ritmo circadiano, sistema immunitario e insufficienza …...ritmo circadiano, sistema immunitario e...

Ritmo circadiano, sistema immunitario e insufficienza surrenalica

Andrea M. Isidori

[email protected]

COMPOUND E and COMPOUND ABetween 1936 and 1938 Kendall’s group isolated and purified 20 chemicaladrenal compounds including Compound E (cortisone). By the same timeReichstein’s group in Basel isolated and purified 28 compounds includingCompound A (desoxycorticosterone).

By 1940 their work had allowed isolation of 28 adrenal steroids includingalso cortisol (Compound F) and corticosterone

And then…. World War II

… and the legend of a miraculous compoundused by pilots of Luftwaffe

The Nobel Prize in Physiology or Medicine 1950Edward C. Kendall, Tadeus Reichstein, Philip S. Hench

The Nobel Prize in

Physiology or Medicine 1950

The Nobel Prize in Physiology or Medicine 1950 was awarded jointly to Edward Calvin Kendall, Tadeus Reichstein and Philip Showalter Hench "for their discoveries relating to the hormones of the adrenal cortex, their structure and biological effects".

Edward C. KendallPrize share: 1/3

Tadeus ReichsteinPrize share: 1/3

Philip Showalter HenchPrize share: 1/3

INSUFFICIENZA CORTICOSURRENALICA

PRIMARIA

Arlt W. Allolio B. Lancet 2003Charmandari et al, Lancet 2014

TERAPIA SOSTITUTIVA CON GLUCOCORTICOIDI

SECONDARIA

TERZIARIA

SCHEMI DI TERAPIA

MANTENIMENTO STRESS MINORI STRESS MAGGIORI

EDUCAZIONE DEL PAZIENTE E DEI FAMILIARI

SCHEMA DI TRATTAMENTO

1. Terapia per la deficienza di glucocorticoidi

Cortisone acetato 25–37·5 mg die (BID-TID) Idrocortisone 15–25 mg die (BID-TID)

Steroide Dose equivalente (mg)

Potenza relativa anti-

infiammatoria

Potenza relativa mineralcorticoidea

Emivita plasmatica (h)

Emivita biologica (h)

Cortisone acetato 25 0.8 2 0.5 8-12

Idrocortisone 20 1 2 1.5-2 8-12

Metilprednisolone 4 5 0 1.5-3 18-36

Prednisone 5 4 1 1 18-36

Prednisolone 5 4 1 2-3.5 18-36

Triamcinolone 4 5 0 3.5-4 18-36

Betametasone 0.6-0.75 20-30 0 5.5 36-54

Desametasone 0.75 20-30 0 2-3.5 36-54

SCHEMA DI TRATTAMENTO

2. Terapia per deficienza di mineralcorticoidi (solo nel M. di Addison)

Fludrocortisone (Florinef) 50-250 mcg/die in dose unica (assunto al mattino)Iniziare con 100 mcg/die

Favorisce il ripristino ed il mantenimento del volume plasmatico e dell’equilibrio idro-salino extra ed intracellulare qualora la terapia cortisonica non sia sufficiente

3. Deidroepiandrosterone (opzionale)Pz con compromissione del tono dell’umoreDonne con segni e sintomi da insufficienza androgenicaDHEA 25–50 mg die in dose unica (assunto al mattino)

Charmandari et al, Lancet 2014

TERAPIA CONVENZIONALE CON GLUCOCORTICOIDI

IDROCORTISONE: farmaco di prima scelta

11• Ormone biologicamente attivo:

idrossilazione in posizione 11

• Concentrazione plasmatica:

picco dopo 1 ora dalla somministrazione;

dosabile nel sangue per circa 2 ore;

i suoi metaboliti sono misurabili fino a 4-7 ore

• Legato a proteine per circa il 90% (CBG e albumina) Forma attiva 5-10%

• Assorbimento intestinale ad opera della glicoproteina P

Farmaci che aumentano i livelli di CBG e che possono falsamente elevare i livelli di cortisolo

Estrogeni

Mitotane

IDROCORTISONE

• La fase I del metabolismo dell’idrocortisone è epatica (citocromo CYP3A4, isoenzima del citocromo P450):

Riduzione e Ossidazione

• La fase II: glucuronidazione e rilascio nel circolo enteroepatico ed eliminato tramite il circolo ematico per via renale (70-80%) e parte nelle feci (20-30%)

Farmaci che accelerano il metabolismo mediante induzione del CYP3A4

Fenobarbitale

Fenitoina

Carbamazepina

Primidone

Rifampicina

Etosuccimide

Pioglitazone

Farmaci che riducono il metabolismo mediante inibizione del CYP3A4

Itraconazolo

Ritonavir

Fluoxetina

Diltiazem

Cimetidina

0 20mg/m2/die

8-10

under-exposure adequate glucocorticoid replacement

androgenssuppression

negative effects on growth

15 17

over-exposure

Dosage titration

Webb EA et al, Current and novel approaches to children and young people with CAH and AI. Best practice and Research Clinical Endocrinologyand Metabolism 2015

safe

CORTISONE ACETATO

PRO FARMACO

Biologicamente attivo solo dopo idrossilazione nel fegato: l’80% è trasformato in cortisolo ad opera della 11β-HSD1 con perdita di circa 1/5 della dose efficace

Segue la stessa via di metabolizzazione dell’idrocortisone

CORTISONE ACETATO versus IDROCORTISONE

• Minore attività glucocorticoide e relativa mineralcorticoide

• Picco di massima concentrazione plasmatica ridotto

• Insufficienza epatica: impossibile idrossilazione nel fegato

• Deficit di 11β-HSD1: impossibile attivare il cortisone (molto rara)

• Sostanze endogene ed esogene (acidi biliari, carbenoxolone, liquirizia) possono inibirne l’attività

ALTRI GLUCOCORTICOIDI

• RITMO CIRCADIANO NON RISPETTATO

• AUMENTO DEGLI EFFETTI COLLATERALI

Steroide Dose equivalente

(mg)

Potenza relativa anti-

infiammatoria

Potenza relativa mineralcorticoidea

Emivita plasmatica (h)

Emivita biologica (h)

Metilprednisolone 4 5 0 1.5-3 18-36

Prednisone 5 4 1 1 18-36

Prednisolone 5 4 1 2-3.5 18-36

Betametasone 0.6-0.75 20-30 0 5.5 36-54

Desametasone 0.75 20-30 0 2-3.5 36-54

Terapia sostitutiva convenzionale con glucocorticoidi

Ridottobenessere

e qualità percepitadella vita

Mortalità

prematura

Ridottadensità minerale

ossea

Alteratoprofilo

metabolico

Steatosi epatica

Bergthorsdottir et al. JCEM 2006, Smans LCCJ et al. ECE 2011, Hahner et al. JCEM 2007 Filipsson et al. JCEM 2007, Zelissen et al. Ann Intern Med 1994; Lövås et al EJE 2009

Alta frequenza di ospedalizzazioni/

infezioni

“Infection was the death cause in 10% (5.1– 14.9) opposed to 6.0% in the general population.”

