Novos Aspectos da Fisiopatologia em Doenças Falciformes e ... · • 0º ao 4º dia...

Novos Aspectos da Novos Aspectos da Novos Aspectos da Novos Aspectos da Fisiopatologia em Doenças Fisiopatologia em Doenças Fisiopatologia em Doenças Fisiopatologia em Doenças Falciformes e Talassemias: Falciformes e Talassemias: Falciformes e Talassemias: Falciformes e Talassemias:

Contribuição de Estudos Contribuição de Estudos Contribuição de Estudos Contribuição de Estudos Brasileiros RecentesBrasileiros RecentesBrasileiros RecentesBrasileiros Recentes

Talassemias

•• a mais comum das doenças monogênicasa mais comum das doenças monogênicas

•• primeiro grupo de doenças caracterizadas do ponto primeiro grupo de doenças caracterizadas do ponto de vista molecularde vista molecular

•• relação entre fenótipo clínico e patologia molecularrelação entre fenótipo clínico e patologia molecular

•• exemplo de como a seleção natural age na exemplo de como a seleção natural age na diversidade humana diversidade humana -- proteção contra maláriaproteção contra malária

•• primeiro diagnóstico préprimeiro diagnóstico pré--natal por DNA natal por DNA recombinanterecombinante

Formas Clínicas da Doença

• talassemia beta heterozigótica

• talassemia intermediária

• talassemia maior

• MODULADORES GENÉTICOS DE GRAVIDADE

AHSPAHSP

((Alpha Alpha HaemoglobinHaemoglobin Stabilizing ProteinStabilizing Protein))

analysis during human analysis during human erythropoiesiserythropoiesis

and in and in ββββββββ--ThalassemiaThalassemia

AHSP

(Alpha Hemoglobin Stabilizing Protein)

• Proteina eritroide-especifica– Liga-se expecificamente a α-globina

– Estabiliza α-globina em excesso – chaperonamoleculas durante a sintese de Hemoglobina

• Modulador de gravidade em β-thalassemia– Relacao entre gravidade clinica e niveis diminuidos de expressao do gene AHSP

Balancing the components of hemoglobin.

Luzzatto and Notaro, 2002

AHSP binds specifically to αααα-hemoglobin, and suggest that it might thereby

serve a dual purpose: to stabilize these chains and to help in delivering newly

formed αααα-chains to ββββ-chains.

Analise genetica populacional

identifica sequencias variantes que

alteram funcao e expressao do gene

AHSP

Camila O. dos Santos, et.al.American Journal of Hematology,in press,2007

50 individuos provenientes da regiao da Papa Nova Guine

46 individuos provenientes de paises do Mediterranean

50 individuos provenientes de paises da India

46 individuos proveniente de Port Olry (Continente Asiatico)

48 individuos provenientes de paises Afro-Caribenhos

126 individuos provenientes do Brasil

Analise do gene AHSP

Polimorfismos no gene AHSP

Haplotipos do gene AHSP

2.04.3G15AGTTH18

12.215.048.56.4G15AATTH17

1.8A18AATTH16

1.80.91.1A18AGTTH15

8.90.93.2A18GATTH14

62.247.06.42.99.6G15GATTH13

4.525.014.9G15GGTTH12

3.2A18GGTTH11

1.8A18AAGTH10

1.15.01.80.9A18GAGTH9

3.03.61.1G15GAGTH8

1.11.01.8A18AGGCH7

1.8G15GGGCH6

2.1A18GGGCH5

1.14.3G15AGGTH4

7.86.037.521.83.2A18AGGTH3

1.12.77.4G15GGGTH2

13.321.044.657.423.539.4A18GGGTH1

1 2 3 4 5 6

P

(n=92)

NG

(n=100)

M

(n=92)

B

(n=252)

AI

(n=100)

AC

(n=96)

Sequencia de ntHaplotipo

Frequencia (%)

AC – Afro caribenhosAI – IndiaB – BrasilM – MediterraneoNG – Papa Nova GuineP – Port Orly

Distribuicao do haplotipo do gene AHSP na

populacao estudada

AHSP N75I é menos efetiva na proteção contra

geração de ROS

Polimorfismo 12391 afeta ligacao do fator de

transcricao Oct1 e reduz expressao do gene AHSP

Conclusoes

• Mutacoes e ou delecoes no gene AHSP sao raras;

• AHSP N75I e menos efetiva na protecao contra geracao de ROS;

