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Chronic Granulomatous Disease: A Defect in NADPH Oxidase

Chronic Granulomatous Disease (CGD), a primary immunodeficiency affecting the innate immune system, is a rare hereditary disorder comprised of a group of four inherited genetic mutations with a common phenotype. It is estimated to affect 1 in 1 million people in the United States, with approximately 67% of CGD patients showing X-linked recessive inheritance. Autosomal recessive forms of CGD also occur in different genes. All forms of CGD are characterized by immunocompromised patients with chronic and recurrent life-threatening bacterial and fungal infections, especially those involving the skin, lungs, lymph nodes and liver, and the formation of abnormal tissue granulomas. In particular, osteomyelitis, gingivitis and perianal and perirectal abscesses are common. Infectious etiology in CGD is largely confined to 5 pathogens: Staphylococcus aureus Burkholderia (Pseudomonas) cepacia Serratia spp. Nocardia spp. Aspergillus spp. The underlying defect in CGD is an inability or reduced ability of phagocytes to produce reactive oxygen species (ROS) as a result of defects in the enzyme NADPH oxidase. NADPH oxidase is composed of several structural and regulatory proteins, including two heme groups. Two of these subunits, gp91phox ( subunit, glycosylated) and p22phox ( subunit, non-glycosylated), are integral membrane proteins in the plasma membrane that form flavocytochrome b558, the electron-transport center of the oxidase. The cytosolic proteins, p40phox, p47phox, and p67phox, and Rac (a GTPase) exist as a complex in the cytosol of resting phagocytes. X-linked recessive CGD inheritance is caused by mutations in the CYBB gene encoding gp91phox. Males who are hemizygous will manifest CGD. Females who are heterozygous for the mutant CYBB gene will have two populations of neutrophils, one normally functional, and the other metabolically defective. If a female is homozygous for the mutant CYBB gene, she will be affected and manifest CGD. Autosomal recessive forms of CGD are caused by mutations in one of three genes: the CYBA (p22phox) gene, the NCF1 (p47phox) gene, and the NCF2 (p67phox) gene. Two mutant copies in either gender will lead to CGD, while heterozygotes will display two populations of neutrophils, similar to the X-linked recessive CGD seen in heterozygous females. The four forms of the disease are referred to as X91, A22, A47 and A67 CGD, with superscripts +, , or added to indicate a normal level, reduced level, or complete absence of the affected subunit.

Innate Immunity
Innate immunity provides early defense, and is in place before infection occurs and reacts rapidly (minutes to hours) after encountering a pathogen. It looks for structures characteristic to and essential to classes of microbes, called pathogen-associated molecular patterns (PAMPs). PAMPs may include ssRNA or dsRNA in viruses, pilin, flagellin, LPS, or lipoteichoic acid in bacteria, and mannan or dectin glucans in fungi. Innate immunity consists of physical and chemical barriers (epithelial & antimicrobial substances secreted), phagocytic cells (neutrophils, macrophages, monocytes, dendritic cells, and NK cells), blood proteins (complement proteins and inflammation response), and cytokines (proteins that coordinate innate immunity). The innate immune system can also recognize characteristics of damaged or stressed cells, such as altered membrane phospholipids or heat shock proteins.

