonin is responsible of the ceasing of firing of serotonergic neurons during REMsleep atonia, then symptoms as sleep paralysis, may reflect a manifestation of cataplexy,sometimes reported in MS, .Mendez MF. Multiple sclerosis presenting as catatonia. Int J Psychiatry Med. 1999;29(4):435-41

Sandyk R. The pineal gland, cataplexy, and multiple sclerosis. Int J Neurosci. 1995 Dec;83(3-4):153-63.

• Also the diurnal variation in vision, reported in MS, maybe related to melatonincircadian secretion as the rapid normalization of visual evocated potential latenciesobtained by the application of picoTesla magnetic field is suggesting the enhancementof neurotransmitter functions rather than a remyelination process.

• In fact, magnetic field stimulates the synthesis of melatonin controlling the release ofmonoamines.

• Moreover, melatonin has been reported to have several roles in vision and it resultssynthesized in the eye.

• Thus a scarce serotonin and melatonin availability maybe responsible of some visionproblems in MS.

Melatonin is linked to anti-inflammatory activity of the brain, too.

Esposito E, Cuzzocrea S. Antinflammatory activity of melatonin in central nervous system. Curr Neuropharmacol.

2010 Sep;8(3):228-42

Recently the role of inflammatory process has been linked to the ability of the brain torepair itself .

Martino G. How the brain repairs itself: new therapeutic strategies in inflammatory and degenerative CNS disorders.

Lancet Neurol 2004;3:372-78

Okatani Y, Wakatsuki A, Reiter RJ. Melatonin suppresses homocysteine enhancement of serotonin-induced

vasoconstriction in the human umbilical artery. J Pineal Res. 2001 Oct;31(3):242-7

Karl Popper (1902-1994) pone al centro dell'epistemologia la fondamentaleasimmetria tra verificazione e falsificazione di una teoria scientifica:

infatti, per quanto numerose possano essere, le osservazionisperimentali a favore di una teoria non possono mai provarladefinitivamente e

basta anche solo una smentita sperimentale per confutarla.

La falsificabilità è anche il criterio di demarcazione tra scienza e non

scienza: una teoria è scientifica se e solo se essa è falsificabile.

Secondo Karl Popper la scienza si sviluppa in un continuo alternarsi diprove ed errori.

La differenza tra un ameba e Einstein è nella critica ai propri tentativi di soluzione:

mentre l’ameba potrebbe perire nel caso non riuscisse a risolvere il problema,Einstein sarebbe predisposto psicologicamente a criticare e falsificare la sua teoria.

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GRAZIE PER L’ATTENZIONEGRAZIE PER L’ATTENZIONEGRAZIE PER L’ATTENZIONEGRAZIE PER L’ATTENZIONE