Urticaria and Angioedema
Urticaria is characterized by the
rapid appearance of wheals
which may be accompanied
by angioedema
A wheal consists of three typical
features:
1. A central swelling of variable
size, almost invariably
surrounded by a reflex
erythema
2. Associated itching or
sometimes burning
3. Fleeting nature, with a
duration of usually 1-24 hours
Angioedema is defined by
• Sudden, pronounced swelling
of the lower dermis and
subcutis
• Sometimes painful rather than
itching
• Frequent involvement of
mucous membranes
• Resolution is slower than for
wheals and can take up to 72
hours
Classification of Urticaria
A) Spontaneous urticaria
B) Physical Urticaria
1 Acute urticaria
2 Chronic urticaria
2a Chronic continuous urticaria
2b Chronic recurrent urticaria
1 Dermographic urticaria
2 Delayed pressure urticaria
3 Cold contact urticaria
4 Heat contact urticaria
5 Solar urticaria
6 Vibratory angioedema
C) Special types of Urticaria
1 Cholinergic urticaria
2 Adrenergic urticaria
3 Contact urticaria
4 Aquagenic urticaria
D) Different diseases related
to Urticaria also for historical
reasons
1 Urticaria pigmentosa
2 Familial cold urticaria
3 Urticarial
Vasculitis
Clinical classification of Urticaria
-Ordinary (recurrent or episodic not in the categories below)
-Physical
•Aquagenic
•Cholinergic
•Cold
•Delayed Pressure
•Dermographism
•Exercise
•Heat
•Solar
•Vibratory angioedema
-Contact
-Urticarial Vasculitis
-Angioedema (without wheals)
Autoimmune
Pseudoallergic
Infection-related
Idiopathic
Physical
Vasculitic
Trasportabilità dell’allergia col siero 1921
Scoperta IgE (Ishizaka K, 1965)
Fattori
Immunologici
Fattori
non
Immunologici
ORTICARIA
Granulociti
Monociti
Linfociti
Fattori Immunologici
Fattori non Immunologici
piccoli vasi
ORTICARIA
Mediatori
flogosi
Fattori Immunologici
Granulociti
Monociti
Linfociti
Aggregazione FceRI
Attivazione C
Citochine
Chemiochine
Fattori non Immunologici
Liberatori istamina
Effetti colinergici
Effetti fisici diretti
Altri
piccoli vasi
Mediatori
flogosi
ORTICARIA/ANGIOEDEMA
FceRI
1
2
Mast
Cell
Membrane
ß
1
2
Allergene
IL-4
IL-4R
IgM
gene C
B
ligando
CD40
CD40
switch
isotipico
IgE
Allergen
IL-4
CD28
B7
peptide
T
TCR
IL-4R
gene C
MHC
II
B
switch
ligand
CD40
CD40
IgE
IgM
Rilascio di
Mediatori
preformati
Sintesi di
mediatori
formati de novo
Proteasi
PGD2
LTC4, D4, PAF
E4
Eparina
Istamina
Citochine IL-3,4,5,6,8,13
GM-CSF, TNFa
ORTICARIA
Classical Pathway
A
C
B
C4b
C1q
C1q
C1q
C1q
C1r
C1s C4b
C1r
C1s
C1s
C1r C4a
C1s
C1r
E
C4b
C4b C3b
C2b
C2b
C2b
C2a
D
C5b,6,7
C9
C5b
C3b
C3a
C5a
C4d
C9
C9
C5b,6,7
C3d
C9 C9 C9 C5b,6,7,8
C9
C9
C9
C9
C9
F
Alternative pathway
C4+C2
activation
C1qrs
C1qrs
DAF
MCP
CR1
C4bp
C1inh
C4b2b
C3
Activating
surface
C3b
C3bBb
D
C3bB
DAF
CR1
MCP
H
C3(H2O)Bb
P
vasodilatation
C5a
permeability
adhesion adhesion
receptors
diapedesis
chemokinesis
permeability
vasodilatation
chemotaxis
histamine
LTB4
NCF
C3a
CHRONIC IDIOPATHIC URTICARIA
•Orticaria persistente con manifestazioni quasi quotidiane
di durata media di circa 3-5 anni
•Ad agente causale non identificabile
•Associata ad angioedema nel 50% dei casi
•Presente nello 0.1% della popolazione
•Rara nell’infanzia, più frequente nelle donne di mezza età
•Pomfi di durata maggiore che nell’orticaria fisica, spesso
sintomi gastro-enterici, grande prurito
CHRONIC IDIOPATHIC URTICARIA
AUTOIMMUNE URTICARIA
•1962: basofili s.p. < in CIU che in
controlli
•1974: < rilascio istamina in vitro
mediante anti-IgE da basofili
CIU
=rilascio da stimoli non
immunologici come 48/80
•FATTORE CIRCOLANTE
CAUSANTE LA
DESENSIBILIZZAZIONE VIA
IgE
•1986: presenza di un fattore sierico che
causa pomfo
all’intradermoreazione autologa
in alcuni pazienti con CIU
CHRONIC IDIOPATHIC URTICARIA
AUTOIMMUNE URTICARIA
•1993: identificazione di
questo fattore
con IgG (1 e 3)
verso catena a
di FceRI
•Fattore causale CIU nel
20%
pazienti CIU
•IgG anti IgE nel 5%
pazienti CIU
Urticaria and autoimmunity
•
Connective tissue disease
•
Vasculitides
•
Cryoglobulinemia
•
Thyroid autoimmunity
Autoimmunità Tiroidea