Neutrophils are granulocytes that originate from the multipotential common myeloid progenitor (CMP) stem cell, which differentiates into granulocyte/monocyte progenitors (GMPs) under the influence of cytokines such as GM-CSF, granulocyte colony-stimulating factor (G-CSF), and IL-3. GM-CSF is a cytokine secreted by endothelial cells, T cells, macrophages, mast cells, and fibroblasts. It stimulates GMP cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. The neutrophil progenitor (NoP) undergoes six morphologically identifiable stages in the process of maturation: The multipotential CMP stem cell also gives rise to the cells that develop along the monocytemacrophage pathway. Monocytes are produced in the bone marrow from a GMP stem cell that can mature into a monocyte or another of the three granulocytic cell lines. In addition, GMP gives rise to dendritic cells. The proliferation and differentiation of CMP into committed GMP is controlled by IL-3. Further progression of monocyte progenitor (MoP) cell lineage depends on the continued presence of PU.1 and Egr-1transcriptionfactors and is stimulated by IL-3 and GM-CSF. The GM-CSF also controls further differentiation into mature cells, which are then released into circulation. The transformation of MoPs to monocytes takes about 55 hours, and the monocytes remain in the circulation for only about 16 hours before emigrating to the tissues where they differentiate under influence of both GM-CSF and M-CSF into tissue macrophages. The phagocytes can be subdivided into 2 groups - macrophages and monocytes, and neutrophils (aka polymorphonuclear leukocytes). Neutrophils typically can be considered the "first responders or EMTs" of the innate immune compartment. However, in submucosal tissue macrophages are the first to encounter pathogens and are later reinforced by neutrophils. All have pattern recognition receptors that will recognize PAMPs. Microbes bind to phagocyte receptors Phagocyte membrane zips up around microbe Microbe ingested into phagosome Fusion of phagosome with lysosome Activation of phagocyte Killing of microbes by lysosomal enzymes, ROS and NO in phagolysosomes

Innate Immune Response Did this section while slightly inebriated, dont judge me, ha Neutrophils and monocytes circulate through the blood. When a monocyte enters damaged tissue through the endothelium of a blood vessel, a process known as the leukocyte extravasation, it undergoes a series of changes to become a tissue-resident macrophage. Dendritic cells and tissue-resident macrophages are present in tissues in contact with the external environment, such as the skin (where there is a specialized dendritic cell type called Langerhans cell and is most prominent in the stratum spinosum) and the inner lining of the nose, lungs, stomach and intestines. Dendritic cells can also be found in an immature state in the blood. Neutrophils are more microbicidal as a result of expression of high amounts of powerful and cytotoxic antimicrobial molecules; consequently, they are potentially dangerous phagocytes strategically