Erichsen MM et al. - Mortality in Addison’s disease - European Journal of Endocrinology (2009) Burman et al - Deaths Among Adult Patients with Hypopituitarism - J Clin Endocrinol Metab 2013

• Piu’di 40 anni per dimostrare gli effetti collaterali della terapia sostitutiva

• Riduzione dell’aspettatitiva di vita e di QoL, aumento della morbidità

• Rischio di crisi surrenaliche associate ad infezioni ed altri stress

• Rischio di sovradosaggio e fallimento nella gestione complessiva del paziente

NUOVE STRATEGIE TERAPEUTICHE

RIDURRE LA DOSE

MODIFICARE LA MODALITA’DI SOMMINISTRAZIONE

90 PZ: 57 PAI (42 F)33 CAH (21 F)

Divisi in 3 gruppi: 1) Non modifiche della dose di HC (N=50) 2) Aumento della dose di HC (N=13)3) Diminuzione della dose di HC (N=27)

Modification of GC replacement should be undertaken with care

Peak M Mah Clinical Endocrinology 2004;61:367–375.

Attempt to reduce AUC of GC exposure by reducing the peak dose might expose the risk toward immune system overreaction and/or chronic inflammatory state

Porter J et al, Immediate release granule formulation of HC, Alkindi, for treatment of pediatric AI. Expert review of endocrinology and metabolism 2018

Novel strategies for dosage titrationthe “old”….

the “new”

Circadian rhythm of cortisol in 33 individuals with 20-minute cortisol profiling

Circadian rhythm of cortisol

Chan S & De Bono M. Replication of cortisol circadian rhythm: new advances in hydrocortisone replacement therapyTher Adv Endocrinol Metab(2010) 1(3)

Il ritmo distingue gli esseri animati

«Circa diem»

• Physiology: circadian control of the immune system and its feedback mechanisms

• Pathophysiology: relevance of glucocorticoid circadian rhythm

• Adrenal Insufficiency: lack of studies on the immune system in patients on life-long treatment with glucocorticoids

• Recent studies: NK in Addison disease and ad interim results from the DREAM trial

• Perspectives: toward Chronopharmacology

Molecular circadian clockwork

Principal or core feedback loop CLOCK and BMAL1, form heterodimers to activate thetranscription of their target genes containing E-box element

These target genes include their negative regulators the Periods (PERs: PER1, PER2 and PER3) and the Cryptochromes (CRYs: CRY1 and CRY2). The concentration of BMAL1 is adjusted by an auxiliary or stabilizing feedback loop formed by the clock-controlled nuclear receptors REVERBα

Gi Hoon Son, Sooyoung Chung, Kyungjin Kim The adrenal peripheral clock: Glucocorticoid and the circadian timing system Frontiers in Neuroendocrinology 32 (2011) 451–465

• About 10% of the entire genome is under circadian control.

• More than 50% of all genes in the mouse genome oscillate with circadian rhythm somewhere in the body.

Zhang et al., PNAS 2015

A circadian gene expression atlas: implications in medicine


Circadian rhythm of cortisol in 33 individuals with 20-minute cortisol profiling

Circadian rhythm of cortisol

Chan S & De Bono M. Replication of cortisol circadian rhythm: new advances in hydrocortisone replacement therapyTher Adv Endocrinol Metab(2010) 1(3)

378 | JULY 2009 | VOLUME 5 www.nature.com/ nrendo

REVIEWS

The pathogenesis of chronic-stress-related disorders

can also be explained by sustained, excessive secretion

and effects of the major mediators of stress and sickness

syndromes, which influence the activities of multiple

homeostatic systems.2,3,9,30–36 These disorders thus rep-

resent chronic, maladaptive effects of two physio logical

processes whose mediators are meant to be secreted in a

quantity-limited and time-limited fashion but have gone

awry. The negative consequences of these effects are both

behavioral and somatic.

Behavioral and somatic consequences

The behavioral consequences of chronic stress result

from continuous or intermittent activation of the stress

and sickness syndromes, and prolonged secretion of

their mediators.2,7,8,12,41–47 Thus, CRH, norepinephrine,

cortisol and other hormones activate the fear system,

which produces anxiety, anorexia or hyperphagia; the

same mediators cause tachyphylaxis of the reward

system, which produces depression and cravings for

food, other substances or stress. These mediators also

suppress the sleep system, which causes insomnia, loss

of sleep and daytime somnolence. On the other hand,

IL-6 and other mediators, possibly in synergy with those

mentioned above, generate fatigue, nausea, headaches

and other pains. Executive and cognitive systems also

malfunction as a result of prolonged, chronic activa-

tion of stress and sickness syndromes and people may

perform and plan suboptimally and make and pursue

the wrong decisions. A vicious cycle is initiated and

sustained, in which be havioral maladjustment leads to

psychosocial problems in the family, peer group, school

and/or work, which sustain or cause further mediator

changes and exacerbate behavioral mal adjustment. The

young, developing brain is particularly vulner able, as it

lacks prior useful experiences to which it can resort.

The somatic consequences of continuous or inter-

mittent activation of the stress and sickness syndromes

can be equally devastating (or even worse) than their

behavioral consequences.2,3,7,8,27,31,41–47 In develop-

ing children, growth may be suppressed as a result of

a hypo functioning growth hor mone axis; in adults,

stress-induced hypo gonadism can manifest as loss of

libido and/or hypo ferti lity, and hyperactivity of the

sympathetic system can lead to essential hypertension.