Pazienti affetti da
Orticaria Cronica
20,68%
Popolazione di controllo
ATOPICI
4,27%
Prevalenza autoimmunità
tiroidea
Orticaria Cronica
Dopo esclusione di tutte le altre possibili
cause di orticaria la percentuale di
positività per autoimmunità tiroidea sale al
38,57% dei pazienti
Maschi
Femmine
16,6%
58,57%
100
90
80
70
60
50
40
30
20
10
0
CU
CIU
CIU Auto-Ab+
UV
HUV
HUVS
Chronic urticaria
Urticaria
Complement
Urticarial vasculitis
Vasculitides
Urticarial Vasculitis
Definition
Urticaria vasculitis (UV) is a clinicopathological condition (entity) characterized
by persistent urticarial wheals, which on
histology show features of a VASCULITIS
(venulitis)
Nomenclature
The variety of cutaneous, systemic and
serological features has resulted in different
nomenclature:
•
•
•
•
•
Hypocomplementemia with cutaneous vasculitis and arthritis
Urticaria and arthralgia with necrotizing angiitis
Hypocomplementemic vasculitic urticarial syndrome
Unusual systemic lupus erythematous-like syndrome
McDuffie Syndrome
Causative factors and associations of
Urticarial vasculitis
Common
Rare
Idiopathic
•Infections: HBV, HCV, EBV, Lyme
disease
LessCommon
•Connective tissue
disease
•SLE
•S.Sjogren
•Complement deficiencies:
C4, nephritic B factor
C3 and
• Immunoglobulin abnormalities: IgG
macroglobulinemia, IgG gammopathy,
IgA myeloma, cryoglobulinemia
•Drugs: diltiazem, cimetidine,
procarbazine, potassium iodide,
fluoxetine
Only 2% of normocomplementemic patients fulfill
diagnostic criteria of SLE, but this percentage raises
to 50% in hypocomplementemic patients.
Hystopathology
Capillaries and Postcapillary venules are
typically involved:
• Endothelial cell swelling and necrosis
• Perivascular infiltrate of inflammatory cells (neutrophils)
• Extravasation of red blood cells
• Leukocytoclasis
• Fibrinoid deposits in perivascular and interstitial locations
Leukocytoclastic vasculitis
Viral hepatitis
Autoimmune (SLE, SS; RA)
Streptococci, staphylococci, and other
Cryoglobulins
Urticarial vasculitis, ulcerative colitis
(especially Henoch-Schonlein purpura)
Lymphoproliferative disease (especially hairy cell leukemia)
Idiopathic causes
Thiazides, phenothiazides
Iodide, Immune serum (blood transfusion, blood products)
Sulfa-based drugs, penicillin, and other antibiotics
Hystopathology
The leukocytic infiltrates in urticarial
vasculitis are often predominantly
composed of Neutrophils, and this
feature is more obvious in
Hypocomplementemic urticaria
vasculitis
Immunopathology
On direct immunofluorescence,
immunoreactants are deposited around
blood vessels and at the basement
membrane in 79% of wheals:
• Immunoglobulins
• C3
• Fibrinogen
Pathophysiology
A type III hypersensivity reaction is involved, altough
the antigen has not been identified (except in hepatitis
B)
Evidence supporting an Immune Complex Disease
includes the presence of:
Circulating immune complexes in 30-75% of patients
with urticarial vasculitis
Activation and deposition of complement and
immunoreactants in blood vessel walls
Mast cell degranulation
Infiltration by acute inflammatory cells
Fibrin deposition
Blood vessel damage
Hypocomplementemic UV (HUV)
Biological Overview
Characterized by a
Complement activation
with antiC1q Ab and a
decrease in C1q levels
ANA are always absent
Rheumathoid serology
is usually negative
Hypocomplementemic UV (HUV) are
characterized by a more frequent systemic
involvement (HUVS) and then they need
of a particular attention and classification
Pathophysiology
In hypocomplementemic
urticaria vasculitis there is
a marked and selective
reduction of serum C1q
In the sera of these patients there are autoantibodies
to the collagen-like region of C1q, which bind to C1q,
activating complement pathway
Possible cross-reactivity with collectins (MBL, BK,
lung surfactant protein A)
C1q
C1 INH
C1r
C1s
C1r
Fc
PA
MBL
C1s
(C1r-C1 INH-C1s)2
C4
C4b
C2
C4b2
C2a
C4b2b
C1q AutoAb
Anti-C1q autoantibodies
are a major criterium for
the diagnosis of HUV. In
SLE, anti C1q Ab are
found in up to 50% of
patients.
