HAE Autumn Symposium

Avner Reshef MD

Allergy, Immunology & Angioedema Center

Sheba Medical Center, Tel Hashomer, Israel

:אַנְגיוֹאֶדֶמָה

הבירור המעבדתי

Angioedema

Tissue swelling caused by extravasation of protein-rich plasma

into the tissues, owing to endothelial hyper-permeability

:הגדרה

של אנדוֹתֶל כלי הדם יתר-מחדירותבצקת הנובעת

ומביאה להצטברות נוזל עשיר בחלבון ברקמות

Endothelial Tight-junctions

Dejana et al . J Cell. Sci. 2008, iedm. Curr Opin Hematol 2012,

• Vasoactive agents (i.e. bradykinin, histamine) induce

phosphorylation of VE-cadherin bridges and cellular

internalization (by cytoskeletal actomyosin)

• Endothelial junctions opens-up, causing temporary

increase of Vascular permeability and tissue edema

Endothelial cells are held by VE-Cadherin bridges

Control of Tight Junctions: Role of VE-Cadherin

Vincent AM, J Physiol Cell Physiol 2004; 286:987,

Hereditary angioedema (HAE)

Described in 1882 by Quincke (“angioneurotic oedema”)

Characterized by recurrent attacks of tissue swelling which include: edema of

limbs, face, tongue, larynx, genitals

Severe attacks of abdominal pains caused by intestinal obstruction

Rare disease (1:50,000). Estimated patients in Israel: ~150-200

Diagnosis is established by personal or family history and laboratory indices

Diagnosis may be delayed for over 20 years, since clinical symptoms are

sometimes misleading

Bradykinin is the main mediator of vascular hyper-permeability in HAE

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ביטויים קליניים של אנגיואדמה

ביטויים קליניים של אנגיואדמה

ביטויים קליניים של אנגיואדמה

9

Abdominal CT during angioedema attack

Edematous intestinal wall with ‘Water-Halo’ sign

Hereditary Angioedema - pedigree

Propositus: Recurrent abdominal pains, swollen hands. Lab: C4= 2.01 (10-40) mg/dl,

AgC1INH= 4.4 mg/dl (19-34), fC1INH= 20% (>50)

Family history of HAE

1940

1966

1959

1940

1958

1954

1951

1947

1981 1984 1990 1996

2010

2010 2003 2005

1971 1972

2012

2014

I

II

III

IV

V

VI 2012

Bissler JJ, et al. Proc Assoc Am Physicians. 1997;109:164-173. Davis AE 3rd. Annu Rev Immunol. 1988;6:595-628. Verpy E, et al. Am J Hum Genet. 1996;59:308-319. Zuraw BL, Herschbach J. J Allergy Clin Immunol. 2000;105:541-546.

HAE is caused by C1-Inhibitor mutations

• SERPING1 gene (“serpin peptidase inhibitor, clade G, member 1”)

• Located on Chr.11q12.1 (bp57,597,553 to bp57,614,852)

• Autosomal dominant trait but 25% are sporadic cases (~250 de-novo

mutations described)

C1 esterase Inhibitor (C1INH)

• SERPIN (serine-protease inhibitor) synthesized by hepatocytes

and blood monocytes

• Major inhibitor of :

Complement serine proteases (C1r, C1s, mannose-binding lectins [MASP] )

Contact-system proteases (coagulation factor XIIa and plasma kallikrein)

Relatively minor inhibitor of the fibrinolytic proteases plasmin and

coagulation factor XIa

• C1INH-Ligand complexes are cleared from the circulation (“suicide complex”)

resulting in decreased enzyme activity in the plasma

• C1INH deficiency leads to activation of Factor XII and accelerated formation of

Bradykinin- a powerful vasoactive chemical causing tissue edema

Function of C1INH

Gooptu B, Annual Review Biochem 2009; 78:147

14

C1INH inhibits autoactivation of C1 complex (C1r,s)

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C1INH

C1INH

C1INH

C1INH and the complement cascade

Pathways inhibited by C1INH

Iwamoto K et al. Journal of Dermatology 2014; 41: 929–932

Mechanisms of Angioedema

Kaplan AP, J Allergy Clin Immunol 2010; 126: 918-25; Joseph K et al. J Allergy Clin Immunol 2013;132:470-5

