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Sara T O Saad
Centro de Hematologia e Hemoterapia, UNICAMP
Anemia falciforme
Anemia Falciforme
• Uma das doenças genéticas com maior freqüência.
• Incidência no Brasil: cerca de 3.500 crianças/ano com a doença ou 1/1.000 nascidos/vivos (PNTN).
• Afeta principalmente os descendentes de africanos.
Patogênese (Orah Plat, NEJM 1995)
Vaso-occlusion in Sickle Cell Disease
Vascular Occlusion
Tissue Infarction Hemolytic Anemia
Pain episodes
Splenic Hypofunction
Infections
Acute chest syndrome Pulmonary hypertension
Stroke Multi-organ damage
HbS
Brain-Adults
• 42 patients com SCD ( without CVA) • 27 W/15M • Median age 38 yo (18-60)
– 33 SS , 6 SC, 3 Sβ
• 49 normal controls • 29W/ 20M • Median age 35yo (25-49)
Results
• SPECT: hipoperfusion (p<0,05) difusely distributed: – Left frontal region – Talamus and basis nucleous – Fronto-parietal transition – Medial occipital region
• Doppler transcranial: median = 93cm/sec
Results: Cognitive functions SCD ( n= 36) • Idade 22-53 anos ( mediana 30) • Escolaridade 0 a 15 anos (
mediana 6) • QI 70-121 ( mediana 80) Controles epilepticos ( n=50) • QI 68-142 ( mediana 98) p<0.05
• Fluência verbal • 45% fraco • 40% regular • 15% bom
Manutenção da atenção (Trail A e B)
• 70% < percentil 10
Formação de conceitos
abstratos ( Wisconsin card sorting test)
• 51% fraco
Fetal Hemoglobin Improves Survival and Decreases HbS Polymerization
(Platt, NEJM 1994) (Schechter, NEJM 1995)
Hydroxyurea can increase HbF in most patients with sickle cell anemia
Hidroxiureia no tratamento da anemia falciforme
Adultos, crianças, bebês ?
2 ou mais crises de dor por ano
Sindrome torácica aguda
Outras complicações
Anemia grave
Profilaxia AVC quando transfusão não é possível
Monitorar Hb, VCM, número de neutrófilos, plaquetas, retics, HbF
Iniciar com 10-15mg/kg até atingir dose máxima tolerada
( Neutrofilos ~2 x 109/L, Plaquetas >100 x 109/L , retics > 50 x 109/L)
Lancet. 2011 May 14;377(9778):1663-72.
Hydroxycarbamide in very young children with sickle-cell anaemia: a multicentre, randomised, controlled trial (BABY HUG).
Wang WC et al BABY HUG investigators.
HbSS or Sβ(0)thalassaemia, aged 9-18 months liquid hydroxycarbamide, 20 mg/kg per day, or placebo for 2 years.
96 hydroxycarbamide – 83 completed the study 97 placebo- 84 completed the study. Significant differences were not seen between groups for the primary endpoints (19 of 70 patients with decreased spleen function at exit in the hydroxycarbamide group vs 28 of 74 patients in the placebo group, p=0·21; and a difference in the mean increase in DTPA glomerular filtration rate in the hydroxycarbamide group versus the placebo group of 2 mL/min per 1·73 m(2), p=0·84). Significantly decreased pain (177 events in 62 patients vs 375 events in 75 patients in the placebo group, p=0·002) and dactylitis (24 events in 14 patients vs 123 events in 42 patients in the placebo group, p<0·0001), with some evidence for decreased acute chest syndrome, hospitalisation rates, and transfusion. Hydroxyurea increased haemoglobin and fetal haemoglobin, and decreased white blood-cell count. Toxicity was limited to mild-to-moderate neutropenia.
Pediatr Blood Cancer. 2011 Jul 8. doi: 10.1002/pbc.23244. [Epub ahead of print] Massive accidental overdose of hydroxyurea in a young child with sickle cell
anemia. Miller ST, Rey K, He J, Flanagan J, Fish BJ, Rogers ZR, Wang WC, Ware RE; for the
BABY HUG Investigators.
Abstract 2-year-old boy ingested at one time an entire 35-day supply of hydroxyurea (612 mg/kg body weight). Despite a serum level of 7,756 µM 4 hours post-ingestion, the only toxicity
was transient mild myelosuppression.
Hydroxyurea Reduces Cumulative Mortality
(Steinberg et al, JAMA 289: 1645, 2003)
HbF (g/L)
Top, <5.0
Bottom,5.0
HbF ACS
(Steinberg et al, JAMA, 2003)
Benefits and Risks of Hydroxyurea
• Reduced pain and ACS
• Mortality reduced 40% • Reduced hemolysis • Reduced
hospitalization • Reduced medical
costs • Improved physical
capacity
• Teratogenicity; neoplasia?