Chronic hypersecretion of stress mediators, in indivi-

duals with a vulnerable background exposed to a permis-

sive environment, may lead to visceral fat accumu lation

as a result of chronic hypercortisolism, reactive insulin

hyper secretion, low growth-hormone secretion and

hypogonadism (Figure 2).2,3,27,47–52 These same hormonal

changes lead to sarcopenia, osteo penia and/or osteo-

porosis. Visceral obesity and sarco penia are associ ated

with manifestations of the metabolic- syndrome, such

as dyslipidemia (elevated levels of total cholesterol, tri-

glycerides and LDL-cholesterol and decreased level

of HDL-cholesterol), hypertension and carbo hydrate

in tolerance or type 2 diabetes mellitus. Genetically or

constitutionally vulnerable women of reproductive age

may develop polycystic ovary syndrome. Stress-related

IL-6 hypersecretion plus adipose-tissue-generated

inf lammatory hypercytokinemia, as well as hyper-

cortisolism, contribute to increased production of

acute-phase reactants and blood hypercoagulation.49–52

Insulin resistance, hypertension, dislipidemia, hyper-

cytokinemia and blood hypercoagulation lead to endo-

thelial dysfunction and consequently atherosclerosis ,

with its cardio vascular and neurovascular sequelae.

Chronic-stress-induced immune dysfunction, pri-

marily the TH1 to T

H2 switch, increases the vulner ability of

indivi duals to certain infections and autoimmune dis-

orders (Figure 1).6–8,29–31,34 For instance, the immune dys-

function observed in individuals who are chronically

stressed might contribute to the persistence of infection

with Helicobacter pylori, granted that this pathogen

Box 3 | Conditions with altered HPA axis activity2

Increased activity of the HPA axis

Cushing syndrome ■

Chronic stress ■

Melancholic depression ■

Anorexia nervosa ■

Obsessive–compulsive disorder ■

Panic disorder ■

Excessive exercise (obligate athleticism) ■

Chronic, active alcoholism ■

Alcohol and narcotic withdrawal ■

Diabetes mellitus ■

Central obesity (metabolic syndrome) ■

Post-traumatic stress disorder in children ■

Hyperthyroidism ■

Pregnancy ■

Decreased activity of HPA axis

Adrenal insufficiency ■

Atypical/ seasonal depression ■

Chronic fatigue syndrome ■

Fibromyalgia ■

Premenstrual tension syndrome ■

Climacteric depression ■

Nicotine withdrawal ■

Following cessation of glucocorticoid therapy ■

Following Cushing syndrome cure ■

Following chronic stress ■

Postpartum period ■

Adult post-traumatic stress disorder ■

Hypothyroidism ■

Rheumatoid arthritis ■

Asthma, eczema ■

Abbreviation: HPA, hypothalamic–pituitary–adrenal.

nrendo_106_JUL09.indd 378 4/6/09 19:04:04

378 | JULY 2009 | VOLUME 5 www.nature.com/ nrendo

REVIEWS

The pathogenesis of chronic-stress-related disorders

can also be explained by sustained, excessive secretion

and effects of the major mediators of stress and sickness

syndromes, which influence the activities of multiple

homeostatic systems.2,3,9,30–36 These disorders thus rep-

resent chronic, maladaptive effects of two physio logical

processes whose mediators are meant to be secreted in a

quantity-limited and time-limited fashion but have gone

awry. The negative consequences of these effects are both

behavioral and somatic.

Behavioral and somatic consequences

The behavioral consequences of chronic stress result

from continuous or intermittent activation of the stress

and sickness syndromes, and prolonged secretion of

their mediators.2,7,8,12,41–47 Thus, CRH, norepinephrine,

cortisol and other hormones activate the fear system,

which produces anxiety, anorexia or hyperphagia; the

same mediators cause tachyphylaxis of the reward

system, which produces depression and cravings for

food, other substances or stress. These mediators also

suppress the sleep system, which causes insomnia, loss

of sleep and daytime somnolence. On the other hand,

IL-6 and other mediators, possibly in synergy with those

mentioned above, generate fatigue, nausea, headaches

and other pains. Executive and cognitive systems also

malfunction as a result of prolonged, chronic activa-

tion of stress and sickness syndromes and people may

perform and plan suboptimally and make and pursue

the wrong decisions. A vicious cycle is initiated and

sustained, in which be havioral maladjustment leads to

psychosocial problems in the family, peer group, school

and/or work, which sustain or cause further mediator

changes and exacerbate behavioral mal adjustment. The

young, developing brain is particularly vulner able, as it

lacks prior useful experiences to which it can resort.

The somatic consequences of continuous or inter-

mittent activation of the stress and sickness syndromes

can be equally devastating (or even worse) than their

behavioral consequences.2,3,7,8,27,31,41–47 In develop-

ing children, growth may be suppressed as a result of

a hypo functioning growth hor mone axis; in adults,

stress-induced hypo gonadism can manifest as loss of

libido and/or hypo ferti lity, and hyperactivity of the

sympathetic system can lead to essential hypertension.

Chronic hypersecretion of stress mediators, in indivi-

duals with a vulnerable background exposed to a permis-

sive environment, may lead to visceral fat accumu lation

as a result of chronic hypercortisolism, reactive insulin

hyper secretion, low growth-hormone secretion and

hypogonadism (Figure 2).2,3,27,47–52 These same hormonal

changes lead to sarcopenia, osteo penia and/or osteo-

porosis. Visceral obesity and sarco penia are associ ated

with manifestations of the metabolic- syndrome, such

as dyslipidemia (elevated levels of total cholesterol, tri-

glycerides and LDL-cholesterol and decreased level

of HDL-cholesterol), hypertension and carbo hydrate

in tolerance or type 2 diabetes mellitus. Genetically or

constitutionally vulnerable women of reproductive age

may develop polycystic ovary syndrome. Stress-related

IL-6 hypersecretion plus adipose-tissue-generated

inf lammatory hypercytokinemia, as well as hyper-

cortisolism, contribute to increased production of

acute-phase reactants and blood hypercoagulation.49–52

Insulin resistance, hypertension, dislipidemia, hyper-

cytokinemia and blood hypercoagulation lead to endo-

thelial dysfunction and consequently atherosclerosis ,

with its cardio vascular and neurovascular sequelae.