FXII (HF) FXIIa (FXIIfr)

Injury, Inflammation, Neg. charged surfaces, gC1qR, Cancer

Kallikrein Bradykinin

PKK

Endothelial Prolyl-CPase

HSP-90+ Zn

C1INH

Contact-Kinin

HMK+PKK

C1INH

C1 Complement

C1r,s

C4, C2

C4 Depletion

C1INH

Angioedema

NO Synthase

Increased vascular permeability BK2 Receptor

C1INH

Plasmin tPA

Plasminogen

Fibrinolysis

C1INH

FXI

Intrinsic coagulation

Thrombin

Fibrin

Responsive to

Anti-Histamines

Non-Responsive to

Anti-Histamines

Cugno, International Immunopharmacol 2003; 3:311

Bradykinin (BK) Nanopeptide generated from from high molecular-weight kininogens

(HMWK) in the activation of the contact/kinin system

Important for normal vascular permeability and vascular tone in normal

homeostasis, immune responses, inflammation

Binds to the B1 and B2 receptors at the endothelial level

Angioedema is primarily mediated through the BK B2 receptor which cause

increased vascular permeability

Increased vascular permeability is primarily mediated by down regulation of

vascular endothelial VE cadherin and increased contraction of the actin

cytoskeleton

The degradation product des-Arg-bradykinin binds to the B1 BK receptor and

has additional activities

Actin stress fibers VE-cadherin

Non stimulated

Stimulated

Increased vascular permeability

Tiruppathi C, et al. Vascul Pharmacol. 2003;39:173-185.

How Does Bradykinin Cause Angioedema?

Skidgel et al. Biol Chem 2006

Bradykinin receptors

Icatibant (Shire/ Jerini, USA) פירזיר

.(א.ח 4-קצר ב) ברדיקיניןשל אנלוג, חומר סינטטי•

BKאינו נפגע על ידי אנזימים המפרקים -עמיד•

דםV-כלי באנדותֶלBK (BKR-B2)חוסם באופן סלקטיבי קולטני •

בינלאומיים מעידים על יעילות ובטיחות גבוהים RCTמחקרי 3•

(2012)ב "וארה( 2011לטיפול עצמי ) 2008אושר באירופה •

ונכלל בסל הבריאות 2009אושר בישראל •

Cicardi M et al. N Engl J Med 2010; 363:532-41 Lumry WR, Li HH, Levy RJ, et al. Ann Allergy Asthma Immunol 2011; 107: 529-37

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אבחנה

Diagnosis of Angioedeama

Complement testing in diagnosis and management

C4- Is the best screening test for HAE and is depressed in 99% of patients

even between attacks. A normal C4 during an attack virtually excludes the

diagnosis of HAE

C1-INH antigen(protein) - low in type I (85%), normal in Type II (15%)

C1-INH functional assay (activity) - low in both type I and type II

C2- often depressed during attacks. Not essential for the diagnosis.

C1q- Should be normal in HAE. Indicated when a patient presents with

angioedema after age 40 (assoc. MGUS, Lymphoma etc.)

CH50- Indicated for complement deficiencies, but not for HAE

C4 is the single best screening test

C4 level is an effective screening tool to detect C1INH deficiency

Occasional (about 1% of HAE patients) false negatives will be encountered,

particularly in patients on anabolic androgens (i.e. Danazol)

Measurement of the activation product C4d may avoid false negative

results (not practical)

False positives may be encountered, especially in C4 deficiency.

C4 level is the best screening test, but cannot be relied upon alone to

make a diagnosis of C1INH deficiency.

Method: Nephelometry

Analyte: protein in serum

Normal values (Israel) mg/dl

1. Sheba: 19-34 2. Ichilov: 21-39 3. Beilinson: 15-33 4. Clalit: 23-42 5. Meuhedet: 19.5-34.5

Cautions:

• Infants: adult C1INH antigenlevels are reached by 6 months of age • Turbidity & particles in the specimen may result in extraneous light scattering

signals, resulting in variable specimen analysis • Quantitation by nephelometry may not be possible in lipemic sera due to the

extreme light scattering properties of the specimen • Specimen should be transferred to the lab within 24-48h

C1-Inhibitor antigen (C1esteraseINH) assay

26

Principle: 1. Chromogenic assay measure the inhibition by plasma C1-inhibitor (C1INH) of

substrate cleavage by C1s (C1esterase), a major target protease of C1INH, inhibited merely by C1INH.