• Use in neonates and young children; how early can HU be used, mortality?
• Is organ damage reversible or preventable?
• Useful in HbSC disease? • Is response predictable? • Why are most adults not
treated?
Aspectos Importantes da Vaso-oclusão
• Adesão celular `na parede vascular
• Estado Inflamatório crônico
• Estresse Oxidativo
• Redução na biodisponibilidade de NO
NO
Hypoxia ROS ET-1
IL-8 IL-1 TNF
TF PAF vWF
Vaso-occlusion pathophysiology: Role of endothelial activation, vascular inflammation and cell adhesion
Adhesion molecule expression
Interações: células endoteliais – células vermelhas
Trombospondina
CD36 IAP
Célula endotelial
ICAM-4
αvβ3 integrina
VCAM-1
VLA-4 integrina
Vaso-occlusion pathophysiology: Role of endothelial activation, vascular inflammation and cell adhesion
Interações células endoteliais - Leucócito
neutrófilo
IgG Autóloga
ICAM-4
ICAM-1
E-selectina
Mac-1integrina
ESL-1
Conran et al., 2009
ICAM-1 Mac-1
Célula Endotelial
LFA-1
Plaquetas e a Anemia Falciforme
• Plaquetas ativadas podem aderir às células endoteliais e aos leucócitos de indivíduos normais.
• Níveis aumentados da integrina GPIIb/IIIa e da P-selectina nas plaquetas de indivíduos falciformes têm sido relatados.
• Relatos de uma adesão aumentada de plaquetas em vênulas cerebrais de camundongos transgênicos falciformes.
Mondoro et al., 2001; Wood et al., 2004
As plaquetas e a vaso-oclusão
Ativação da coagulação
Produção de trombina ?
Villagra et al., 2007; Proença-Ferreira et al., 2010
Hemácia
Ativação Plaquetária
↓ NO
Integrina GPIIb/IIIa ativada P-selectina
fosfatidilserina
↓ AMPc
Anemia Falciforme e o Estado Inflamatório Crônico
Interações adesivas com o endotélio
Lesões ao endotélio por hemólise
Isquemia-reperfusão
Ativação do endotélio - aumento na expressão de moléculas de adesão
E-selectin, VCAM-1, ICAM-1
Adesão de células brancas e vermelhas ao endotélio
Aumento dos níveis circulatórios de citocinas e
quimiocinas
IL-1, TNFα, GM-CSF, IL-8
Conran et al., 2009
IL-8
Plasma Inflammatory Proteins in SCD
Lanaro et al., 2009; Garrido, in submissão
Targeting Vascular Inflammation:
Control SCA SCAHU0
10
20
30
40
50
60
P<0.0001
28 44 35
IL-8
(pg/
ml)
Hidroxiureia reduz o estado de hipercoagulabilidade de pacientes com anemia falciforme
Marina P.Colella, Erich V. dePaula, Nicola Conran, João A. Machado-Neto,Susan Quaino,Joyce M Annicchino-Bizzacchi, Fernando F. Costa, Sara T Olalla Saad, Fabiola Traina
Controls SS SS-HU0
10
20
30
n= 18 n= 15n= 9
A
*** ****
Rel
ativ
e ex
pres
sion
ofT
F/β-
Act
in
Controls SS SS-HU0
100
200
300
400
n= 10 n= 14 n = 18
B
** **
TF p
lasm
a le
vels
(pg/
mL)
Controls SS SS-HU0
10
20
30
40
50
60
70
n= 23 n= 19 n= 22
**
A
********
TAT
plas
ma
leve
ls ( µ
g/L)
Controls SS SS-HU0
100
200
300
400
500
600
700
n= 10 n= 14 n= 17
******
B
F 1+
2 pl
asm
a le
vels
(pm
ol/L
)
Controls SS SS-HU0
1
2
3
4
5
6
7
8
n= 25 n= 22 n= 21
*** **C
Solu
ble
thro
mbo
mod
ulin
plas
ma
leve
ls (n
g/m
l)
Controls SS SS-HU
0.5
1.5
2.5
3.5
4.5
5.5
6.5
n= 21 n= 17 n= 23
**** ***D
TNF-α
ser
um le
vels
(pg/
mL)
Redução da expressão de fator tecidual:
Redução de marcadores de geração de trombina:
Redução de trombomodulina solúvel e TNF-α:
% HbF correlaciona-se negativamente com expressão de: Fator tecidual TAT F1+2 TNF-α
Targeting Vascular Inflammation: Pro-coagulant factors in SCD
Endothelial Adhesion Molecules
Targeting Vascular Inflammation:
Control SCA SCAHU0
100
200
300
400
P<0.05 P<0.05
500600700
sIC
AM
-1 (n
g/m
l)
Control SCA SCAHU0
500
1000
1500
2000
2500
P<0.001
30004000
sVC
AM