Chronic-stress-induced immune dysfunction, pri-

marily the TH1 to T

H2 switch, increases the vulner ability of

indivi duals to certain infections and autoimmune dis-

orders (Figure 1).6–8,29–31,34 For instance, the immune dys-

function observed in individuals who are chronically

stressed might contribute to the persistence of infection

with Helicobacter pylori, granted that this pathogen

Box 3 | Conditions with altered HPA axis activity2

Increased activity of the HPA axis

Cushing syndrome ■

Chronic stress ■

Melancholic depression ■

Anorexia nervosa ■

Obsessive–compulsive disorder ■

Panic disorder ■

Excessive exercise (obligate athleticism) ■

Chronic, active alcoholism ■

Alcohol and narcotic withdrawal ■

Diabetes mellitus ■

Central obesity (metabolic syndrome) ■

Post-traumatic stress disorder in children ■

Hyperthyroidism ■

Pregnancy ■

Decreased activity of HPA axis

Adrenal insufficiency ■

Atypical/ seasonal depression ■

Chronic fatigue syndrome ■

Fibromyalgia ■

Premenstrual tension syndrome ■

Climacteric depression ■

Nicotine withdrawal ■

Following cessation of glucocorticoid therapy ■

Following Cushing syndrome cure ■

Following chronic stress ■

Postpartum period ■

Adult post-traumatic stress disorder ■

Hypothyroidism ■

Rheumatoid arthritis ■

Asthma, eczema ■

Abbreviation: HPA, hypothalamic–pituitary–adrenal.

nrendo_106_JUL09.indd 378 4/6/09 19:04:04

Conditions with altered HPA axis activity

Chrousos GP. Stress and disorders of the stress system. Nat Rev Endocrinol 2009;5:374–381

Alterations in its rhythmicity are frequently found in many human diseases

Although the precise significance of the daily GC rhythm is not yet well established, a growing body of evidence does point to its clinical importance.

Circadian control of the immune system:- cyclical release for the haematopoietic niche- cyclical recruitment of immune cells to tissues

Rest Activity Activity Rest

24/0 12 24/0

Recruitment of HSCs (haematopoietic stem cell) and

mature immune cells to tissues

Numbers of HSCs and mature immune cells in blood

Cortisol

1. Clock’s gene expression2. Oscillations in adhesion molecules and

chemokines by endothelial cells 3. Oscillations in pro-migratory factors

(homing to tissues at specific stages of circadian cycle)

CXCL12 levels in bone marrow

P-selectin, E-selectin,VCAM1 and ICAM1

levels on endothelial cells

Rest Activity Activity Rest

24/0 12 24/0

Adapted from: Scheiermann C et al. Nat Rev Immunol 2013;13:190.Lightman SL et al. Nat Rev Neurosci. 2010 Oct;11:710.

4. Hormonal regulation and sympathetic nerves stimulation

HPA control of immune circadian rhythm

Scheiermann C et al. Nat Rev Immunol 2013;13:190.

CRH

ACTH, adrenocorticotropic hormone; SNS, sympathetic nervous system; MIF, macrophage inhibitory factor; CRH, Corticotropin-releasing hormone; SCN, suprachiasmatic nuclei

Interleukins

The HPA-immunity counter-regulatory feedback (1)

Light activates the adrenal gland

A. Ishida, T. Mutoh, T. Ueyama, H. Bando, S. Masubuchi, D. Nakahara, G. Tsujimoto, H. Okamura, Light activates the adrenal gland: timing of gene expression and glucocorticoid release, Cell Metab. 2 (2005) 297–307.

Per1-luc luminescence in visceral organs

HOW?

Circadian Metabolism

in the Light of Evolution

Endocr Rev. 2015;36(3):289-304

Sunlight, temperature, physical activity, and food intake serve as basic entraining cues, or zeitgebers, to reset

the master clock (A)

in the hypothalamic SCN each day.

Dexamethasone induces circadian gene expression1

GRAlfpCre mice with a liver-specific disruption of the GR gene

The glucocorticoid receptor (GR) is required for dexamethasone-induced Per1 phase shifting1

Cortisol synchronizes peripheral clocks

1. Balsalobre A et al. Science 2000;289:2344-2347; 2. Gi Hoon Son et al. Frontiers in Neuroendocrinology 2011;32:451–465.

DBP, TBP

4h shift

Nader, N., Chrousos, G. P., Kino, T. Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications. FASEB J. 23, 1572–1583 (2009)

The clockwork influenceresponsiveness to

glucocorticoids

The Clock-HPA axis counter-regulatory feedback (2)

Clock/Bmal1 physically interact with the ligand-binding domain of the GR through a regionenclosed in the C-terminal part of the Clock protein, and suppressed GR-inducedtranscriptional activity

The clockwork influence responsiveness to GC

Nancy Nader et al Trends Endocrinol Metab. 2010 May ; 21(5): 277–286.

By disrupting the clock -> we can alter the efficacy (side effects) of GC

Figure 2.

The circadian CLOCK system and the HPA axis influence each other’s activity at multiple

levels. The central CLOCK under the regulation of the light input controls the HPA axis and

produces regular diurnal secretion of glucocorticoid hormones from the adrenal glands, while

the peripheral CLOCKs, which is located in the adrenal glands and other components of the

HPA axis and are regulated by the central CLOCK through the sympathetic nervous system,

also contribute to the rhythmic glucocorticoid secretion from these organs. Secreted

glucocorticoids in turn reset and phase-delay circadian rhythm of the peripheral CLOCKs by

stimulating the expression of several CLOCK-related genes; this is especially important for

temporal adjustment of body’s activity against stress. The peripheral CLOCKs also regulate

glucocorticoid effect in local tissues through interaction between Clock/Bmal1 and GR,

providing a local counter regulatory feedback loop to the effect of central CLOCK on the HPA

axis.

Nader et al. Page 12

Trends Endocrinol Metab. Author manuscript; available in PMC 2010 May 4.

NIH

-PA

Au

tho

r Ma

nu

scrip

tN

IH-P

A A

uth

or M

an

uscrip

tN

IH-P

A A

uth

or M

an

uscrip

t

Interactions of the Circadian CLOCK System and the HPA Axis

Nancy Nader et al Trends Endocrinol Metab. 2010 May ; 21(5): 277–286.

MMIF

Arjona A & Sarkar DK Neurochem Res 2008;33:708–718.