stored as reserve pools during steady-state conditions as resting cells in bone marrow and blood, activated and used only in emergency situations, when, where, and while needed. Macrophages and dendritic cells express Toll-Like Receptors (TLRs), a type of PRR, to recognize PAMPs found on subsets of pathogens. Once a microbe is recognized, the macrophage or immature dendritic cell ingests the pathogen, and pathogen becomes trapped in a phagosome, which then fuses with a lysosome. Within the phagolysosome, enzymes and toxic peroxides digest the pathogen, and upon maturation present those fragments at their cell surface using MHC Class II molecules. Simultaneously, the dendritic cells and macrophages secret chemokines to recruit neutrophils (CXCL8, CXCL1, CXCL2, and CXCL3) and monocytes (CCL2, CCL3, CCL4), which become activated and recruit inflammatory phagocytes and attracting additional inflammatory neutrophils and monocytes. The recruited monocytes give rise to inflammatory macrophages. Activated neutrophils release granules and antimicrobial granule proteins that directly kill pathogens free in the extracellular space. Infected macrophages ingest neutrophils and released neutrophil granule proteins to enhance their antimicrobial capacity. The recruited neutrophils and monocytes get to the site of infection through dilation of capillaries and chemoattractants. When a microbe enters or skin (for example) is injured, epithelial cells and bacteria release chemokines. Mast cells present near the epithelial cells will be stimulated to release histamine and heparin. Histamine and heparin cause vasodilation, and the endothelial cells become more loosely arranged. Vasodilation and increased endothelial permeability occur causing increased hydrostatic pressure and decreased plasma osmotic pressure by the outflow of fluid rich in proteins. The fluid loss results in a high concentration of red cells and increase blood viscosity, leaving the flow slower (stasis), and contributing to the leukocytes (especially neutrophils) to move to the most peripheral layers of the bloodstream, initiating the so called leukocyte margination along the vascular endothelium. The endothelial cells lining postcapillary venules express selectins on their surfaces that mediate low-affinity binding (fast off-rate due to shear force of blood flow) to leukocytes passing through. P-selectin is stored in cytoplasmic granules of endothelial cells and is rapidly redistributed to the surface in response to microbial products, cytokines, histamine from mast cells, and thrombin generated during blood coagulation. E-selectin is expressed on endothelial cell surfaces within 1-2 hours in response to IL-1 and TNF, as well as microbial products like LPS. Ligands on leukocytes that bind E- and P-selectin are sialylated carbohydrate groups related to the Lewis X family. L-selectin is expressed on leukocytes (but not endothelial cells), and their ligands are present on high endothelial venules (HEVs) called peripheral node addressin (PNAd). Leukocytes also express the ligands for E- and P-selectin on the tips of their microvilli. The binding of selectin ligands on microvilli of leukocytes and selectins on endothelial cells is transient, seen as rolling. Chemokines produced in tissues bind heparin sulfate on endothelial cells that line postcapillary venules and are displayed like this to circulating leukocytes and lymphocytes that have bound the endothelial surface through adhesion molecule interactions. When they have bound to chemokines, chemokine receptor signaling occurs, activating integrins on rolling leukocytes, which increases the affinity for ligands on endothelial cells via a conformational change in the extracellular domain of the integrin cytoplasmic tail, as well as membrane clustering of integrins. This increases avidity of integrins to ligands on endothelial cell surfaces. Endothelial cell ligand (VCAM-1, ICAM-1) expression increases by TNF and IL-1. ICAM-1 is expressed on cytokine-activated endothelial cells, dendritic cells, and macrophages. ICAM-2 is expressed on endothelial cells, and ICAM-3 is expressed on lymphocytes. VCAM-1 is expressed on cytokineactivated endothelial cells in some tissues. LFA-1 on leukocytes (Leukocyte Function Associated Antigen 1) binds ICAM-1 on the endothelial cells. Mac-1 and VLA-4, present on monocytes, binds ICAM-1 and mediated adhesion to endothelium. This allows the leukocytes to firmly attach to endothelial cells, followed by reorganization of their cytoskeleton and spreading out on the endothelial cell surface. The leukocytes undergo diapedesis (paracellular

transmigration) to reach extravascular tissues, mediated by CD31 which transiently and reversibly disrupts adherens junctions VE-cadherin complex between endothelial cells. The endothelial cells were also previously loosened by heparin and histamine released by mast cells. The macrophages and dendritic cells also upregulate CCR7, a chemotactic receptor that induces the cells to travel through the blood stream to the spleen or through the lymphatic system to a lymph node (which produce CCL19 and CCL21 that attracts CCR7). Here they act as antigen-presenting cells: they activate helper T-cells and killer T-cells as well as B-cells by presenting them with antigens derived from the pathogen, alongside non-antigen specific costimulatory signals. Upon effective control of the infection, the presence of potentially dangerous neutrophils at infectious/inflammatory foci is terminated quickly through the removal of senescent/apoptotic neutrophils by macrophages before lysis and associated tissue damage.

CGD MoA: A Deficient Respiratory Burst

After ingestion of microbes into phagosomes, the cytosolic proteins translocate to the membrane to complex with flavocytochrome b558 to form activated NADPH oxidase. The subunit (p22phox) of NADPH oxidase is required for stabilization of the enzyme. The subunit (gp91phox) of NADPH oxidase transfers an electron from bound NADPH on the cytosolic side to FAD (flavin), which then transfers the electron to Fe-heme, and finally to O2 on the vacuolar or extracellular side, generating superoxide anion (O2-), a ROS, a major microbicidal mechanism of phagocytes called the Respiratory (or Oxidative) Burst. Superoxide can then generate hydrogen peroxide, which leads to production of more ROS to kill microbes. Figure 1. A model of phagocyte NADPH oxidase (respiratory burst) activation. Binding of the cytosolic components activates the flavocytochrome to catalyze the transfer of electrons from NADPH to oxygen, via the flavin (FAD) and heme redox centers in gp91phox, to form O2 (red dashed line). The resulting rapid consumption of oxygen is known as the respiratory burst. The compartment labeled inside is the cytoplasmic space; outside refers either to the extracellular or phagosomal space.