-1 (n
g/m
l)
Garrido et al., in submission; Conran et al., 2004
Plasma ICAM-1 Plasma VCAM-1
Targeting Vascular Inflammation:
Platelet-derived inflammatory proteins
Plasma CD40L
CON SCA SCAHU0
25
50
75
100
125
N=29 45 40
150
200
250
Plas
ma
sLIG
HT (p
g/m
l)
P<0.001CON SCA SCAHU
0
1000
2000
3000
N=29 45 40
5000
7000
P<0.0001
Plas
ma
CD
40L
(pg/
ml)
Plasma LIGHT
Garrido et al., in submission; Lee et al., 2006
Plasma Inflammatory Modulators in SCD
Lanaro et al., 2009; Conran et al., 2007
Targeting Vascular Inflammation:
PGE1
Control SCA SCAHU0
100
200
300
400
PGE 2
(pg/
ml)
Control SCD SCDHU0
25
50
75
Plas
ma
PGE1
(ng/
ml)
PGE2
↑ NFκB
eNOS function
↑ XO ↓ BH4
Cell Adhesion
Adhesion Molecule Expression
IL-1, IL-8,
GM-CSF,
IL-6
vWF, TXA2,
PAF, Tissue Factor
Inflammation
Coagulation
TNF-α
Platelets CD40L
LIGHT
Leukocyte Activation
CRP
Thrombin
ET-1 ET-3
Vasoconstriction
HU Therapy and Endothelial Interactions
Reduce Hemolysis
Reduce Inflammation
Reduce Endothelial Activation
Reduce Red Cell Adhesion
Reduce Neutrophil Adhesion
Increase NO Bioavailability and cGMP signalling
Reduce Oxidative Stress
Targeting Vaso-occlusion:
Basal Sim TNF TNF/Sim0
1020304050607080
***
••
% S
CD
neu
toph
il ad
hesi
on
Statins reduce SCD neutrophil adhesion to endothelial cells in vitro
Canalli et al., 2011
Basal SIM TNF TNF/SIM0
1
2
3
4
5
6 **
##
B
% V
CA
M-1
Exp
ress
ion
Basal SIM TNF TNF/SIM0
25000
50000
75000
100000 ***
* ##
A
ICA
M-1
Exp
ress
ion
(MFI
)Statins reduce SCD neutrophil adhesion to
endothelial cells in vitro
Role for Adhesion Molecule Expression
ICAM-1 expression on HUVEC VCAM-1 expression on HUVEC
Canalli et al., 2011
Basal Sim TNF TNF/Sim TNF/Sim/L-NAME0
1020304050607080
***••
≈≈
% S
CD
neu
toph
il ad
hesi
on
Statins reduce SCD neutrophil adhesion to endothelial cells in vitro
Role for NO production
Canalli et al., 2011
Reduce Hemolysis
Reduce Inflammation
Reduce Endothelial Activation
Reduce Red Cell Adhesion
Reduce Neutrophil Adhesion
Increase NO Bioavailability and cGMP signalling
Reduce Oxidative Stress
Targeting Vaso-occlusion:
Hemólise Intravascular como um Mecanismo Importante na Anemia Falciforme
Leva à uma biodisponibilidade diminuída de NO.
Reiter et al., Nature Medicine, vol 8, 2002
NO-cGMP pathway in SCD
PKG
NO
Soluble guanylate cyclase (sGC)
GTP cGMP
γ -globin
HbF
Cell adhesion
Almeida et al., 2008; Cokic et al., 2003; Ikuta et al., 2001
RED CELLS
WHITE CELLS
BASAL
DEANO
BASAL
DEANO0
5
10
15
Control SCD
***
##
% A
dhes
ion
to F
N
Basal
DEANO Bas
al
DEANO 0
5
10
15
20
25
Control SCD
**
##
% A
dhes
ion
to IC
AM
-1
NO donors reduce SCD neutrophil adhesive properties in vitro
Canalli et al., 2008
Hydroxyurea
PKG
NO
Soluble guanylate cyclase (sGC)
GTP cGMP
Erythrocytic lineage cells
↑ γ-globin
HbF
Leukocytes Cell adhesion
The cGMP-dependent pathway as a potential therapeutic target for SCA vaso-occlusion
King, 2004
Hydroxyurea
PKG
NO
Soluble guanylate cyclase (sGC)
GTP cGMP PDE 9
Erythrocytic lineage cells
↑ γ-globin
HbF
Leukocytes Cell adhesion
The cGMP-dependent pathway as a potential therapeutic target for SCA vaso-occlusion
Almeida et al., 2008
The cGMP-dependent pathway as a potential therapeutic target for SCA vaso-occlusion
Almeida et al., 2008
Ret
icul
ocyt
es
Neu
trop
hils
K56
2
- ery
thro
id c
ells
+C
D34
Bra
in
T98G
colo
n
Hea
rt
Kid
ney
Live
r
Lung
Lym
phno
de
Mam
mar
y
Ova
ry
Skel
etal
Mus
cle
Skin
Sple
en
Test
icle
Ute
rus0
20
40
60
80
100
120
140
Rel
ativ
ePD
E9A
Gen
e Ex
pres
sion
BAY 73-6691
Hydroxyurea
PKG
NO
Soluble guanylate cyclase (sGC)