More than 95% of the
patients with lupus
nephritis have such
autoantibodies
AntiC1q Ab bind
with high affinity to
the
collagenous
region of C1q, most
likely with their Fab
fragments. The titer
of C1q Ab correlates
inversely with the
concentrations
of
C1q and C4 antigens
Clinical Features
• The majority of patients are
middle-aged women
Classic description
includes:
• Wheals tend to persist for
24 hours, and often 3-7
days
•Purpura
• Wheals vary in size and
some extend gradually over
several days into large
plaques
•Residual
hyperpigmentation
• Most lesions are itchy and
may be associated with
burning sensation, pain,
tenderness
•Ecchymoses
but recent studies
demonstrated that these
symptoms are
uncommon (35%)
Clinical Features
Wheals
may
occur
anywhere,
and
in
contrast to palpable
purpura
they do not show any
predilection to lower
legs
Rarer cutaneous
include:
Associated
Angioedema
occurs in up to 42% of
patients and infrequently
may leave a residual bruised
appearance
manifestations
•Livedo reticularis
•Erythema multiforme-like lesions
•Raynaud’s phenomenon
•Bullous lesions
Systemic Involvement in UV
Common involvement
Muskoloskeletal:
• Arthralgia, Arthritis
Systemic Involvement in UV - HUVS
Less common involvement
Respiratory
Gastrointestinal
Renal disease
Diagnosis of UV
Diagnosis is first
established on
HISTOLOGICAL
EXAMINATION
A positive direct
immunofluorescence
with C3, Ig or
fibrinogen is present
in up to 79% of
patients
Minimum criterion for
Urticarial Vasculitis is
LEUKOCYTOCLASIS
with or without fibrinoid
deposits around blood
vessels
Investigations on UV
Confirmation of clinical diagnosis
Biopsy of early lesions
Serum Complement:
•
•
•
•
C3
C4
CH50
C1q
HUVS
Diagnostic criteria
Chronic
Hypocomplementemic
Urticaria with at least two of the
following symptoms/signs:
•
•
•
•
•
•
Dermic venulitis (biopsy)
Arthralgia or Arthritis
Glomerulonephritis
Uveitis or Episcleritis
Recidivant Abdominal Pain
Presence of
Anti
C1q
Autoantibodies
Exclusion criteria:
•
•
•
•
•
•
Cryoglobulinemia
ANA
Anti ds-DNA
HBV
C1inh deficit
Complement deficit
TERAPIA
RIMOZIONE CAUSE IDENTIFICABILI
TERAPIA
NON-FARMACOLOGICA
TERAPIA
FARMACOLOGICA
TERAPIA NON-FARMACOLOGICA
•Informazione del paziente
•Evitare fattori aggravanti (Aspirina,FANS, oppiacei,
ACE inib.)