Defective production of ROS results in a failure to kill phagocytosed microbes, and recurrent infections with catalase-producing intracellular bacteria and fungi occur from early childhood. Catalase, produced by many organisms that are troublesome in CGD patients, destroys microbicidal hydrogen peroxide (H2O2) produced by host phagocytes and many microbes themselves. In contrast, infections with catalase negative microbes allow the generation of H2O2 by the microbe which is harnessed by host phagocytes and can compensate for the defective NADPH oxidase enzyme in CGD patients. Reactive Oxygen Species (ROS) is a phrase used to describe a number of reactive molecules and free radicals derived from molecular oxygen. Cells have a variety of defense mechanisms to ameliorate the harmful effects of ROS. Superoxide dismutase (SOD) catalyzes the conversion of two superoxide anions into a molecule of hydrogen peroxide (H2O2) and oxygen (O2). In the peroxisomes of eukaryotic cells, the enzyme catalase converts H2O2 to water and oxygen,

and thus completes the detoxification initiated by SOD (Eq. 2). Glutathione peroxidase is a group of enzymes containing selenium, which also catalyze the degradation of hydrogen peroxide, as well as organic peroxides to alcohols. There are a number of non-enzymatic small molecule antioxidants that play a role in detoxification. Glutathione may be the most important intra-cellular defense against the deleterious effects of reactive oxygen species. This tripeptide (glutamyl-cysteinyl-glycine) provides an exposed sulphhydryl group, which serves as an abundant target for attack. Reactions with ROS molecules oxidize glutathione, but the reduced form is regenerated in a redox by an NADPH-dependent reductase. Vitamin C or ascorbic acid is a water soluble molecule capable of reducing ROS, while vitamin E (-tocopherol) is a lipid soluble molecule that has been suggested as playing a similar role in membranes. The ratio of the oxidized form of glutathione (GSSG) and the reduced form (GSH) is a dynamic indicator of the oxidative stress of an organism. Lipid peroxidation is one of the most widely used indicators of free radical formation, a key indicator of oxidative stress. Unsaturated fatty acids such as those present in cellular membranes are a common target for free radicals. Reactions typically occur as a chain reaction where a free radical will capture a hydrogen moiety from an unsaturated carbon to form water. This leaves an unpaired electron on the fatty acid that is then capable of capturing oxygen, forming a peroxy radical (Figure 4). Lipid peroxides are unstable and decompose to form a complex series of compounds, which include reactive carbonyl compounds, such as malondialdehyde (MDA). The free radical nitric oxide (NO) is produced by a number of different cell types with a variety of biological functions. Nitric oxide is a product of the oxidation of L-arginine to L-citrulline in a two step process catalyzed by the enzyme nitric oxide synthase (NOS). Two major isoforms of nitric oxide synthase have been identified. The constitutive isoform found in neurons and endothelial cells, produces very low amounts of nitric oxide in a calciumand calmodulin-dependent fashion. NO activates soluble guanlyate cyclase in target cells, resulting in increased levels of cGMP, which in turn facilitates neuronal transmission and vascular relaxation, and inhibits platelet aggregation [41]. The inducible isoform, found in macrophages, fibroblasts, and hepatocytes, produces NO in relatively large amounts in response to inflammatory or mitogenic stimuli and acts in a host defensive role through its oxidative toxicity [42]. Regardless of the source or role, the free radical NO has a very short half life (t= 4 seconds), reacting with several different molecules normally present to form either nitrate (NO3-) or nitrite (NO2.-)