Circadian gene disruption on the immune system

Several GREs elements

Glucocorticoids

Clock-genes

CCG genes:• STAT3, • STAT5, • NF-κB

v

vv

vv

v

Some monocytes got rhythm22- Circadian timing in disease manifestations3,4

Disease or model Oscillating parameter AcrophaseRheumatoid arthritis Stiffness, pain, serum TNF, serum IL-6 CT5–CT8 Allergic rhinitis Sneezing, nasal congestion CT6 Bronchial asthma Bronchoconstriction CT6

Sputum eosinophils, serum IL-5 CT7 Myocardial infarction Pain CT9 Ischaemic stroke Hypertension CT6–CT12Sickle cell vaso-occlusion Crisis (hospitalization) CT18

1- Circadian response to acute inflammatory insults1,2

In animals it is proven that over-reaction to pathogens (and their by-products) lead to increased lethality in circadian related mechanism!

Is immune circadian rhythm relevant?

1. Shackelford et al. Science 1973;182:285–287. 2. House SD et al. Life Sci 1997;60:2023–2034, Khoa D Nguyen et al. Science 2013;341:1483. Druzd D & Scheiermann C Science 2013;341:1462.3. Gibbs J E et al. Proc Natl Acad Sci USA 2012;109:582–587.4. Scheiermann C et al. Nat Rev Immunol 2013;13:190.

Symptoms of circadian clock mutant animals and human glucocorticoid dysregulation

Gi Hoon Son, Sooyoung Chung, Kyungjin Kim. Frontiers in Neuroendocrinology 2011;32:451–465.

Primary adrenal insuficiency was

associated with a 2–3 fold increased

mortality rate compared with the

background population

Mortality risk in patients with addison’s disease

• The idea that GCs are only immunosuppressive is over simplistic:

1. Circadian rhythm in immune cells regulates cells formation and extravasation to target tissues

2. GCs synchronize endogenous clocks in immune and endothelial cells

3. Circadian rhythm in immune cells controls anticipatory response and pathogen-driven response preventing overreaction

• Disruption of circadian rhythm in immune cells could be linked to time-clustered diseases (myocardial infarction, atheroma plaque rupture, autoimmune exacerbations…)

GC clocks immunity

Scheiermann C et al. Nat Rev Immunol 2013;13:190.Lightman SL et al . Nat Rev Neurosci. 2010 Oct;11:710House SD et al. Life Sci 1997;60:2023–2034Khoa D Nguyen et al. Science 2013;341:1483.

Druzd D & Scheiermann C Science 2013;341:1462.Gibbs J E et al. Proc Natl Acad Sci USA 2012;109:582–587.Scheiermann C et al. Nat Rev Immunol 2013;13:190.Gi Hoon Son et al. Front. in Neuroendocrinol. 2011;32:451–465

Immune function in adrenal insufficiency: neglected area of research

185 related to immunological pathogenesis of autoimmune Addison

9 describing the immune phenotype in AI (miRNA, cytokines)

0 describing myeloid subpopulations

NUOVE STRATEGIE TERAPEUTICHE

Dual-release hydrocortisone preparations

Rivestimento esterno

Nucleo centraleØ 8 mm

LI=liberazione

Immediata

LL= Liberazione Lenta

G. Johannsson et al 2012A.Falorni et al 2013

Unica somministrazione giornaliera per os la mattina al risveglio

La compressa non è divisibile e non deve subire alterazioni chimico-fisiche

Dose di mantenimento 20-30 mg

Dosaggio inferiore in pz con una minima quota di cortisolo endogeno

40 mg dose massima studiata

Clock time 06.00 12.00 18.00 24.0000.00

0

200

400

600

800 Immediate release hydrocortisone tablet

Dual-release HC tablet

Improved Serum Cortisol Profilewith Dual-Release HC tablet

0-24 h

(8 AM-8

AM)

Total exposure 19%

lower on Dual-release

than TID

0-4 h

(8 AM-

12.00)

Morning exposure 6%

higher on Dual-

release than TID

4-12 h

(12.00-8

PM)

Afternoon and early

evening exposure

38% lower on Dual-

release than TID

12-24 h

(8 PM-

8AM)

Night exposure 41%

lower on Dual-release

than TID

Cortisol conc. (nM)

Johannsson G et al. J Clin Endocrinol Metab 2012;97:473–481

The DREAM trial - NCT02277587

Dual RElease Hydrocortisone Versus conventionAl Glucocorticoid replaceMent

Therapy in Hypocortisolism

MCEL: molecular and cellular endocrinology lab, 1Sapienza University of Rome, Rome, Italy

2Università Federico II, Naples, Italy

Spontaneous non-sponsored trial

Controls

Secondaryadrenal insuf.

Primaryadrenal insuf.

Randomisation

Standard Multiple times daily GC

Once-daily MR hydrocortisone

FACsSerumDEXA

Epicardial fatHepatic fat

FACsserum

FACsserum

FACsserum

FACsserumDEXA

Epicardial fatHepatic fat

T=0 1 month

2 months

3 months

6 months

Once-daily MR hydrocortisone

Standard Multiple times daily GC

Standard GC could be IRHC TID or BID or cortisone acetate

Isolation of peripheral blood mononuclear cells

CD14/CD16/ CD56/CD3 labelling

Flow cytometry

analysis

CD

16

CD14+ CD16-Classical monocytes(pro-inflammatory)

CD16+CD14-

Isidori AM et al. Abstract presented at ENDO 2015: PP17-4.

Monocytes

Lymphocytes

CD16+ CD14+(Anti-inflammatory) monocytes

CD14

Healthy Subject

Isidori Venneri et al. The Lancet. Diabetes & Endocrinology, 2017

Endocr Rev. 2015;36(3):289-304Isidori et al. Lancet Diabetes Endocrinol. 2018 Mar;6(3):173-185.

4 wks of DR hydrocortisone

ADDISON’S disease

Male patients #1

Leuko: 6.6 X 103

Neutro: 41.4%Mono: 7.3%Lympho: 44.5%Eosi: 4.8%Baso: 0.7%Luc: 1.2%ESR: 3Fibr: 2.13PCR: 600 ml/dl

Leuko: 5.46 X 103

Neutro: 49.8%Mono: 6.1%Lympho: 36.5%Eosi: 5.5%Baso: 0.7%Luc: 1.4%ESR: 2Fibr: 1.81PCR: 800 nm/dl

HC 15 + 5 mgDual-release

hydrocortisone 20 mg

19%

10.5% 2.9%

56%

0.4% 6.6%

48%

0.2% 3.6%

Before switching treatment

12%

19% 2%



Isidori AM et al. Abstract presented at ENDO 2015: PP17-4. DREAM trial ad interim analysis.