•Evitare stress, riscaldamento ambientale eccessivo,
alcol
•Esclusione di alimenti, coloranti, conservanti dopo
opportuna diagnostica
•Dieta a basso contenuto di pseudoallergeni in caso di
non risposta ai farmaci
TERAPIA FARMACOLOGICA
•H1 antistaminici
•Eventualmente aggiungere altro antistaminico
•Eventualmente aggiungere H2 antagonisti
________________________________________________
•Corticosteroidi per brevi periodi (per ordinaria severa o da
pressione)
•Adrenalina (per edema glottide o anafilassi)
•Altri farmaci
________________________________________________
•Terapia immunomodulante-immunosoppressiva
ANTISTAMINICI
•Mainstay della terapia dell’orticaria
•Risposta in circa il 50% dei pazienti, miglioramento nel 15%
•Non eccedere la dose terapeutica
•Non effetti avversi importanti dopo il ritiro di Astemizolo e
Terfenadina
•Uso in gravidanza possibile (Clorfenamina)
•In casi gravi aggiunta di antistaminici sedativi come
Clorfenamina, Idrossizina
1937 (Bovet e Staub)
ASSOCIAZIONE ANTI-H1
ANTI-H2
CORTICOSTEROIDI
ANTILEUCOTRIENI
TERAPIE ALTERNATIVE
NELLE FORME
RESISTENTI
IVIg
CICLOSPORINA
PLASMAFERESI
TIROXINA
DANAZOL
ANTIMALARICI
CICLOFOSFAMIDE
SULFASALAZINA
Il mapping della catena alfa del recettore per le
IgE porterà allo sviluppo di peptidi basati sulla
struttura che potranno essere impiegati per una
specifica e selettiva immunoterapia nell’orticaria
autoimmune
Un altro sviluppo promettente la vaccinazione con
DNA plasmidi per indurre tolleranza per la catena
alfa
Da considerare i trials con anti IgE, anti TNF alfa,
anti IL-5
URTICARIA (non FceRI dependent)
•
Complement dependent
congenital: C1INH, I
acquired: C activation by IC
anti-C1 INH, -C1q,-C4
•
Role of Cytokines and Chemokynes
HAE
QUINCKE H: On acute localized edema of the skin
Monatshefte Prakt.Dermatol. 1:129, 1882
FALCONE T: Edema acuto angioneurotico ereditario
Gazzetta degli Ospitali 7:16, 1886
OSLER W.: Hereditary angioneurotic edema
Am.J.Med.Sci. 95:362,1888
LANDERMAN N.S. et al.: Hereditary angioneurotic edema:
II. Deficiency of inhibitor for serum globulin permeability factor
and/or plasma kallicrein
J.Allergy 33:330, 1962
DONALDSON V.H., EVANS R.R.: A biochemical abnormality in
hereditary angioneurotic edema: absence of serum inhibitor of
C1-esterase
Am.J.Med. 35:37, 1963
•HEREDITARY
•TYPE 1
(Heterozygous for deficiency of C1 INH)
C1
INHIBITOR
DEFICIENCY
•TYPE 2
(Heterozigous for dysfunctional C1 INH)
•1/10,000-1/50,000 people worldwide
ACQUIRED
•TYPE 1
(Lymphoproliferative disorders)
•TYPE 2
(Autoantibodies to C1 INH)
• Hereditary angioedema is caused by mutation in
the C1 inhibitor gene (C1INH; OMIM 606860)
The C1NH gene is located on the chromosome 11,
11q11-q13.1.
• It is comprised of 8 exons and 7 introns, and is
17,159 kb long. The first exon contains only the 5’
non-coding region, while exon 8 codes for the
reactive center of C1 INH.
C1 INH
SITES OF SYNTHESIS
•liver
•amniotic epithelial cells
•endothelial cells
•fibroblasts
•macrophages
•megakaryocytes
•microglial cells
•monocytes
•placenta
•platelets
•pyramidal neurons
C1 INH
HORMONES
•Androgens
•DHEA
C1 INH
CYTOKINES
•IFN-g
•CSF-1
•IFN-b
•IL-6
•TNF-a
HF
HFa
COAGULATION
FIBRINOLYSIS
KININOGEN SYSTEM
COMPLEMENT SYSTEM
COMPLEMENT ACTIVATION
C1
C1
PLASMIN
PG
KK
KININS
PK
KG
TRAUMA
(KG)
C1 INH
HFa
PTAa
HF
PTA
THROMBIN
COMPLEMENT ACTIVATION
C1q
C1 INH
C1r
C1s
C1r
Fc
PA
MBL
C1s
(C1r-C1 INH-C1s)2
C4
C4b
C2
C4b2
C2a
C4b2b
ANGIOEDEMA
HAE
-Recurrent circumscribed non pitting, evanescent,
subepithelial edema
-No erythema nor pruritus; sometime pain caused by
distension of the skin and of subcutaneous tissue
-Cutaneous angioedema may develop over several hours at
any given site and may be preceded by a faint macular or
serpiginous erythema
-May occur in the mucosa of the respiratory and
gastrointestinal tracts
-Patients are frequently aware of a peculiar tingling or