– Possivel implicacoes em β-talassemia

• Ampliacao dos estudos em individuos provenientes do continete Mediterraneo

• Polimorfismo 12391 diminui os niveis de expressao do gene AHSP

– Possivel implicacoes em β-talassemia

CASE 1

ββββ-thalassemia

AHSP N75I

αα/αα5.2410.419676.8N75I/N75Nβ39/βApatient

αα/αα0.75.719.76310.8N75N/N75Nβ39/βAmother

αα/αα0.22.029.789.614.5N75I/N75NβA/βAfather

AA/AA0.22.029.789.614.5N75I/N75NBA/BAfather

AA/AA0.75.719.76310.8N75N/N75NB39/BAmother

AA/AA5.2410.419676.8N75I/N75NB39/BApatient

Family 1

α-globin genesHbFHbA2(%)

Ret (%)

MCH (pg)

MCV (fL)Hb(g/dL)

AHSP geneβ-globin genesSample

Expressão gênica durante diferenciação

eritróide

Cultura de Células em Duas FasesCélulas mononucleares isoladas à partir de sangue de doadores por centrifugação em gradiente de Ficoll-Hypaque. Consiste em cultura primária (+ 7 dias) e em cultura secundária com adição de eritropoetina (+15 dias).

– Coletas para citospin – morfologia celular;

– Coletas para citometria de fluxo – diferenciação;

– Coletas para extração de RNA – cDNA

dos Santos CO et al. Exp. Hematol. 32 (2):157-62, 2004.

Expressão gênica durante diferenciação

eritróide

Cultura de Células em Duas Fases

– Foram analisadas 5 culturas de células, onde as coletas de cada cultura foram agrupadas em:• 0º ao 4º dia – predominância de pró-eritroblastos

• 5º ao 7º dia – presença de eritroblastos basofílicos

• 8º ao 10º dia – presença de eritroblastos policromatófilos e alguns ortocromáticos

• 11º ao 14º dia – predominância de eritroblastos ortocromáticos

– Análise estatística – Teste não paramétrico de Wilcoxon-Mann-Whitney, sendo significativo se p igual ou menor que 0,05.

0-4º dia 5º-7º dia

8º-10º dia 11º-14º dia

Expressão gênica durante diferenciação eritróide

Slides Citospin

Expressão do gene AHSP na diferencição

eritróide

0

20

40

60

80

100

0-4 days 5-7 days 8-10 days 11-14 days

Diferenciação Celular (Dias)

Exp

ress

ão Gên

ica (fo

ld cha

nge)

0-4º dia 5º-7º dia 8º-10º dia 11º-14º diap <0,05n=5

*


Expressão do gene Alfa-globina na diferenciação

eritróide

0

100

200

300

400

500

0-4 days 5-7 days 8-10 days 11-14 days

Diferenciação celular (Dias)

Exp

ress

ão Gên

ica (fold chan

ge)

0-4º dia 5º-7º dia 8º-10º dia 11º-14º diap <0,05n=5

*



Razão da expressão dos genes AHSP / Alfa-globina na

diferenciação eritróide

0

0,2

0,4

0,6

0,8

1

1-4 dias 5-7 dias 8-10 dias 11-14 dias

Diferenciação Celular (Dias)

Razão

N.S.

0-4º dia 5º-7º dia 8º-10º dia 11º-14º dia

Expressão gênica em pacientes com

ββββ-Talassemia e em controles

Razão da expressão dos genes AHSP/Alfa-globina em pacientes

com B-talassemia e em controles

0,000

0,020

0,040

Intermediaria Maior Controle

Pacientes

Razão

p > 0.05

*

0.04

0.02

0.00

MAIN HYPOTHESIS

Lower AHSP production

severe clinical status

Higher α-globin degradation and precipitation

Consequences of reduction of AHSP

synthesis in hemin-induced K562 cells

and EPO-induced CD34+ cells

The relationship between Hb formation and

alterations in AHSP expression, which may

affect human erythropoiesis, has not yet been

described in human cells.

The aim of this is to examine the effects of

AHSP knockdown in hemin-induced K562 and

EPO-induced CD34+ cells with particular

reference to cellular aspects and gene

expression.

ResultadosResultados

Expressão do gene AHSP

0

1

2

3

4

5

6

7

8

Antes da

transfecção

0h sem

hemina

0h com

hemina

24h com

hemina

48h com

hemina

72h com

hemina

96h com

hemina

Cultura de células K562 transfectadas por Eletroporação

Unidades Arbitrárias

Controle sem Vetor

Vetor Negativo - 15ug

Vetor de RNAi para AHSP - 15ug

(A) AHSP expression profile and the representative AHSP knockdown in human erythroid cells in culture at end stages of maturation.