GTP cGMP PDE 9
Erythrocytic lineage cells
↑ γ-globin
HbF
Leukocytes Cell adhesion
The cGMP-dependent pathway as a potential therapeutic target for SCA vaso-occlusion
Almeida et al., 2008
Basal BAY73 Basal BAY730
10
20
30
CONTROL SCD
***
••
A
•••
% A
dhes
ion
to F
N
Inhibition of PDE9 decreases neutrophil adhesion to FN, in vitro
Sickle Cell Mouse Inflammatory Vaso-occlusive Model
Almeida et al., 2011
Blood flow
3h post -TNF-α
Cremaster Muscle Measure: leukocyte Adhesion, rolling
and extravasation
Effects of HU and BAY73-6691 on leukocyte recruitment in TNF-α-treated SCD mice
Almeida et al., 2011
HU
BAY73-6691
HU + BAY73-6691
Vehicle
Effects of HU and BAY73-6691 on leukocyte recruitment in TNF-α-treated SCD mice
Blood flow
10μm Almeida et al., 2011
BAY73-6691 decreases WBC-RBC interactions
Almeida et al., 2011
Co-administration of HU and BAY73-6691 improves SCD mouse survival in the setting of vaso-occlusion
Almeida et al., 2011
HbF in Sickle Cell Anemia: What Modulates the Modulator?
Random forest analysis of joint association of multiple SNPs with % HbF
TOX binding sites within the LCR and Ggamma-globin gene promoter
6q 8q
Xp
(Ma et al, 2007)
(Wyszynski et al, Cell Mol Biol 2004)
(Dover et al, Blood 1992)
Hemoglobinopatia SC
• Hemoglobina C ativa cotransporte de K-Cl – ( possivelmente pela carga positiva da
Hemoglobina C) – Células são mais desidratadas – Inibidores de canal de Gardos ou de co-
transporte K/Cl • Hemoconcentração
– Sangrias
Perspectivas
Inflamação contribue para vaso-oclusão ativando células endotelias e células do sangue
• Reduzir a inflamação vascular deve ser alvo terapêutico;
•Inibição da adesão dos leucócitos à parede do vaso é importante para prevenção de vaso-oclusão
• Drogas sinérgicas a HU na via cGMP-dependente podem ser úteis no tratamento
• Inibição da PDE9 tem potencial de aumentar a produção de HbF, diminuir a adesão de leucócitos e é relativamente tecido-específica
Tratamento
• Transplante de medula óssea
Bone Marrow Transplantation , (18 April 2011) Allogeneic cellular gene therapy in hemoglobinopathies—evaluation of hematopoietic SCT in sickle cell anemia G Lucarelli, J Gaziev, A Isgrò, P Sodani, K Paciaroni, C Alfieri, G De Angelis, M Marziali, M D Simone, C Gallucci, A Roveda, F Saltarelli, F Torelli and M Andreani 11 SCA median age of 12 years (range, 2–16) Hematopoietic SCT from HLA-identical, related donors following a myeloablative-conditioning regimen. Indications for transplantation:vaso-occlusive crisis, acute chest syndrome, avascular bone necrosis, chronic RBC transfusions, or hemorrhagic stroke. All patients had sustained engraftment. One patient - stable mixed chimera with 25% of donor cells at 4 years after transplantation. One patient died at 1 year after transplantation. The probability of survival, SCA-free survival and TRM at 5 years after transplant were 90, 90 and 10%, respectively. All 10 surviving patients remained free of any SCA-related events after transplantation.
Agradecimentos • Allan Santos • Andre Fattori • Carmen S P Lima • Eduardo Rocha • Erich Vinicius de
Paula • Leonardo de Deus • Nicola Conran • Oswaldo Ueti • Regina Aoki • Valder R Arruda
•Assistentes sociais • Equipe de enfermagem
•Equipe de Fisioterapia
•Equipe de Odontologia
Hematology and Hemotherapy Center Fernando F. Costa
Camila Almeida Carla Franco-Penteado
Andreia Canalli Lediana Miguel
Vanessa Garrido Kleber Fertrin Fabiola Traina
New York: Albert Einstein College of Medicine Paul Frenette Christoph Scheiermman Jungan Jang
