Transcript Snímek 1

Phagocytosis Is Always the Best Time of Day
Burst test using FagoFlow in patients with CGD and
hematopeotic stem cell transplantation
Andrea Poloučková, M.D.
Department of Immunology, Charles University, 2nd Medical Faculty and University Hospital
Motol, Prague
CGD diagnosis
• NBT (nitro-blue tetrazolium chlorid)
• Chemiluminiscence
• Flow cytometry – Fago Flow test is based on the
measurement of respiratory (oxidative) burst of granulocytes after their
stimulation with E. coli bacteria. After the ingestion of bacteria, phagocytes
activate the NADP oxidase producing reactive oxidative intermediates
(respiratory burst). Reactive intermediates inside phagocytes oxidize
dihydrorhodamine 123 (DHR123) into fluorescent rhodamine123 which is
detected by a flow cytometer
• Enzyme activity (MPO)
• Molecular - genetic analysis of the defect
genes
CGD therapy
• Antibiotic and antifungal profylaxis and
therapy
• Neutrophil infusions
• Ɣ-INF
• Hematopoetic stem cell transplantation
–
Goudemand J, Anssens R, Delmas-Marsalet Y, Farriaux JP, Fontaine G: [Attempt to treat a
case of chronic familial granulomatous disease by allogenic bone marrow
transplantation]. Arch Fr Pediatr 1976, 33(2):121-129.
– HLA identical family donor, complicated course of the disease
• Gene therapy
–
Ott, M. G. et al. Correction of X-linked chronic granulomatous disease by gene therapy,
augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nature Med. 2 Apr
2006
Patient 1
(PB, 2006)
•
FH:negative
•
from 9 months – respiratory infections
18 months - multiple liver foci – susp. on
malignancy, transfer to our hospital
- laparotomy – multiple liver
abscesses (Staph. epidermidis)
•
Patient 1- CGD
Lab. findings:
•
NBT: 0
Molecular-genetic analysis:
gp91-phox mutation (CYBB, exon 4 – genotype 334:T>C, S112P)
• mother - carrier
•
Treatment:
ATB: cefotaxim, oxacilin, amikacin
meropenem (24 days), teicoplanin (76), ciprofloxacin (59)
• corticosteroids, γ – interferon
• granulocyte infusions
•
Before SCT:
•21
months – without clinical and laboratory signs of infection,
USG – significant regression of liver abscesses
Patient 1- SCT
7.11.2007 (aged 21 months)
• donor: MUD, 10/10, BM
•
conditioning: Fludarabine, Melfalan, MabCampath
• GVHD prophylaxis: CsA, Methylprednisolone 1mg/kg/day
•
•
engraftment: ANC D+12, Plt D+16
•
D+16: liver USG - without focal finding
complications: bilateral interstitial pneumonitis
increasing mixed chimerism
•D+61 – NBT 26
•
•
aGVHD: gr II (gut) D+63 - corticosteroids
•
discharge from BMT unit: D+83 (aged 24 months)
Patient 1- 16 months after SCT
without IS treatment, no signs of GVHD
• permanent increase of the mixed chimerism (D+70 29%
autolougous chimerism, 1 year after Tx 75%
autologous chimerism)
• decrease in NBT and FagoFlow
• NBT test: 12 - 7 – 6 - 4 (normal range > 9)
• FagoFlow test: 65% - 77% - 22% (normal range > 76)
• without clinical problems
• therapy: penicilin
•
Hemopoetic chimerism
% of allogeneic hemopoiesis
100
80
Granulocytes
66% .. 41%
60
withdrawal of
MP D+109
40
20
withdrawal of
CsA D+147
0
14
42
70
98
days after SCT
126
154
182
Patient 1 - FagoFlow
Patient 2
(LK, 2006)
FH: negative
6 months – cervical lymphadenopathy,
fever, hepatomegaly, splenomegaly – therapy
with ATB
•
7 months – abscess of soft tissue of the
head with osteomyelitis and intracranial
penetration (Serratia marcescens)
•
Patient 2 - CGD
Lab. findings:
•
NBT: 0
Molecular- genetic analysis:
gp91-phox mutation (CYBB, exon 4 – genotype 91:CGA>TGA, R91X)
• mother - carrier
•
Treatment:
ATB: amikacin, ciprofloxacin, linezolid + itraconazol, trimetoprim
• corticosteroids
• γ – interferon
•
Before SCT:
•11
•
months – without clinical and laboratory signs of infection,
MRI of CNS - mild residual changes
Patient 2 - HSCT
19.9.2007 (aged 12 months)
• donor: MUD, 9/10 (Cw), BM
•
conditioning: Busulfan, Cyclophosphamide, Thymoglobulin
• GVHD prophylaxis: CsA, Methotrexate
•
•
engraftment: ANC D+19, Plt D+42
complications: ascites, hepatopathy and nefropathy D+15
(imunopathological ?) – corticosteroids
bilateral interstitial pneumonitis
CMV infection
• aGVHD: gr II (gut) D+62 - corticosteroids, MMF
•
•
discharge from BMT unit: D+110 (aged 15 months)
Patient 2 - 19 months after SCT
10 months after Tx: no immunosupression, no
signs of GVHD
• stable mixed chimerism (9O% allogeneic)
• 11 month after Tx – viral infection – aneamia
(Coombs +++) – AIHA
• Therapy: Mabthera (7 months), prednison, IVIG
•
Nowadays: autologous chimerism 10-15%
• NBT test: 16 (n. 9)
• flow cytometry (FagoFlow): 61%
• therapy: Prednison, clotrimoxazol, penicilin,
Sporanox
•
Patient 2 - FagoFlow
Conclusion
FagoFlow – very rapid and easy test, recognize
the defects of NADPH system
Mixed chimerism
Dynamics, interpretation
of the results
Greek mythology:
Chimera – the
creature belching
the flame with the
head of the lion, the
body of the goat, the
tail of the snake
(Bader et al., 2005).
Thanks
Patients and their families
Prof. Anna Šedivá, M.D., PhD.
Renata Formánková, M.D., PhD.
Doc. Petr Sedláček, M.D., PhD.
Prof. Jan Starý, M.D., PhD.
Aleš Janda, M.D., PhD.
Jarmila Grecová