(Β) α-globin gene expression profile in human erythroid cells in culture at end stages of maturation.

(C) γ-globin gene expression profile in human erythroid cells in culture at end stages of maturation.

Control Negative Control AHSP sh-RNA cells

♦♦ ♦♦KWOWAV (p<0.05)

I

γγ γγ-globinexpression levels

0

0.5

1

1.5

αα αα-globinexpression levels H

0

0.5

1.5

1

G

AHSP expression levels

0

0.2

1

0.6

C10 C13

♦

♦

DCF fluorescence intensity

C10

C13C

10C13

A B CI

γγ γγ-globinexpression levels

0

0.5

1

1.5

αα αα-globinexpression levels H

0

0.5

1.5

1

G

AHSP expression levels

0

0.2

1

0.6

C10 C13

♦

♦


C10

C13C

10C13

A B C

K

Annexin-and PI-labeled cells (%)

0

5

1 0

1 5 ♦♦

L

HbFfluorescence intensity

0

1 , 0 0 0

1 , 5 0 0

5 0 0

♦

J


0

1 0 0

3 0 0

2 0 0♦

♦

HbFfluorescence intensity

0

expression ratioB

1

1 . 5

B T H * H7 2

H1 2 0

H1 6 8

C1 0

C1 3C

1 0C1 3C

1 0C1 31 3

A B C

Control Negative Control AHSP sh-RNA cells

(A) Indirect identification of ROS through DCF fluorescence detection and increase in ROS expression in human AHSP-shRNA mature erythroid cells.

(B) Increase in Annexin and PI labeling in human AHSP-shRNA erythroid cells in culture at end stages of maturation.

(C) Decrease in fetal hemoglobin (HbF) formation in human AHSP-shRNA erythroid cells in culture at end stages of maturation.

♦♦ ♦♦KWOWAV (p<0.05)

ResultadosResultados

Anti-Alfa Anti-BAC

DAPI DAPI

Anti-Alfa Anti-BAC

Eletroporação

Controle Negativo RNAiEletroporação

RNAi para o gene AHSP

• (D) Alpha hemoglobin chain precipitation in AHSP-shRNA mature erythroid cells (bottom and left) in relation to negative control cells (top and left), the inclusion bodies are clearly identifiable (white arrow).

αααα-globinOverlay

with DAPIββββ-actin

3 dyes together

Negative

Control

AHSPshRNA

D αααα-globinOverlay


3 dyes together

Negative

Control

AHSPshRNA



3 dyes together

Negative

Control

AHSPshRNA



3 dyes together

Negative

Control

AHSPshRNA

D

Conclusions• The AHSP knockdown, induced in our study, indicates that AHSP

in human erythroid cells performs the same function as previously

identified in mice and in vitro assays.

• The data suggest that AHSP is important for the prevention of the

apoptosis of erythroid precursors in the human erythropoiesis, as

indicated by the results for both types of culture.

• These findings demonstrate that the α-globin precipitation is not

related to α-globin overexpression, but to the AHSP knockdown.

• AHSP-shRNA showed an evident decrease in fetal hemoglobin

formation.

Conclusions

• Taken together, our data show for the first time a

possibly highly significant role for AHSP in normal

hemoglobin synthesis and cell survival in human

cells. The possibility that AHSP acts as a genetic

modifier in human disease remains to be

demonstrated; however our data strengthen and

support studies regarding this possibility.

PHHF tipo Brasileira

• Mutação –195 C�G promotor γA (Costa e cols., 1990 e Bordin e

col., 1998).

• Experimentos preliminares utilizando gene repórter

da luciferase (Schreiber e cols., 2001).

– Não demonstraram ↑ de expressão na presença da

mutação.

– Hipótese: ausência do LCR

Fatores de Transcrição

Vetores de expressãoTakahashi, T. et. al.Eur J.

Haematol,2003

Quantificação de luciferase na célula K562

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

2,2

2,4

LucHS2normal

LucHS2-195

LucHS2-175

Luc-195

Luc normal

High levels of human γγγγ-globin gene expression in adult mice carrying

a transgene of Brazilian type of Hereditary Persistence of Fetal

Hemoglobin

(Aγγγγ -195 C →→→→G )

• The term, hereditary persistence of fetal hemoglobin (HPFH), describes a

hereditary benign disease, characterized by an increase in fetal hemoglobin

(HbF) during adult life.