Female patients #8

Leuko: 6.8 X 103

Neutro: 48.1%Mono: 3.1%Lympho: 44.7%Eosi: 1.3%Baso: 0.3%Luc: 1.7%ESR: 9Fibr: 4.45 g/LPCR: 500 mg/dl

Leuko: 8.8 X 103


HC 15 + 5 mg

63 %

0.1% 1 %

18.5 %

11% 4.3 %

50 %

0.1% 0.3 %


12.5 %

7% 4 %




Dual-release hydrocortisone 20 mg

ADDISON’S disease


Male patients #5


Secondary AI (hypopit)

51% 55%

4.1% 0% 1%14.0%

24%

11.1% 3.1%

Leuko: 6.28 X 103


Leuko: 6.9 X 103


Cortisone acetate

29%

10.6% 3.6%

Dual-release hydrocortisone 20 mg





Controls

sAI

CD14+ CD16- monocytes are increased in AI patients

CD14+ CD16- (% cells)

Classical monocytes

(pro-inflammatory)

sAI on CGC

pAI on CGC

CD16+ CD14- (% cells)

NK and NKT

Isidori et al. Lancet Diabetes Endocrinol. 2018 Mar;6(3):173-185.

CD16 expression on NK cells defined as CD56+CD3-

CD

56

CD16

Controls

AI patients

Total numberof CD56+ NK cells does not differ amongthe different groups

CD16+ NK cellsreduction

How NK cells Kill?

-Antibody dependent cytotoxicity-Recognition of altered surface molecules

Mediated by:-Perforin-Granzymes:-TNF-alfa

Beltran Front. Immunol., 2014

Shift timing of GC replacement affect circulating monocytes and NK cells in AI patients

Calssical (inflammatory)Monocytes

% of CD16+ NK cells….OKAY, ….but the mechanism?

Once-daily

Multiple-daily

Isidori et al. Lancet Diabetes Endocrinol. 2018 Mar;6(3):173-185.

CD16 regulation in NK cells

Romee et al. Blood 2013

ADAM17

Soluble CD16

…then is the CD16 Lost?

Isidori - Venneri et al. The Lancet. Diabetes & Endocrinology, 2017

Does it matters?

Correlation between immune changes and infection score

Isidori - Venneri et al. The Lancet. Diabetes & Endocrinology, 2017

Core clockwork in innate immunity

Scheiermann et al, Nature Reviews Immnunoly 2018

Importance of a functional clock in regulating the daily flux of inflammatory cellsbetween bone marrow, blood and immune organs under homeostatic conditions

Total PBMCs

Sorted by cell type:- Lympho- Mono

Pooled patientsby treatment group

Individually analysed Patients

PCR amplification in Real time PCR array predesigned 96-well panel for use with SYBR® Green Circadian rhythms (SAB Target List) H96

Heat map of the transcriptome at baseline

Clock gene group CREB signaling gene group

Transcription gene group Other Circadian Related

19 genes displayed a statistically different level of expression in PBMCs drawn from healthy vs AI subjects

Impact of switching glucocorticoids replacement therapy

12-weeks posttreatment switch:

multiple once daily glucocorticoid

administration

***

Differentially modulated genes in all groups at baseline and after treatment

PER3 plays an interesting role inadipogenesis homeostasis byregulating Klf15, a pivotal genein adipocyte differentiation.

Aggarwal et al Cell Rep. 2017


***

Perforin forms membrane pores that

allow the release of granzymes and

subsequent cytolysis of target cells.

PRF1 deficiency -> IMMUNE DEFECT


***

PRF1 Adipose Tissue Inflammation

PER3 Impaired Adipogenesis


CREB is induced by a variety of growth factors and inflammatory signals; it can promote anti-inflammatory immune responses, through inhibition of NF-kB, induction of IL-10, but it is also considered a nutrient-sensing transcriptional regulatory proteins (with FOXO-p300, PGC-1, and SP1 family members).

Wen A e al. J Immunol 2010

**

J Clin Endocrinol Metab, August 2018, 103(8):2998–

3009

Resetting the expression () of 9.00 AM clock-related genes correlates with the improvement in clinical outcomes (metabolism, inflammation, infections).

Perspectives: toward better Chronopharmacology

Non-physiological cortisol replacement in adrenal insufficiency

Peak M Mah Clinical Endocrinology 2004;61:367–375.

The non-physiological circadian profile rather than the dose could be the major explanation for the AI outcome

FrequentAbnormal

RE-SYNC ? ! ?

Immediate release HC (TID) in patients with AI does not mimick cortisol rhythm by giving multiple peaks and nadirs

Single Pulse GC

Immune cell oscillation

Multiple Pulses GC

Immune cell oscillation

In-phase amplified oscillation

Out-phase disrupted oscillation

Fixing the broken clock?

J Clin Endocrinol Metab, September 2018, 103(9):3511–

3513

GC fluctuations postively and negatively affect memory

COSA BISOGNA SAPERE SUL PLENADREN

Per il passaggio da terapia convenzionale a plenadren è di aiutol’applicazione CORTICONVERTER

Nella pratica clinica il passaggio da Idrocortisone BID o TIDa plenadren prevede lo stesso dosaggio

A causa della minore biodisponibilità del plenadren bisogna monitorare sempre la risposta clinica ed eventualmente

personalizzare la dose

Nei pazienti affetti da Insufficienza corticosurrenalica secondaria, in trattamento conaltri ormoni, ad esempio GH, la posologia del plenadren non va modificata

In presenza di IPOTIROIDISMO, iniziare prima la terapia con Plenadren e successivamente con Levotiroxina sodica


MALATTIE INTERCORRENTI

NON sottovalutare il rischio di crisi surrenalica

Raddoppiare il dosaggio di glucocorticoidi in caso di FEBBRE o di altre patologie, triplicare per temperatura corporea > 39°assumendo un’altra compressa di Plenadren dopo almeno 8 ore dalla precedente senza modificare la dose del mattino

•In alternativa somministrare compresse di Idrocortisone a rilascio immediato

In caso di vomito persistente e/o diarrea bisogna somministrareGlucocorticoidi per via i.m. o endovena

NON SOTTOVALUTARE MAI LA PRESENZA DI INFEZIONI

A.Falorni et al 2013G. Johannsson 2015


INTERVENTI CHIRURGICI MINORI

La dose sostitutiva giornaliera deve essere aumentata termporaneamente

INTERVENTI CHIRURGICI MAGGIORI

Richiedono la somministrazione diGlucocorticoidi per via endovenosao intramuscolare

