hae autumn symposium
TRANSCRIPT
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
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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
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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
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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
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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)
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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
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-אימונולוגיה והמרכז לאנגיואדמה, היחידה לאלרגיהתל השומר, שיבא