• Non-deletional forms of HPFH are characterized by single-base mutations in

the promoter region (most of them between -114 and -202 from the cap site) of

either the Gγ or Aγ -globin gene, resulting in an increase of HbF ranging from 3 to 20% in heterozygotes.

• Many point mutations in this region have been described, including the Aγ -195 C →G mutation that causes the Brazilian type of HPFH (HPFH-B).

LCR ε γG γA ψβ δ β

Embrião

Feto

Adulto


Embrião

Feto

Adulto

Embrião

Feto

Adulto


Embrião

Feto

Adulto

Aγγγγ -195 C →→→→G

Expressão de gama

globina no adulto


Embrião

Feto

Adulto

Aγγγγ -195 C →→→→G

Expressão de gama

globina no adulto

Normal developing

Brazilian type of Hereditary

Persistence of Fetal

Hemoglobin

(HPFH-B)

Embryo

Fetus

Adult

Embryo

Fetus

Adult

Gamma globin

expression during

adult life

RPA (RNAse Protection

Assay)

Single cellembryo Embryos

Transgenicselection

HPFH-BTransgenic mouse

pseudo-pregnant female

F1

Aγγγγ -195 C →→→→G

HPFH-B transgenic mice construction

α

γβ

α

α

RNAse Protection Assay

mRNA levels of human gamma-globin

in fetal liver of transgenic mice

containing mutation were clearly

higher as compared with control

transgenic mice bearing cosmid

construct with wild type sequence

gamma promoter.

Conclusions

• A higher increase in gamma globin transcripts were found in a adult mice

carrying a transgene of Brazilian type of Hereditary Persistence of Fetal

Hemoglobin (Aγ -195 C →G ) showing that the mutation is the unique cause of elevation of HbF.

• These results may help develop the novel clinical approaches for the

increase of the expression of gamma globin in patients with abnormal beta

globin genes, such as sickle cell (HBS) and beta-thalassemia diseases

Anemia FalciformeAnemia Falciforme

Os Leucócitos e a Vaso-oclusão

Frenette & Atweh (2007) J Clin Invest 117: 850

Efeitos do ambiente na Efeitos do ambiente na

evolução clínica da anemia evolução clínica da anemia

falciformefalciforme

Clinical, hematological, andmolecular characterization of sicklecell anemia pediatric patients from

two different cities in Brazil

Caracterização clínica, hematológica e molecular de crianças portadoras

da anemia falciforme em duas diferentes cidades do Brasil

Lyra I. et al. Cadernos de Saúde Pública(FIOCRUZ); 21: (04), 1287-1290, 2005.

100.056100.070Total

8.563.02Atypical

55.03148.034CAR

34.01948.034Ben

São Paulo

n %

Salvador

n %

Chromosome

Chromosome distribution in the cities of Salvador

and São Paulo, Brazil

Table 1

7538124524074TOTAL

125900411Atypical

00151058Ben/Ben

32142810414CAR/CAR

311882322741Ben/CAR

LithiasisStrokeHospital

admissions

infection

Hospital

admissions vaso-

occlusion

LithiasisStrokeHospital

admissions

infection

Hospital

admissions vaso-

occlusion

São PauloSalvadorHaplotype

Frequency of clinical manifestations in different ββββs globin genotypes in Brazilian sickle cell anemia

Table2

Pacientes falcêmicos dos estados da Bahia e São Paulo e características laboratoriais hematológicas. Resultados do teste de Mann - Whitney (z) e do teste "t" de Student ("t").

Característica Ba SP Análiselaboratorial estatística

Ht 23,27 ± 3,21 24,38 ± 3,73 z cal .= 0,91 N.S.

Hb 7,48 1,11 7.8 1,17 "t"cal .= 0,96N.S.

V.C.M. 91,89 8,07 88,84 8,27 "t"cal.=1,5 N.S.

H.C.M. 29,71 3,12 28,61 3,0 "t"cal.=1,43 N.S.

C.H.C.M. 32,28 1,71 31,89 2,25 "t"cal. =0,76N .S.

Hb A 2,41 ± 0,60 2,86 ± 0,64 z cal. =3,12 *

Hb F 6,56 ± 3,22 6,25 ± 3,76 z cal. =0,43N.S.