twitching sensation in the affected area
-Within hours the skin will swell progressively, returning to
normal in 24 to 48 hours
CAUSES OF MORTALITY BASED ON
INFORMATION PROVIDED BY PATIENTS
AND REGARDING THEIR RELATIVES
AFFECTED WITH HAE
laryngeal edema
23%
mainly women
5 mothers and 2 daughter died during spontaneous
parturition
SURGICAL TREATMENT IN PATIENTS
appendicectomy
N=10
hysterectomy
N=5
annexectomy
N=5
CLINICAL FEATURES OF
PATIENTS
PRECIPITANTING FACTORS
Trauma
Emotional stress
%
60
40
CLINICAL FEATURES OF PATIENTS
SIGN AND SYMPTOMS
Recurrent abdominal pain
Extremities angioedema
Nausea &/or vomiting
Face angioedema
Larynx angioedema
Neurologic disorders
Diarrhoea
Cutaneous rash
%
98
90
70
60
40
30
20
20
C1 INH DEFICIENCY
Associated diseases
•Systemic sclerosis
•Pituitary adenoma
•Membranoproliferative
glomerulonephritis (Type III)
•SLE
•Sjogren Syndrome
AGENTS USED TO TREAT HAE
agent
dose
ATTENUATED ANDROGENS
1-4 mg/die
•Stanozolol
50-400 mg/die
•Danazol
ANTIFIBRINOLYTICS
•EACA
•Tranexanic acid
ANTI-SEROTONIN
•Cinnarizine
C1 INH CONCENTRATE
7-10 g/die
1-2 g/die
50-150 mg/die
500-1000 U
TREATMENT OF HAE
•
LONG TERM THERAPY
attenuated androgens
antifibrinolytics
antiserotonin
•
SHORT TERM PROPHYLACTIC THERAPY
attenuated androgens
C1-INH concentrate
•
TREATMENT OF ACUTE ATTACKS
maintain airway
maintain intravascular volume
C1-INH concentrate
LOOKING AT TOMORROW
•Heparin
•Designed molecules with C1-INH activity
•Recombinant C1-INH
•Hormones
Heparin
•Efficacy of nebulized heparin therapy in HAE
(Levine et al., Immunol All Pract, 1992)
•Commercial heparin (either subcutaneously injected
or inhaled) is ineffective in preventing exacerbations
of HAE (Weiler et al., JACI, 2002)
•Intravenous heparin did not prevent exacerbations
of HAE in a patient on maintenance hemodialysis
(Perricone et al., JACI)
Designed molecules with C1-INH activity
Dyax
DX-88
•A highly specific and potent inhibitor of
kallikrein,
•DX-88 may have fewer side effects and greater
effectiveness than recombinant or plasma
derived C1-INH.
•Currently, there is no marketed therapy for HAE
in the United States. Plasma derived C1-INH is
the current standard-of-care in Europe but is
unavailable in the United States.
Designed molecules with C1-INH activity
DX-88
Clinical Status
Normal Volunteers
A Phase I trial of DX-88 in normal healthy subjects has been
completed and DX-88 was shown to be well tolerated. The
trial showed that DX-88 is active in the plasma of dosed
subjects.
•Hereditary Angioedema
A multi-center Phase II dose-escalation study of DX-88 in
hereditary angioedema patients was initiated by Dyax and its
collaborator Genzyme in 2001 at several European sites. A
second multi-center Phase II dose-escalation study of DX-88
in hereditary angioedema has began enrolling patients in the
U.S. and Europe in September 2002.
Recombinant human C1 inhibitor
Pharming's
recombinant human C1 inhibitor in the
milk of transgenic rabbits.
CLINICAL FEATURES OF
PATIENTS
PRECIPITANTING FACTORS
Trauma
Emotional stress
%
60
40
ACTH
100
fmol/ml
75
50
25
0
HAE
Controls
160
HAE
Controls
140
fmol/ml
120
100
80
60
40
20
0
bE
bL ME
bE
bL ME
CLINICAL FEATURES OF PATIENTS
SIGN AND SYMPTOMS
Recurrent abdominal pain
Extremities angioedema
Nausea &/or vomiting
Face angioedema
Larynx angioedema
Neurologic disorders
Diarrhoea
Cutaneous rash
%
98
90
70
60
40
30
20
20
SURGICAL TREATMENT IN PATIENTS
appendicectomy
N=10
hysterectomy
N=5
annexectomy
N=5
Policystic ovary syndrome
•Polycystic ovaries with associated increase in size and
functional activity of stromal tissue
•Abnormal gonadotropin secretion
•LH exaggerated surge
•LH/FSH ratio >2
•High beta-endorphin concentrations with normal ACTH
•Increased testosterone
Multifollicular ovaries
•Presence of multiple cysts with no increase in stromal tissue
Plasma BE concentrations and PBMC BE in HAE patients
Patient
1
2
3
4*
5
6
7
2
Means±S.E.M.