2. A plasma test sample is incubated with an excess of C1s for 5–15min to complex all C1INH to C1s.

3. The remaining C1s activity is subsequently measured by addition of a chromogenic substrate (*) measuring absorbance at 405nm in a kinetic test.

4. The difference between the amount of C1s added and the esterolytic activity measured reflects the amount of C1INH activity present in the sample.

C1INH sample+ C1s excess [C1INH - C1s] + C1s remained

*MeOC-Lys(e-Cbo)Gly-Arg-pNA MeOC-Lys(e-Cbo) Gly-Arg-OH+ p-nitroaniline

C1s remained

Berichrom C1-Inhibitor functional assay

27 (*MethylOxyCarbonyl-Lysyl (e-carbobenzoxy) Glycyl-L-Arginyl-p-nitrolailide)

C1-Inhibitor functional assay

28

1. Plasma should be collected to the 2 citrated PT tubes

2. Centrifuge immediately at no less 1500x g for at least 10 min.

3. Stability of the samples:

@ -20ºc => 1 month

@ +2ºc - 8ºc => 2 days

@ +15ºc -25ºc => 6 hours

4. Plasma stored at -20ºc can be thawed within 10 min at 37ºc, assay should

be performed within 2 hours

Range (variable within laboratories):

Normal >67% of normal Equivocal 41-67% of normal Abnormal <41% of normal

29 Wagenaar-Bos IGA, et al. J Immunol Meth 2008; 338: 14–20

Chromogenic vs. Complex-ELISA fC1INH assays

Chromogenic assay (i.e. Berichrome)

Complex-ELISA assay*

* The complex assay is based on detection of complexes formed between C1Inh and C1s following addition of exogenous biotinylated C1s to the serum test sample

HAE disease severity correlates with C1INH Function

Kelemen Z Clin Immunology 2010 p354

C4 level C1INH function

Bork, Lancet 2000

Hereditary Angioedema with normal C1INH: Type III

1. A newly described entity found mainly in women with family history of angioedema.

2. In ~20% attacks are triggered by hormones (estrogen) 3. A mutation in factor XII gene is found in 1:4 patients

Other tests

• Sequencing of SERPING1 gene or factor XII mutations can be done,

however this approach is rarely needed

• Measurement of complement C3 levels or CH50 is not useful

• Many patients with acquired C1INH deficiency (AAE) have anti-C1INH

autoantibodies

• Cleaved High-Molecular Kininogen (cHMWK) was recently proposed as a

diagnostic parameter for acute attacks of HAE1

• D-dimers (DD) were found elevated during acute attacks of HAE2

1. Suffritti C, et al. High-molecular-weight kininogen cleavage correlates with disease states in the bradykinin-mediated

angioedema due to hereditary C1-inhibitor deficiency. Clin Exp Allergy 2014; 44(12):1503-14

2. Reshef A, et al. elevated D-Dimers in attacks of hereditary angioedema are not associated with increased thrombotic risk.

Allergy. 2015 Jan 30 doi.

C1est INH (antigen)

Normal (>50%)

Low (<50%)

Func. C1-INH (activity)

HAE Type II (15%)

nC1INH AE Familial (“Type III”)

Idiopathic, ACE-I-induced

Normal

(19-34mg/dl)

Laboratory evaluation of Angioedema

C4

Low (<10mg/dl)

Normal Repeat during attacks

Low (<19mg/dl)

HAE Type I (85%)

HAE Diagnosis: Low C4+ low C1INH (specificity 98%, NPV 96%)

C1q

Acquired (AAE)

Normal Low

+Late onset (>40)

• Recurrent edema attacks • Family history • No rash, no urticaria • No anaphylaxis • Not responding to AH’s • ACE-I use

Acquired Angioedema (AAE)

• First report in 1972 (Caldwell et al)

• Rare disease: <200 cases described (est. prevalence: 1:100-500,000)

• Attacks of tissue swelling caused by vascular hyper-permeability and

extravasation of protein-rich plasma

• Clinical picture identical to HAE (exc. older age at diagnosis!)