Idade 9,3 ± 3,91 9,3 ± 4,54 “ t" cal = 0,01 N.S.

molecular modulators of clinicalseverity

UDP – glucunosyltransferase 1 (UGT1A)

Table 1. Laboratory and clinical data of patients with sicklecell anemia subdivided by their UDP-glucuronosyltransferase 1 (UGT1A)

UGT1Agenotype

Mean serum total bilirubin levels (mg/dl)

TA6/ TA6

TA6/ TA7

TA7/ TA7

3.33 = 0.87

3.95 = 1.07

6.88 = 1.52

Mean serumunconjugated bilirubin

levels (mg/dl)

2.42 ± 1.62

3.12 ± 0.94

6.03 ± 1.48

Frequency of cholelithiasis (%)

58.8 (10/17)

70.0 (14/20)

80.0 (8/10)

Frequency of cholecystectomy (%)

5.5 (1/18)

36.8 (7/19)

10.0 (1/10)

Fertrin . et al. Clinical Genetics 64:160-162, 2003.

ASSOCIATION OF THE G-463A MYELOPEROXIDASE

POLYMORPHISM WITH INFECTION IN SICKLE CELL ANEMIA

� Infections constitute a principal cause of morbidity

� and mortality in sickle cell anemia (SCA). Here

� we present evidence to suggest that a polymorphism

� (G-463A MPO) in the gene encoding the

� myeloperoxidase (MPO) enzyme, important for the

� host defense system, may significantly increase

� susceptibility to infection in SCA.

Costa, R.N.P. et al. Haematologica, 90: (07), 977-979, 2005.

Table 2. MPO genotype and allele frequency in SCA patients not experiencing infections and in those having one or more infections during their clinical follow-up.

Genotype Frequency (%)

Allele Frequency (%)

A/A A/G G/G A G Reference Group (n=50)

3

(6.0)

17

(34.0)

30

(60.0)

23.0 77.0

SCA without infections (n=63)

2

(3.2)

20

(31.7)

41

(65.1)

19.0 81.0

SCA with one or more infections (n=28)

2

(7.1)

17

(60.7)

9

(32.1)

37.5 62.5

p=0.0112 p=0.0096 The reference group was formed of racially-matched healthy individuals. Fisher’s exact test was used for the statistical

analyses. p values refer to the comparison of the underlined groups.

Coagulação e Trombose

• Variante da protombina (alelo 20.210 A)

• Fator V de Leiden (677 C→T)

• Variante termolabil da metilenotetrahidrofolatoredutase (MTHFR)

• Plaquetas

Human Platelet Antigen (HPA)-5 system polymorphism is a riskfactor for occlusive vascular complications in Sickle Cell

Anemia patients

Castro,V.et. al.Vox Sang 2004

Table 2- Genotypes, number of alleles and allelic frequencies of HPA-1, 2, 5 systems,

GpIa C807T polymorphism and 5T/C Kozak sequence between sickle cell anemia

patients with (SCA-VC) or without (SCA-N) occlusive vascular complications.

SCA-VC (n=34) SCA-N (n=63)

Genotype (%) N of

alleles

allele

frequency Genotype (%)

N of

alleles

allele

frequency

aa ab bb a b a b aa ab bb a b a b P(*)

HPA-1 64.7 32.4 2.9 55 13 0.809 0.191 73.0 25.4 1.6 108 18 0.857 0.143 NS

HPA-2 76.5 20.6 2.9 59 09 0.868 0.132 60.3 34.9 4.8 98 28 0.778 0.222 NS

HPA-5 44.1 47.1 8.8 46 22 0.676 0.324 77.8 22.2 0.0 112 14 0.889 0.111 0.0002

CC CT TT C T C T CC CT TT C T C T

C807T 61.8 26.5 11.8 51 17 0.750 0.250 46.0 36.5 17.5 81 45 0.643 0.357 NS

Kozak 0.0 41.7 58.3 10 38 0.208 0.792 6.0 32.0 62.0 22 78 0.220 0.780 NS

* Qui-Square P values obtained comparing allele frequencies of each polymorphism between the groups

Epinefrina

Citocinas?AMPc

Célula Endotelial Hemácia

falciforme

BCAM/LU

Laminina

Trombospondina

Fibronectina

VCAM-1 VLA-4

CD36

A Hemácia e a Vaso-oclusão

Therapy with hydroxyurea reduces adhesion molecule gene and protein expression in sickle red cells with a concomitant

reduction in adhesive properties

Gambero et al. (2007) Eu J Haematol 78:144

Aim

Compare the expressions of the CD36 and the VLA-4 integrin subunit genes on

reticulocytes from steady-state SCA patients on/off hyroxyurea therapy.