The phagocytes are unable to control the infection, and as a result, chronic cell-mediated immune responses are stimulated, leading T cells to activate macrophages which form granulomas to try, unsuccessfully, to eliminate the infection. CGD is often fatal, even with aggressive antibiotic therapy. A granuloma is tiny collection of macrophages. Granulomas form when the immune system attempts to wall off substances that it perceives as foreign but is unable to eliminate. Macrophages often, but not invariably, fuse to form multinucleated giant cells. The macrophages in granulomas are often referred to as "epithelioid. This term refers to the vague resemblance of these macrophages to epithelial cells. Epithelioid macrophages differ from ordinary macrophages in that they have elongated nuclei that often resemble the sole of a slipper or shoe. They also have larger nuclei than ordinary macrophages and their cytoplasm is typically more pink when stained with eosin. These changes are thought to be a consequence of "activation" of the macrophage by the offending antigen. Granulomatous reactions occur if the microbial antigens persist in the tissues and continue to stimulate host reactivity. (1) IL-1 and IL-8 are released in response and attract an inflammatory cell influx. (2) IL-4 and IFN-g promote the retention of macrophages and cause the fusion of monocytes at the site, leading to an epithelioid cell granuloma derived from macrophages, histiocytes, and epithelioid cells.

Staphylococcus aureus
Gram-positive, cluster-forming coccus nonmotile, non-spore-forming facultative anaerobe fermentation of glucose produces mainly lactic acid ferments mannitol (distinguishes from S. epidermidis) catalase positive coagulase positive golden yellow colony on agar normal flora of humans found on nasal passages, skin and mucous membranes pathogen of humans, causes a wide range of suppurative infections, as well as food poisoning and toxic shock syndrome

S. aureus expresses many potential virulence factors: (1) surface proteins that promote colonization of host tissues; (2) invasins that promote bacterial spread in tissues (leukocidin, kinases, hyaluronidase); (3) surface factors that inhibit phagocytic engulfment (capsule, Protein A); (4) biochemical properties that enhance their survival in phagocytes (carotenoids, catalase production); (5) immunological disguises (Protein A, coagulase); (6) membranedamaging toxins that lyse eucaryotic cell membranes (hemolysins, leukotoxin, leukocidin; (7) exotoxins that damage host tissues or otherwise provoke symptoms of disease (SEA-G, TSST, ET); and (8) inherent and acquired resistance to antimicrobial agents. Human staphylococcal infections are frequent, but usually remain localized at the portal of entry by the normal host defenses. The portal may be a hair follicle, but usually it is a break in the skin which may be a minute needle-stick or a surgical wound. Foreign bodies, including sutures, are readily colonized by staphylococci, which may make infections difficult to control. Another portal of entry is the respiratory tract. Staphylococcal pneumonia is a frequent complication of influenza. The localized host response to staphylococcal infection is inflammation, characterized by an elevated temperature at the site, swelling, the accumulation of pus, and necrosis of tissue. Around the inflamed area, a fibrin clot may form, walling off the bacteria and leukocytes as a characteristic pus-filled boil or abscess. More serious infections of the skin may occur, such as furuncles or impetigo. Localized infection of the bone is called osteomyelitis. Serious consequences of staphylococcal infections occur when the bacteria invade the blood stream. A resulting septicemia may be rapidly fatal; a