Terapia per os una volta che il pazientesi alimenta

Assumere un’altra compressa di Plenadren dopo almeno 8 ore dalla precedente senza modificare la dose del mattino


POPOLAZIONI SPECIALI

ANZIANI: dose inferiore soprattutto in relazione a peso ed età

INSUFFICIENZA RENALE: LIEVE/MODERATA, nessun aggiustamento della dose GRAVE, monitoraggio clinico stretto ed eventuale aggiustamento della dose

INSUFFICIENZA EPATICA: LIEVE/MODERATA, nessun aggiustamento della dose

GRAVE, monitoraggio clinico stretto

ed eventuale aggiustamento della dose

HIV: Aggiustamento posologia in relazione al dosaggio di farmaci antiretrovirali

FERTILITA’, GRAVIDANZA E ALLATTAMENTOIl plenadren non ha nessun effetto negativo sulla fertilità e puòessere usato in gravidanza e durante l’allattamento

formulation, DIURF-006, when given asa twice-daily ‘toothbrush’

regimen, 20 mg at night (23:00 h) and 10 mg in the morning

(07:00 h) provided cortisol exposure similar to that seen in

physiological cortisol levels in a healthy reference population

and also to that seen in dexamethasone-suppressed healthy volun-

teers after a single dose of 30-mg hydrocortisone. The relative

(a)

(b)

(c)

Fig. 4 (a) Dose–response of DIURF-006; 5,

10 and 20 mg (Geomean SEM). (b) Dose

proportionality for DIURF-006; 5, 10 and 20 mg

AUC (Geomean SD). Dotted line represents the

regression line: Slope: 0 69, 95% CI 0 65–0 73. (c)

Twice-daily dosing at 23:00 and 07:00 h with

DIURF-006; 20 and 10 mg in dexamethasone-

suppressed healthy male volunteers.

© 2013 John Wiley & Sons Ltd

Clinical Endocrinology (2014), 80, 554–561

Modified-release hydrocortisone mimics cortisol rhythm 559

Twice-daily dosing at 23:00 and 07:00 h with DIURF-006; 20 and 10 mg in dexamethasone- suppressed healthy male volunteers.

DIURF-006

Dose–response of DIURF-006; 5, 10 and 20 mg

Chronocort formulation,DIURF-006,when given as a twice-daily‘toothbrush’ regimen, 20 mg at night (23:00 h) and 10 mg in the morning(07:00 h) provided cortisol exposure similar to that seen in physiological cortisol levels in a healthy reference population and also to thatseen in dexamethasone-suppressed healthy volunteers after a single dose of 30-mg hydrocortisone.

Modified-release hydrocortisone tablet to provide circadian profile

Migliore esposizionesistemica al cortisolonelle prime ore notturne

• Microinfusore sottocutaneo: idrocortisone 50mg/ml (durata 3 giorni)

• 10 mg/m2 per superficie corporea die

• Simulazione della normale secrezione circadiana di cortisolo

• Replicando il normale ritmo del cortisolo:

riduzione di ACTH e miglioramento QoL

• Costo: pz selezionati in cui non si riesce ad ottenere un compenso con la tradizionale terapia

LA CRISI SURRENALICA

• Fattori precipitanti: patologie GI (32.6%) e altre cause infettive soprattutto simil-influenzali (24.3%)

• 1 su 12 ha una crisi surrenalica che mette a rischio la sua vita nel corso di un anno

Hahner S. et al. EJE. 2010;162:597– 602White K. Et al. EJE. 2010; 162:115–120 Hahner S. et al .JCEM. 2015;100:407– 416

Articolo - Autore N N° crisi surrenaliche

Hahner S. et al. EJE. 2010

444 6.6/100 anno

White K. Et al 2010 841 8/100 anno

Hahner S. et al. JCEM, 2015

768 8.3/100 anno

PRESENTAZIONE CLINICA

• Ipotensione severa

• Ipovolemia clinicamente evidente

• Anomalie ECG o cardiomiopatia

• Paziente esausto e depresso

• Severa mancanza di energie

• Anoressia nausea e vomito

(errato confondimento con malattia GI)

• Mialgia, dolori articolari

• Dolore addominale suggestivo di iniziale peritonite

• Febbre spesso presente

• Stadio avanzato: disturbi cognitivi, sonnolenza, shock ipovolemico

Hahner S. et al. EJE 2010 162 597–602White K. Et al. EJE 2010 162 115–120

ESAME OBIETTIVO –LABORATORIO

• Approfondito esame obiettivo

• Parametri vitali

• ECG

• EGA

• Sodio

• Potassio

• Glicemia

• Emocromo

• Cortisolemia (ACTH)

• PCR

Hahner S. et al. EJE 2010 162 597–602White K. Et al. EJE 2010 162 115–120

DEFINIZIONE DI CRISI SURRENALICA

A) Severo peggioramento delle condizioni generali e almeno 2 dei seguenti segni/sintomi

Ipotensione (sistolica <100 mmHg)

Nausea o vomito

Fatica severa

Febbre

Sonnolenza

Ipoglicemia (> bambini)

Iponatremia (Na < 132 mmoli/l) o Iperkaliemia

B) Somministrazione parenterale di GCs (HC) seguita da miglioramento clinico

GRADING SYSTEM (a seconda del setting necessario per il trattamento e dell’outcome)

Grado 1: gestione ambulatoriale

Grado 2: gestione ospedaliera in reparto generale

Grado 3: unità di terapia intensiva

Grado 4: morte per crisi surrenalica

Hahner S. et al. JCEM 2014

EVENTI PRECIPITANTI CRISI SURRENALICA

• Infezioni, in particolare gastroenteriti

• Interventi chirurgici

• Esercizio fisico intenso, stress emotivo

• Incidenti

• Interruzione di terapia con GCs

Il timing di sviluppo è molto variabile, più rapidamente nei bambini che negli

adulti (range 5 minuti - 7 giorni)