Adhesion of red cells from these subjects was compared using static adhesion assays

Surface protein expression was compared by flow cytometry.

0

2

4

6

8

10

12

14

16

18

20

AA EH SS SS+HU

% de adesão para FN

**

##

n=9 n=9 n=9n=4

AA: 50.9 x 106 cells/ml

EH: 360.2 x 106 cells/ml

SS: 370.9 x 106 cells/ml

SSHU: 288.2 x 106 cells/ml

The adhesion of SS RBC to FN is significantly greater than that of other patient groups, irrespective of red cell number

The gene expressions of CD36, CD49d and CD29 are significantly reduced in SSHU RBC

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

SS SS+HU

Expressão de CD36

*

n = 10 n = 7

0

0,5

1

1,5

2

2,5

3

3,5

4

SS SS+HU

Expressão de CD49d

*

n = 10 n = 7

0

0,5

1

1,5

2

2,5

3

SS SS+HU

Expressão de CD29

*

n = 10 n = 6 * p < 0.05. Mann-Witney test.

Conclusions

• The increased adhesive properties of SSRBC are reversed in patients on HU

therapy;

• This decrease could not be justified solely by altered reticulocyte numbers in

this population

• The decrease in adhesion of SSHU RBC was accompanied by a reduction in the

protein expression of the CD36 and VLA-4 adhesion molecules.

• Our data suggest that these HU-mediated reductions, both in protein

expression and in adhesive properties, may be mediated, in part, by the

decrease in the gene expression of these molecules.

Inhibition of spontaneous apoptosis

in neutrophils from sickle cell disease patients

Conran et al. (2007) Br J Haematol. (in press)

• SCD is a chronic inflammatory condition.

• Leukocytes may play an initiating and propagating role in vaso-occlusion

• Removal of inflammatory cells by apoptosis is fundamental for the resolution of inflammation

• We aimed to determine whether leukocyte apoptosis is altered in SCD.

Leucocytosis

Vascular Inflammation

Prostaglandins

(E1/E2)GM-CSF IL-8

Cytokine Production

IL-6

Intracellular AMPc



Conran et al., 2007 Br J. Haematol

Conran et al., 2007 Annals Hematol

Neutrophil

Intracellular AMPc



Neutrophil

Decreased cell death

in vitro

LeukocytosisDelay in the resolution of Inflammation

InflammationInflammation

VasoVaso--occlusionocclusion

Role for cAMP-Protein Kinase A signalling in

augmented neutrophil adhesion and chemotaxis in sickle cell disease

Canalli, Franco-Penteado et al., (2007) Eur J Haematol (in press)

• Leukocytes adhere to the vascular endothelium and play an initiating and propagating role in SCD vaso-occlusion

• What are the signaling pathways that mediate these increased adhesiveproperties?

• Are leukocyte chemotactic properties altered?

Basal

KT5720

Basal

KT 5720

KT 5823

DMSO

0

5

10

15

20

***

SCD

###

Control

A

% adhesion to FN

Basal

IL-8

IL-8+KT5720

Basal

IL-8

IL-8+KT 5720

IL-8+KT 5823

0

5

10

15

20

25

###

B

⊗⊗⊗⊗⊗⊗⊗⊗

SCD

+

Control

% adhesion to FN



Fig. 1. Increased SCD neutrophil adhesion is

abrogated by inhibition of PKA.

(A) Basal adhesion of control and SCD neutrophils

adhesion of neutrophils to fibronectin

In presence of: KT5720 (PKA inhibitor; 2.5 µM)KT5823 (PKG inhibitor;1 µM)

(B) IL-8 stimulated control and SCD neutrophil

adhesion to FN:

In the presence of: KT5720 (2.5 µM) KT5823 (1 µM)

Spont KT 5720 Spont KT 5720 KT 58230

5

10

15

Control SCD

**

#

A

Neutrophils x105/ml

IL-8 KT5720 KT5823 IL-8 KT5720 KT58230

5

10

15

20 B

Control SCD

+++

⊗⊗⊗⊗⊗⊗⊗⊗⊗⊗⊗⊗

+

Neutrophils x105/ml



Fig. 2. Spontaneous and IL-8-stimulated chemotaxis

is increased in SCD neutrophils and reversed by

the PKA inhibitor, KT5720.