bacteremia may result in seeding other internal abscesses, other skin lesions, or infections in the lung, kidney, heart, skeletal muscle or meninges. Membrane-damaging toxins alpha toxin (alpha-hemolysin) The best characterized and most potent membrane-damaging toxin of S. aureus is alpha toxin. It is expressed as a monomer that binds to the membrane of susceptible cells. Subunits then oligomerize to form heptameric rings with a central pore through which cellular contents leak. In humans, platelets and monocytes are particularly sensitive to alpha toxin. Susceptible cells have a specific receptor for alpha toxin which allows the toxin to bind causing small pores through which monovalent cations can pass. The mode of action of alpha hemolysin is likely by osmotic lysis. -toxin is a sphingomyelinase which damages membranes rich in this lipid. The classical test for -toxin is lysis of sheep erythrocytes. The majority of human isolates of S. aureus do not express -toxin. A lysogenic bacteriophage is known to encode the toxin. delta toxin is a very small peptide toxin produced by most strains of S. aureus. It is also produced by S. epidermidis. The role of delta toxin in disease is unknown. Leukocidin is a multicomponent protein toxin produced as separate components which act together to damage membranes. Leukocidin forms a hetero-oligomeric transmembrane pore composed of four LukF and four LukS subunits, thereby forming an octameric pore in the affected membrane. Leukocidin is hemolytic, but less so than alpha hemolysin. Only 2% of all of S. aureus isolates express leukocidin, but nearly 90% of the strains isolated from severe dermonecrotic lesions express this toxin, which suggests that it is an important factor in necrotizing skin infections. Coagulase and clumping factor Coagulase is an extracellular protein which binds to prothrombin in the host to form a complex called staphylothrombin. The protease activity characteristic of thrombin is activated in the complex, resulting in the conversion of fibrinogen to fibrin. Coagulase is a traditional marker for identifying S aureus in the clinical microbiology laboratory. However, there is no overwhelming evidence that it is a virulence factor, although it is reasonable to speculate that the bacteria could protect themselves from phagocytic and immune defenses by causing localized clotting. There is some confusion in the literature concerning coagulase and clumping factor, the fibrinogen-binding determinant on the S. aureus cell surface. Partly the confusion results from the fact that a small amount of coagulase is tightly bound on the bacterial cell surface where it can react with prothrombin leading to fibrin clotting. However, genetic studies have shown unequivocally that coagulase and clumping factor are distinct entities. Specific mutants lacking coagulase retain clumping factor activity, while clumping factor mutants express coagulase normally. Staphylokinase Many strains of S aureus express a plasminogen activator called staphylokinase. This factor lyses fibrin. The genetic determinant is associated with lysogenic bacteriophages. A complex formed between staphylokinase and plasminogen activates plasmin-like proteolytic activity which causes dissolution of fibrin clots. The mechanism is identical to streptokinase, which is used in medicine to treat patients suffering from coronary thrombosis. As with coagulase, there is no strong evidence that staphylokinase is a virulence factor, although it seems reasonable to imagine that localized fibrinolysis might aid in bacterial spreading. Other extracellular enzymes S. aureus can express proteases, a lipase, a deoxyribonuclease (DNase) and a fatty acid modifying enzyme (FAME). The first three probably provide nutrients for the bacteria, and it is unlikely that they have anything but a minor role in pathogenesis. However, the FAME enzyme may be important in abscesses, where it could modify anti-bacterial lipids and prolong bacterial survival. Possible virulence determinants expressed in the pathogenesis of Staphylococcus aureus infections boils and pimples (folliculitis) Colonization: cell-bound (protein) adhesins Invasion: Invasins: staphylokinase Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.) Resistance to phagocytosis: coagulase, leukocidin Resistance to immune responses: coagulase Toxigenesis: cytotoxic toxins (hemolysins and leukocidin)

