1. 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 Phagocytosis Is Always the Best Time of Day

2. 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

3. 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

4. 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)

5. 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

6. 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)

7. 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

8. Hemopoetic chimerism days after SCT % of allogeneic hemopoiesis withdrawal of MP D+109 withdrawal of CsA D+147 Granulocytes 66%.. 41%

9. Patient 1 - FagoFlow

10. 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)

11. 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

12. 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)

13. 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

14. Patient 2 - FagoFlow

15. 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).

16. 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