(A) Spontaneous chemotaxis of control and SCD

neutrophils

In the presence of: KT5720 (PKA inhibitor,2.5 µM) KT5823 (PKG inhibitor,1 µM)

(B) IL-8-stimulated chemotaxis of control and SCD

neutrophils

In the presence of: KT5720 (PKA inhibitor,2.5 µM) KT5823 (PKG inhibitor,1 µM) .

Intracellular AMPc

Neutrophil

Role for cAMP-Protein Kinase A signalling

in augmented neutrophil adhesion and chemotaxis in sickle cell disease

IncreasedIncreased adhesiveadhesive

propertiesproperties

Role in

Vaso-occlusion

Increased

Chemotaxis

Pulmonary

Inflammation?

� Leukocytes in sickle cell disease (SCD)

important role in vaso-occlusive process

Increased number of leukocytes in SCD

� Nitric oxide (NO)

regulation of vaso-relaxation

platelet aggregation

important in SCD pathophysiology

plasma bioavailability is decreased in SCD

Inhibition of Increased Sickle Neutrophil Adhesion to ICAM-1

by Nitric Oxide Donors and Activation of cGMP Signalling

Canalli et al sumitted by Hematologica in august 2007

� To compare nitric oxide metabolites and cGMP levels (second

messenger for NO) in neutrophils from normal subjects and

neutrophils from SCD patients (SCD neutrophils)

� To compare the adhesive properties of these cells and looked at

the effect of NO donating agents on this adhesion.

Study Design

0

5

10

15

20

25

control SCD

SNP0 0 SNP

neutrophil adhesion

to ICAM-1 (%)

0

5

10

15

20

25

control SCD

0 Deano Deano0

neutrophil adhesion

to ICAM-1 (%) Co-incubation of SCD neutrophils with SNP

(10µµµµM), DEANO (1µµµµM) and Bay 412272 (150 nM)

(reduced their increased adhesion to ICAM-1. In

contrast, these drugs did not significantly affect

normal neutrophil adhesion to ICAM-1

Effect of Nitric Oxide Donors (SNP; DEANO) and Guanylate

Cyclase Activator (Bay 412272) on SCD Neutrophil

Adhesion to ICAM-1

n ≥8

**p=0.009

t- test

n ≥3

**p=0.002

t- test

**

**

0

5

10

15

20

25

0 0

control SCD

Bay Bay

neutrophil adhesion

to ICAM-1 (%)

n ≥4

*p=0.01

t- test

*

�NO bioavailability may be decreased in SCD; however, our data

indicate that NO dynamics are not significantly altered in the

leukocytes of SCD patients (data not show)

� Increased neutrophil adhesive properties in SCD may not be the

consequence of decreased NO bioavailability;

� Other factors, such as altered cytokine levels may mediate

increased cell adhesion.

Conclusions

� However, NO donors were able to reduce increased SCD

neutrophil adhesion to ICAM-1, possibly via a cGMP-dependent

pathway;

� HU appears to decrease SCD neutrophil adhesive properties and

this may be mediated by an increase in intracellular cGMP;

� NO donating drugs may benefit SCD patients by reducing

increased leukocyte adhesion, a mechanism important for the

vaso-occlusive process.

Conclusions

Increased Adhesive Properties of

Eosinophils in Sickle Cell DiseaseCanalli et al; Exp Hematol, 2004.

� Vaso-occlusion of blood vessels is the hallmark of sickle cell

disease (SCD), accounting for much of its morbidity and mortality.

� The pathophysiology of the vaso-occlusive episodes involving

polymerization of the hemoglobin S, interactions between SS

erythrocytes, endothelium, platelets, leukocytes and plasma

constituents.

� Polymorphonuclear cell counts are often higher in SCD patients

and leukocytosis has been correlated with an increased rate of early

death, acute chest syndrome and stroke.

Objective

� The neutrophils of these patients demonstrate increased

adhesion to fibronectin and endothelial layers in static

adhesion assays.

Here we investigate a possible role for the eosinophil in

sickle cell disease (SCD) in process of vaso-occlusion.