FISIOPATOLOGIA DELLA CRISI SURRENALICA

Permissive Soppressive

GCs

Stimolatorie

Sapolsky RM et al. Endocrine Reviews 2000 21 55–89Besedovsky H et al. Science 1986 233 652–654Barber AE et al. Journal of Immunology 1993 150 1999–2006Webster JC et al. PNAS 2001 98 6865–6870

alterata attivazione e responsività delsistema CV

AI non ancora diagnosticata

Ipotensione

deplezione di volume: Na e della ritenzione di liquidi(mancata azione dei mineralcorticoidi)

manca l’azione soppressiva dell’aumentata secrezione di GCs che previene effetti dannosi di una esagerata difesa immunitaria mediata da citochine pro-infiammatorie

perdita dell’azione permissiva dei GCs sui recettoriadrenergici

FebbreInfezioni

Interazione di GCs e citochinein corso di infezioni maggiori

Interazione di GCs e citochine in corso di infezioni maggiori in A) Soggetti sani B) Pz con AI in terapia sostitutiva standard senza modifiche delle dosi di GCs

La crisi surrenalica si autoamplifica etutti i sistemi lavorano senzacoordinamento a volte non arrestabile

La crisi surrenalica non è mai dovuta alla sola carenza di GCs

La carenza di GCs non è condizionesufficiente per crisi surrenalica ma siverifica dopo l’esposizione a triggers non è prevedibile

CORTISONICI Adulti Idrocortisone (HC) 100 mg e.v. immediatamente, seguito da 200 mg nell’arco delle 24 h (infusione continua e.v. o iniezioni da 50 mg ogni 6 h), poi HC 100 mg/die il giorno successivo

Bambini HC 50 mg/m2 e.v. in bolo,seguito da HC 50–100 mg/m2 nell’arco delle 24 h (infusione continua e.v. o iniezioni ogni 6 h)

Se HC non disponibile: Prednisolone (o Desametasone solo se altri GCs non disponibili)

FLUIDI Adulti SF 0.9% 1000 ml e.v. in infusione rapida nei primi 60 min o SG 5% in isotonica poi infusione salina continua e.v. in base a clinica del pz

Bambini SF 0.9% 20 ml/kg e.v. in bolo rapido da potersi ripetere fino a un totale di 60 ml/kg nella 1° ora poi infusione salina continua e.v. in base a clinica del pz

In base a severità crisi e a pat intercorrenti

Monitoraggio cardiaco, profilassi antitrombotica, antibiotici, ricovero in terapia intensiva

• Generalmente guarigione clinica in 24h • Se non c’è miglioramento considerare altre

cause

TRATTAMENTO

Bornstein S.R: et al. JCEM 2016, 101(2):364 –389

• Il sospetto richiede immediata azione terapeutica, il trattamento precede la conferma biochimica della diagnosi

• Nel sospetto di crisi surrenalica:

Mineralcorticoidi non richiesti se HC > 50 mg/24 h

PREVENZIONE DELLA CRISI SURRENALICA

• dose GCs non hanno l’obiettivo di mimare l’incremento mediano di cortisolo nei soggetti sani durante stress ma di mimare il massimo aumento di cortisolo che si può avere nei soggetti eusurrenalici stressati da tali procedure al fine di coprire necessità inaspettate

• L’eccesso di GCs somministrati a questi pazienti nel breve termine non sembra associarsi a rischi significativi

Udelsman R. et al. Journal of Clinical Investigation 1986 77 1377–1381Salem M. et al.Annals of Surgery 1994 219 416–425

Chirurgia elezioneStress fisico prolungato

rapidi aggiustamenti in risposta a bisogni inaspettati

GESTIONE DELL’ AI IN CONDIZIONI SPECIFICHE - PREVENZIONE DELLA CRISI

Condizione Azione suggerita

Gestione domiciliare della febbre Raddoppiare GCs se TC>38°C o triplicare se > 39°C fino a guarigione (di solito 2-3 gg), aumentare consumo di liquidi con elettroliti

Pz non in grado di tollerare farmaci per o.s. (gastroenterite, trauma o digiuno es. per colonscopia)

Adulti: HC 100 mg i.m.Bambini HC 50 mg/m2 i.m.; età infantile: 25 mg, età scolare 50 mg, adolescenti 100 mg

Stress chirurgico lieve o moderato Adulti: HC 25-75 mg/24h (per 1-2 gg)Bambini: HC 50 mg/m2 o HC dose raddoppiata o triplicata

Chirurgia maggiore con anestesia generale, trauma, parto o patologie che richiedono la terapia intensiva

HC 100 mg e.v. seguito da infusione continua di 200mg/24h (o 50 mg e.v. o i.m. ogni 6h)Bambini: HC 50 mg/m2 e.v. poi 50–100 mg/m2 nell’arco delle 24 h (iniezioni ogni 6 h)

Ridurre il dosaggio e passare a terapia orale quanto prima in base alla clinica

Bornstein S.R: et al. JCEM 2016, 101(2):364 –389

EDUCAZIONE DEL PAZIENTE ALLA GESTIONE DELLA CRISI

• Deve essere rivolta a paziente e familiari

• Aumenta la consapevolezza della propria patologia e nemigliora l’outcome

• Il paziente e i suoi familiari, se ben informati, guidano allagestione più idonea e in tempi più rapidi il personaleparamedico/medico

Il ruolo dello SPECIALISTA:

• Dedicare al paziente il tempo necessario alla spiegazione delle situazionispecifiche e alla gestione delle emergenze/urgenze

• Spiegare il razionale dell’aumento di dose

• Focalizzare l’attenzione sull’educazione del paziente ad ogni controllo(almeno annuale! e indagare la comparsa e modalità di gestione di crisi)


Il ruolo del MEDICO DI MEDICINA GENERALE• Gestione del pz nel complesso delle comorbidità• Ruolo essenziale nel supporto alle situazioni di urgenza/ emergenza• Coordinamento del management multidisciplinare


Ogni pz dovrebbe portare sempre con se:

•Una steroid emergency card

•Medical alert bracelet or necklace:

“Insufficienza surrenalica – Somministrare cortisonici!”

•Leaflet con informazioni chiare sulle condizioni in cui

aumentare la terapia con GCs in situazioni particolari

o di emergenza

•Un kit di emergenza (idrocortisone 100 mg fiale + siringa ed ago)

utilizzabile da personale paramedico o parenti addestrati

KIT DI EMERGENZA

Thanks to all my LAB and CLINICAL Staff:

Mary A. Venneri, Daniele GianfrilliElisa GiannettaCarlotta PozzaEmilia SbardellaRiccardo PofiAlessia CozzolinoValeria HaisenmajerGiulia PulianiTiziana FeolaChiara GraziadioMarianna Minnetti

Sapienza University of Rome – Italy

and Andrea Lenzi

ritmo circadiano, sistema immunitario e insufficienza …...ritmo circadiano, sistema immunitario e...

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