CHRONIC GRANULOMATOUS DISEASE

1. CHRONIC GRANULOMATOUS DISEASE (CGD)Pathogenesis and DiagnosisAdel Almogren, BCLS, MS, PhDAssistant Professor of Immunology

2. Topics to be considered Phagocytes & Pahgocytosis Review of NADPH Oxidase Pathway Mutations in CGD Patients Pathogenesis in CGD and clinical characteristics Diagnosis of CGD Treatment of CGD Case Study

3. Chronic Granulomatous Disease Rare (1/200,000) inherited disorder of the reduced nicotinamide dinucleotide phosphate oxidase complex (NADPH) - may be higher Phagocytes defective in production of microbicidal reactive oxidant superoxide anion and metabolites (hydrogen peroxide, hydroxyl anion, & hypohalous acid Patients suffer from life-threatening bacterial & fungal infections. Dramatic inflamatory response leads to granuloma formation

4. CGD - History First described in 1957 by Good et. al. As fatal granulomatous disease of childhood CGD granulomas had similarities to other granuloma forming diseases (Wegener granulomatosis, brucellosis, sarcoidosis, Hodgkin�s disease, etc.) Combination of recurrent suppurative and inflammatory complications in childhood recognized - distinct clinical syndrome Originally thought to be X-linked as only diagnosed in boys

5. History continued 1960�s CGD established as a disease of phagocytes Neutrophils exhibit normal phagocytic activity Bacteriocidal activity against S. aureus impaired 1990�s to present - Actual defects elucidated CGD genetically heterogeneous, caused by mutation in any one of four structural components of NADPH oxidase complex Mouse knockout models created Gene therapy trials now underway

9. Current Cellular Phenotype & Genotype in CGD

10. Mutations in CGD In X91 103 specific mutations identified in 13 exons of gp91phox CYBB gene Approximately 90% come from X linked carriers X linked CDG = Heterogeneous spectrum of mutations Two-thirds of cases are X-linked, affecting the gp91 unit of cytochrome b558 (gene CYBB), mutations are unique to individual families. Similar heterogeneity for the p22 phox gene CYBA and p67 phox CGD gene NCF2. In contrast, p47 phox CGD - majority due to GT deletion (exon two in NCF1 gene) - frameshift yielding premature stop codon at amino acid 51.

12. Pathogens in CGD Recurrent infections with catalase producing pathogens (Staph. Aureus, Serratia marcescans, Nocardia Aspergillus sp. ) occurring at sites acting as environmental barriers: Skin -Lymph nodes Liver or spleen -Lungs Gastrointestinal tract Catalase negative pathogens can not degrade their own H2O2 providing exogenous source hypohalous acid formation Other factors besides catalase Not susceptible to all catalase + organisms CGD knockout mice - Catalase-deletant strains of Aspergillus nidulans as virulent as wild type

13. Leucocyte Adhesion Def. Type1 Decreased or absence of CD11/CD18 expressionLeucocyte Adhesion Def. Type1 Decreased or absence of CD11/CD18 expression

14. Pathogens in CGD

15. Pathogens in CGD

18. Inflammatory Complications in CGD Chronic inflammation skin ulceration, pneumonitis, inflammatory bowel disease. Exuberant & persistent tissue granuloma formation Mechanism of abnormal inflammation - largely unknown Inability of CGD neutrophils to inactivate proinflammatory chemoattractants via reactive oxidants

19. Aspergillus pneumoina & liver abscessAspergillus pneumoina & liver abscess

20. Exuberant granuloma formation in chronic granulomatous disease (CGD). Wound dehiscence and impaired wound healing at surgical incision sites due to dysregulated inflammatory responses in an X-linked CGD patient.Wounds take a very long time to heal in these patients. They must be constantly washed and cared for every day. Exuberant granuloma formation in chronic granulomatous disease (CGD). Wound dehiscence and impaired wound healing at surgical incision sites due to dysregulated inflammatory responses in an X-linked CGD patient.Wounds take a very long time to heal in these patients. They must be constantly washed and cared for every day.

22. A)normal, B) carrier has both populations C) CGD pateintA)normal, B) carrier has both populations C) CGD pateint

23. CGD by Flow Cytometry Small whole blood sample is stimulated to undergo the oxidative burst with phorbol myristate acetate in the presence of Dye. Dihydrorhodamine123 when oxidized is fluorescent. Fluorescence is quantitated. Results expressed as ratio of Fl. Stimulated over Fl. Unstimulated. CGD test by Flow Cytometry can detect CGD patients, carriers, and can suggest the genotype of the CGD patient.

24. Case Study

26. Treatment of CGD Prophylaxis with antibiotics trimethoprim-sulfamethoxazole itraconazole Interferon-gamma does not increase NADPH oxidase (1st yes, 2nd large NIH study - no) Augmentation of oxidant-independent antimicrobial pathways TNF, granule protein synthesis, MHC II expression, FC gamma receptors - all increased

27. Granulocyte transfusion Has been used in life threatening infections No prospective studies Many possible adverse reactions Bone marrow transplantation Curative 60% of time Due to morbidity and mortality - not routine use May be revisited with stem cell transplantation Treatment of CGD

28. Gene Therapy Ideal candidate for hematopoietic stem cell gene therapy engraftment of stable normal myeloid stem cell is curative only 3-10% normal neutrophils required for normal protection Treatment of CGD

29. NIH Gene Therapy Clinical Trial 5 patients with p47phox deficient CGD Autologous CD34 peripheral blood stem cells transduced in vitro with retrovirus vector containing p47phox cDNA Infused without bone marrow conditioning .004 to .05% circulating corrected granulocytes 2 of 5 patients had detectable corrected granulocytes after 6 months Treatment of CGD