Normal SCA0.0

0.5

1.0

1.5

Absolute eosinophil

number x 109cells/L

Normal SCA0

5

10

15

*

% adhesion to

fibronectin

Elevated Absolute Eosinophil Numbers and

Adhesion of Sickle Cell Anemia Patient

Eosinophils to Fibronectin

0.0 0.1 0

5

10

15

20

25

[GM-CSF] ηηηη g/ml

% adhesion to

fibronectin

A

0.0 0.1 0

5

10

15

20

25 B

[GM-CSF]]]] ηηηη g/ml

% adhesion to

fibronectin

**

AA

SS

***

p<0.0001

p=0.02P<0.01

Conclusions

� We demonstrate, that the eosinophils of SCD individuals, in addition

to being found in increased numbers in the circulation of patients,

present augmented adhesive properties

� These enhanced adhesive properties are mediated, at least in part, by

the ββββ1 and ββββ2 integrins, VLA-4, LFA-1 and Mac-1 and appear to be the

consequence of changes in integrin affinity or avidity rather than

integrin expression (Flow cytometry indicated that expression of

these integrins is unaltered on the surface of SCD eosinophils)

� Further studies are required to determine whether the eosinophil

contributes to vasoocclusion in SCD.

GENE EXPRESSION PROFILES OF BONE

MARROW CELLS CHARACTERISE

SEVERAL MECHANISMS OF THE

ACTION OF HYDROXYUREA IN SICKLE

CELL ANAEMIA

British Journal of Haematology, 2007 Jan;136(2):333-42.

HBS – WITHOUT HU HBSHU – WITH HU

95 85338

146repressed

192induced

HbSHUHbS

differentially expressed tags

Pvalor>0,05Fold>5

Results

Total 518CLASSIFICATION HbS HbSHU

Total Tags 45.014 49.814

Unique Tags 15.357 16.506

Identified genes 8.143 (53%) 9.749 (59%)

ORFs 3.422 (22.3%) 2.685 (16.26%)

Non-identified tags 4.053 (24.7%) 4.074 (24.6%)

Conclusions

� First study showing global gene expression induced by HU in bone marrow of Sickle cell anemia patient

�The results strongly suggest that HU produces significant changes in the gene expression pattern of human bone marrow cells in a SCA patient.

� Our data suggest a recruitment of transcriptional machinery and pathways involved in signal transduction during HU treatment. These pathways may have a direct effect on induction of γ globin gene expression at the transcriptional level.

� Conversely, pathway stimulation associated with channel or pore class transporter activity indicates a possible interference with the decrease in the dehydration status of cells, interfering with the rheological properties of the cell.

�The study of genes associated with these pathways and the description of new genes, up or down regulated by HU, may also represent a potential tool to identify new targets for SCA patient therapy.

Sara T.O. SaadValder R. Arruda

Fernando Lopes AlbertoNicola C. ZorzettoAndreia A. Canali

Camila Oresco dos SantosLuciana HollandaSheley Gambero

Carla F.Franco-PenteadoAnderson CunhaAndre Fattori

PRDX-6 HBG

HSPE-1 EGR-1

EGR-1HSPE-1

PRDX-6

Reticulocyte

Leukocyte

A.

B.

W/o HU With HU

W/o HU With HU

W /o HU With HU

W /o HU With HU

W /o HU With HU W /o HU With HU

W /o HU With HU

PRDX-6 HBG

HSPE-1 EGR-1

EGR-1HSPE-1

PRDX-6

Reticulocyte

Leukocyte

A.

B.

W/o HU With HU

W/o HU With HU

W /o HU With HU

W /o HU With HU

W /o HU With HU W /o HU With HU

W /o HU With HU

Expresssions of EGR1, PRDX-6, HSPE1 and HBG

genes in reticulocytes (A) and EGR1, PRDX-6 and

HSPE1 genes in leukocytes (B) from the

preripheral blood of sickle cell patients on (n=4)

and off (n=4) HC therapy.BAC

HBG

EDF1

EGR1

HSPE1

PRDX6

- HU

+ HUBMC

BAC

HBG

EDF1

EGR1

HSPE1

PRDX6

K562

ERH

STAT5A

A.

B.

1:8 1:16 1:4 1:2 1:2 1:4

1:64 1:64 1:16 1:4 1:16 1:8 1:2 1:64Dilution

Dilution

- HU

+ HU

Results

Semi quantitative RT-PCR analysis of the genes in bone marrow cells

(HBMC) from a SCA patient before and after HC treatment (A) and in

K562 cells lines treated and not treated with HC (B). The cDNAs were

quantified and equal amounts of cDNA were used in RT-PCR with 7

different dilutions for both samples. The dilutions that demonstrated a

difference between samples were loaded on 2% agarose gels and

stained with ethidium bromide. The dilutions are indicated at the

bottom of each figure.

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