• Mechanism: uninhibited activation & consumption of complement due to

reduced C1esterase Inhibitor (C1INH) in the plasma (low C1q)

34

Caldwell JR et al. Clin Immunol Immunopathol 1972; 1: 39-52 Geha RS, et al. N Engl J Med 1985; 312:534-40 Breitbart SI & Bielory L. Allergy Ast Proc 2010; 31: 428-34

Cicardi M et al. Medicine 2003; 82: 274-81

Cicardi M & Zanicelli A. Allergy, Asthma Clin Immunol 2010; 6: 14-19

Acquired Angioedema (AAE)

Associated with: Multiple myeloma, Waldenström macroglobulinemia, CLL

Sjögren’s syndrome, SLE, carcinoma, hepatitis, Churg-Strauss vasculitis

36 Immunofixation

Fraction 9.0%

Protein Electrophoresis

Serum (mg/dl):

IgG = 1090

IgA = 278

IgM = 1690

Kappa = 2500

Lambda = 582

Urine (mg/dl):

Protein = 380 mg/24h

BJP=Positive

Kappa = 19.9 mg/dl

Lambda <5.0 mg/dl

0

10

20

30

40

50

60

70

80

90

12-Jul-02 17 Sept 02 29-Apr-03 01-May-03 26-Jun-03 17-Aug-03 07-Oct-03 15-Jan-04 09-May-04

C3

C4

C1INH

* *

A case of Acquired Angioedema (AAE) 79 YO man with 2 episodes of laryngeal edema

התאוששות 27/1/2013

.כשלוש שנים Ramipril-ד מטופל ב"יתר ל. איש מכירות, 62בן •

, הופיעה נפיחות של הלשון( עקב אירוע של אבוד הכרה)במהלך המתנה בחדר מיון •

ציאנוזיס , קושי בנשימה

עבר צנרור קנה דחוף•

ACE-Inhibitor induced angioedema

ערירי, רווק, 57גבר בן

מחלת , ספיקת כליות כרונית-אי, סכרת, יתר לחץ דםGraves

קושי בדיבור ונשימה, נפיחות בלשון - 27/12/13חדר מיון

נוטלTritace (ramipril) מזה מספר שנים

אירוע קודם של נפיחות בלשון לפני מספר שבועות

יום 60ושיקום במשך נ"בטאשפוז , מוות קליני, עבר אינטובציה קשה

ACE-Inhibitor induced angioedema

Bradykinin and the Renin/Angiotensin system

ACE Inhibitors

(ACEI)

AT-1 receptor

blockers (ARB)

Degradation

Bradykinin

Inactive peptieds

(BK 1-7)

Angioedema

ACE (Kininase II)

Activation

Renin

Angiotensinogen

Angiotensin I

Angiotensin II

Hypertension

Chymase

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באנגיואדמההטיפול

Treatment of Angioedeama

Drugs Targeting HAE Pathways

FXII FXIIa C1INH

Modified from: Kaplan AP, J Allergy Clin Immunol 2010; 126: 918-25

Injury, Inflammation, (-)charged surfaces, gC1qR, Cancer

C1

Complement

System

C1r,s

C4, C2

C4 Depletion

C1INH

Angioedema

NO Synthase

Increased vascular permeability BK Receptor

Kallikrein Bradykinin

PreKallikrein

Endothelial

Prolyl-CP/HSP-90

C1INH

Contact-Kinin

System

HMK+PK

C1INH

C1INH Plasmin

tPA Plasminogen

Fibrinolytic

System

C1Inhibitor: 1.Attenuated androgens

(Danazol) 2.C1INH Concentrates

(human, recombinant)

Kalbitor (eccalantide), Dyax DX-2930, Biocryst

BCX4161, ISIS-PKK

Firazyr (icatibant)

Hexacapron (tranexamic acid)

"שיבא"המרכז לאנגיואדמה

רפואי לחולים ומשפחותיהם-ייעוץ מקצועי

(התקפים וטיפול מונע)יום -מתן טיפולי

הדרכה למתן טיפולי בית( תרכיזי עירוייC1INH , עורית-תתהזרקות)

סוציאלית-תמיכה סיעודית ופסיכו

חדשות למחקרים בתרופות- HAE

ונשאיםמשפחות -מחקר גנטי

שרותי מעבדה((Complement, fC1INH, D-dimers

43

-אימונולוגיה והמרכז לאנגיואדמה, היחידה לאלרגיהתל השומר, שיבא