Normal values
Controls
Plasma BE
(fmol/ml)
PBMC BE
(pg/106 uncultured cells)
300
282
228
0
0
44
88
500
155
50
110
120
60
90
50
160
180.25±62.86
0-30
13.33±3.67
99.37±15.71
p<0.02
18±4.93
Perricone et al.: Immunopharmacology 22:21,1991
*hypophysectomized
Clinical characteristics, ovaries and menstrual pattern in HAE
women
Patient Age Ovaries Associated Oligomenorrhoea
pathology
G.C.
C.P.
C.E.*
T.M.
B.B.
V.M.
M.C.
C.A.
C.A.M.
M.D.
P.R.*
A.L.
A.N.
20
24
32
18
38
18
20
30
35
16
30
27
38
PCO
PCO
PCO
PCO
PCO
MFO
MFO
MFO
MFO
MFO
MFO
MFO
Normal
FBD**
FBD**
FBD**
-
Perricone et al.:Clin. Exp. Immunol.,
90:401, 1992
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Hirsutism
Yes
Yes
Yes
Yes
Yes
-
*Type 2 hereditary angioedema
** Fibrocystic breast disease
Patient Ovaries
PCO
G.C.
PCO
C.P.
PCO
C.E.
PCO
T.M.
PCO
B.B.
MFO
V.M.
MFO
M.C.
MFO
C.A.
C.A.M. MFO
MFO
M.D.
MFO
P.R.
MFO
A.L.
Normal
A.N.
LH
FSH
LH/FSH Testosterone
(mlU/ml) (mlU/ml)
ratio
(ng/ml)
19.2
8
10.5
12.7
13
10.7
12.2
26.2
11
3.4
10
15.2
25.4
13.4
6.1
7.7
10.5
8.1
10.5
7.6
5.2
8.5
5.4
7.1
7.1
15.7
1.4
1.3
1.3
1.2
1.6
1
1.6
5
1.3
0.6
1.4
2.1
1.6
HAE, n=13
means±s.d.
13.6±6.5 8.7±3.1 1.6±1.1
Controls, n=20
means±s.d.
13.4±4.4 25.8±18.3 0.6±0.4
P
ns
<0.001 <0.001
Perricone et al.:Clin. Exp. Immunol., 90:401, 1992
BE
(pg/ml)
1.09
0.94
0.30
0.58
0.58
1.10
0.55
0.52
0.58
0.21
0.35
0.75
0.50
81
144
52
65
80
87
66
25
28
50
60
20
13
0.62±0.28
59.3±35.2
0.55±0.4
ns
3.6±2.8
<0.001
HAE
•Presence of polycystic ovaries in about 40%,
multifollicular ovaries in about 50% of the HAE
patients of reproductive age (n=18)
•not under oral contraceptives
•not under danazol treatment
•Low FSH
•Normal LH, testosterone and ACTH concentrations
•High beta-endorphin concentrations
•High LH/FSH ratio
•Rare cystic ovaries (1/9) in HAE patients of
reproductive age under danazol treatment
•Normalization of the ultrasound pattern in
HAE patients previously affected with cystic
ovaries who underwent danazol treatment
(5/5)
GONADOTROPINS
PLASMINOGEN
ACTIVATOR
PLASMINOGEN
PLASMIN
LATENT
COLLAGENASE
COLLAGENASE
POLYMORPHONUCLEAR
LEUKOCYTES
COLLAGEN
TELOPEPTIDEFREE
COLLAGEN
FOLLICLE
RUPTURE
•Presence of functionally active complement in
human ovarian follicular fluid
•THC, CPA, APA, factors C1-9, B, C1INH, H, I are
present with values within the normal serum range
•Presence of complement cleavage fragments
•Presence of anaphylatoxins
•Activation of FF complement after the addition of
urokinase and seminal plasma
GONADOTROPINS
PLASMINOGEN
ACTIVATOR
PLASMINOGEN
PLASMIN
LATENT
COLLAGENASE
C
COLLAGENASE
C
POLYMORPHONUCLEAR
LEUKOCYTES
COLLAGEN
TELOPEPTIDEFREE
COLLAGEN
FOLLICLE
RUPTURE
Study of follicular fluid complement
In HAE
HAE
patient
FF or
Serum (S)
C3
(mg/dl)
C1INH
(mg/dl)
C4
(mg/dl)
C4/TP
ratio*
B
(%)**
C3
A
FF1
60
n.d.
12
1.93
100
-
A
FF2
62
n.d.
10
1.61
90
-
A
S
100
8
10
1.54
100
-
B
FF1
78
n.d.
8
1.34
80
+
B
FF2
70
n.d.
9
1.55
80
+
B
S
90
7
12
1.76
110
-
a
FF1,2
90
25
18
3
120
+
a
S
110
20
28
4.17
100
-
b
FF1,2
132
28
23
3.44
103
+
b
S
150
28
30
3.66
110
-
c
FF1-3
125
22
21
3.4
108
+
c
S
145
28
27
3.56
115
-
d
FF1
92
32
24
4.66
98
+
d
S
130
32
35
4.79
110
-
e
FF1-3
107
28
20
3.21
87
+
e
S
128
30
28
3.96
90
-
FF(n=9)
97+26
28+4
22+6
3.77+1
111+8
+
S(n=20)
120+30
29+7
27+7
3.88+1.09
105+10
-
cleavage
fragments
+/-
Controls
Previously
studied women
[2] (Mean + s.d.)
HAE
patient
THC
(U/ml)
THC/TP
ratio*
CPA
(U/ml)
hC4
(%)**
APA
(U/ml)
2sAPA
(U/ml)
HB
(%)**
A
FF or
Serum
(S)
FF1
25
0.40
n.d.
n.d.
20
25
100
A
FF2
30
0.48
n.d.
n.d.
22
22
90
A
S
40
0.61
50
30
20
22
90
B
FF1
30
0.52
40
35
18
23
80
B
FF2
25
0.43
38
40
20
24
90
B
S
40
0.60
48
45
25
25
95
a
FF1,2
142
2.35
200
90
22
24
90
a
S
130
1.93
255
120
30
32
110
b
FF1,2
151
2.26
220
85
25
30
120
b
S
160
1.95
230
130
33
28
90
c
FF1-3
133
2.15
180
90
25
28
110
c
S
150
1.98
220
90
25
32
130
d
FF1
120
2.33
210
110
30
30
100
d
S
155
2.12
240
100
35
36
120
e
FF1-3
135
2.17
210
110
20
20
90
e
S
140
1.98
250
110
28
26
100
Previously
studied
women [2]
FF(n=9)
133+10
2.25+0.17
200+14
99+10
25+0.5
25+1
113+7
(Mean + s.d.)
S(n=20)
140+18
1.97+0.29
240+28
105+40
29+6.2
28+6
100+20
Controls
AN IMPAIRED COMPLEMENT ACTIVITY
IN HAE PATIENTS’ FF MAY RESULT
•IN A MORE DIFFICULT RUPTURE OF
THE FOLLICLE
•IN AN ALTERED INTRAOVARIC
REGULATION BY ATRETIC FOLLICLES
•IN PCO OR IN MFO
•IN ABDOMINAL PAIN
Danazol
Synthetic isoxazol derivative of
ethisterone
•Inhibits gonadotropin secretion
•Prevents mestrual bleeding
•Mildy androgenic
•Androgenic effects
•Acne vulgaris
•Flushes
•Sweats
•Edema
•Libido changes
•Blood clotting disorders
•Liver disease
•Tumor caused by too much male
hormones
•Tumor on the genitals or
•Unusual bleeding from the vagina
Indicated in the treatment
of
•pain and/or infertility due
to endometriosis
•fibrocystic breast disease
•HAE
•not indicated in the
treatment of cystic ovaries
Danazol is capable of improving these
abnormal ovarian conditions
IU/ml
EFFECTS OF LH-RH ON LH
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
HAE
Controls
0
50
100
time (min)
200
300
400
U/ml
EFFECTS OF LH-RH ON THC
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
HAE
Controls
0
50
100
time (min)
200
300
400
Patients treated
20
Hormones
LH-RH modulating treatment of HAE
DANAZOL:
10
LEUPROLIDE
ACETATE
10
200mg daily
for 6 months
3.75mg/month
for 6 months
Prel.res. At Perricone et al.: Molecular Immunology 30:43, 1993.
HAE and PREGNANCY
• KALLIKREIN-KININOGEN-KININ
SYSTEM IS BIOLOGICALLY ACTIVE
DURING ESTABLISHMENT OF
PREGNANCY
• Factor XII and H-kininogen gene
expression are similar across the days of the
estrous cycle and early pregnancy
HAE and PREGNANCY
Since attacks often occur during delivery we
studied a 30 years old woman, with a
previous diagnosis of HAE, along all
pregnancy and during the caesarian section.
Mother
Pre-delivery
Mother postdelivery and post
C1INH infusion
Newborn
6.4
20.6
7.2
27.8
37.7
33.5
75.8
163.0
119.0
C3 (mg/dl) 122.0
117.0
67.0
C4 (mg/dl) 6.0
6.0
9.0
C1INH
(mg/dl)
C1INH f
(%)
CH50 (%)
•We found low levels of C1INH and of C4 in the baby
with values suggesting the diagnosis of HAE.
•During and soon after delivery a HAE attack
became evident. We treated the patient soon after
parturition with 1000 U.I. of C1INH e.v.
•Mother’s CH50 values were increased, as well as
C1INH values.
•The increase in CH50 values after delivery can be
explained as a possible result of the phenomenon
called “Reactive Lysis”.
•In this patient this phenomenon is very evident
possibly because of the sparing effects on C5b-a of
C1INH deficiency.
•HEREDITARY
•TYPE 1
(Heterozygous for deficiency of C1 INH)
C1
INHIBITOR
DEFICIENCY
•TYPE 2
(Heterozigous for dysfunctional C1 INH)
•1/10,000-1/50,000 people worldwide
ACQUIRED
•TYPE 1
(Lymphoproliferative disorders)
•TYPE 2
(Autoantibodies to C1 INH)
• Hereditary angioedema is caused by mutation in
the C1 inhibitor gene (C1INH; OMIM 606860)
The C1NH gene is located on the chromosome 11,
11q11-q13.1.
• It is comprised of 8 exons and 7 introns, and is
17,159 kb long. The first exon contains only the 5’
non-coding region, while exon 8 codes for the
reactive center of C1 INH.
DHPLC
•DHPLC (Denaturing High
Performance Liquid
Chromatography) is a rapid and
accurate method to reveal the
presence or absence of
mutations.
•After genomic DNA amplification
by PCR (polymerase chain
reaction), no additional procedure
is required except loading the
sample onto the DHPLC system.
•The DHPLC run time is less than
10 min.
• DHPLC is a novel,
nongel-based method
that is very sensitive
for detection of DNA
sequence variations.
• Detection is based on
differences in the
retention of
heteroduplexes
containing one
mismatched basepair
respect to
homoduplex.
•
In the preliminary study we
have enrolled 11 families
affected with HAE
Table of mutation detect
exon
mutation
Cange
Type
n° patients
3
c.365 C>A
S115X
NONSENSE
3
3
C376 G>T
N126Y
MISSENSE
3
IVI3
g.5528G>A
Ins. Ala 162
5
c.656G>C
R219P
8
c.1214T>C
L405P
8
c.1372C>A
V458M
MISSENSE
1
8
c.1376T>C
L459P
MISSENSE
1
8
c.1330G>A
R444H
MISSENSE
1
8
c.1330C>T
R444C
MISSENSE
3
8
delG1412
G471fs stop at
545
FRAMESHIFT
3
MISSENSE
MISSENSE
MISSENSE
7
1
1
GERMINAL MOSAICISM AND HAE
•We have studied two little brothers of 5
and 2 years affected with recurrent non
pitting attacks of oedema of extremities and
one of them also with laryngeal oedema.
•There was no family history of HAE, none
of the parents have had bouts of oedema,
nor grandparents, nor other relatives.
•DNA paternity was performed and resulted
in a confirmation of legal parents.
COMPLEMENT ANALYSIS
Baby1
Baby2
mother
father
C1INH <0.048
<0.048
0.24
0.24
C4
3
7
24
24
CH50
31.8
114.7
80
85
C1INH
f
17.04
30.19
85.3
94.74
•We performed DNA molecular studies in the two babies and in the
parents.
•We did enhance in the babies the same nonsense mutation 531C>G
(STOP codon Y177X

protein without 302 aa).
•This same mutation wasn’t found in the two parents, so we hypothesized
the presence of a germinal mosaicism in one of the parents.
•We performed linkage analysis and we found the same aplotype in both
babies.
•We found by this test the maternal germinal origin of the mutation, that
was confirmed by the consequent molecular studies of other maternal
tessues (urin, sputum, skin) where mutation is obviously absent.
•This is the first case of germinal mosaicism in HAE.
Thank You