pneumonia Colonization: cell-bound (protein) adhesins Invasion: Invasins: staphylokinase, hyaluronidase Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.) Resistance to phagocytosis: coagulase, leukocidin, hemolysins, carotenoids, superoxide dismutase, catalase, growth at low pH Resistance to immune responses: coagulase, antigenic variation Toxigenesis: Cytotoxic toxins (hemolysins and leukocidin) food poisoning (emesis or vomiting) Toxigenesis: Enterotoxins A-G septicemia (invasion of the bloodstream) Invasion: Invasins: staphylokinase, hyaluronidase Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.) Resistance to phagocytosis: coagulase, protein A, leukocidin, hemolysins, carotenoids, superoxide dismutase, catalase, growth at low pH Resistance to immune responses: coagulase, protein A, antigenic variation Toxigenesis: cytotoxic toxins (hemolysins and leukocidin) osteomyelitis (invasion of bone) Colonization: cell-bound (protein) adhesins Invasion: Invasins: staphylokinase, hyaluronidase Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.) Resistance to phagocytosis: coagulase, protein A, leukocidin, hemolysins, carotenoids, superoxide dismutase, catalase, growth at low pH Resistance to immune responses: coagulase, protein A, antigenic variation Toxigenesis: cytotoxic toxins (hemolysins and leukocidin) toxic shock syndrome Colonization: cell-bound (protein) adhesins Resistance to immune responses: coagulase, antigenic variation Toxigenesis: TSST toxin, Enterotoxins A-G surgical wound infections Colonization: cell-bound (protein) adhesins Invasion: Invasins: staphylokinase, hyaluronidase Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.) Resistance to phagocytosis: coagulase, protein A, leukocidin, hemolysins, carotenoids, superoxide dismutase, catalase, growth at low pH Resistance to immune responses: coagulase, protein A, antigenic variation Toxigenesis: cytotoxic toxins (hemolysins and leukocidin) scalded skin syndrome Colonization: cell-bound (protein) adhesins Invasion: Invasins: staphylokinase, hyaluronidase Other extracellular enzymes (proteases, lipases, nucleases, collagenase, elastase. etc.) Resistance to phagocytosis: coagulase, leukocidin, hemolysins Resistance to immune responses: coagulase, antigenic variation Toxigenesis: Exfoliatin toxin

Testing for CGD

There are a few different methods available for the laboratory testing of neutrophil function as a diagnostic test for CGD. The primary endpoint of lab testing in CGD is the assessment of the neutrophil oxidative burst since the molecular defects are confined to this enzyme complex. Tests for clinical diagnostic purposes in CGD include:

Reduction of Nitro Blue-Tetrazolium (NBT) performed on slides o The NBT test is a semiquantitative method for evaluating neutrophil oxidative burst dysfunction in CGD patients. o The panel on the right demonstrates that neutrophils ingest the dye, nitroblue tetrazolium, and in the presence of reactive oxygen species, the yellow colored NBT compound is converted to the purple-blue formazan compound. o The panel on the left demonstrates that neutrophils from CGD patients fail to generate reactive oxygen species and therefore, cannot reduce the yellow NBT dye and thus there is no change in the color produced. Measurement of ROS production by flow cytometry, fluorometry or chemiluminescence o The dihydrorhodamine flow cytometry based assay is the more commonly used diagnostic screening test for CGD in reference laboratories and larger medical centers. o This test is based on the principle that nonfluorescent DHR (dihydrorhodamine) 123 when phagocytosed by normal activated neutrophils (after stimulation with PMA phorbol myristate acetate) can be oxidized by hydrogen peroxide, produced during the activated neutrophil respiratory oxidative burst, to rhodamine 123, a green fluorescent compound, which can be detected by flow cytometry. o Therefore, the detected fluorescence is an indirect measure of neutrophil function and oxidative burst. The top left-hand panel demonstrates normal neutrophil activation resulting in a robust oxidative burst and a complete shift of the peak (containing the cells) to the right indicating DHR oxidation to rhodamine. o The top right panel depicts an X-linked CGD female carrier demonstrating two populations one with no fluorescent shift, ie no oxidative burst and conversion of DHR to rhodamine, and the second normal peak, showing a typical oxidative burst. This would be expected in carrier females of X-linked diseases, since one allele is mutant and the other allele is normal. CGD is one of the PIDs where there is significantly skewed lyonization (random X-chromosome inactivation) resulting in female carriers with a clinical phenotype of disease. While it has been reported that even the presence of ~10% neutrophils with normal oxidative burst is sufficient to prevent a clinical phenotype, sometimes that is not always the case and therefore, clinical correlations should be performed with the DHR assay results. o The lower left panel demonstrates the typical pattern of oxidative burst seen in patients with the autosomal recessive forms of CGD. This tends to be more challenging to interpret since there is not a complete absence of oxidative burst as is seen in the classic X-linked CGD patient (lower right panel). Rather, the autosomal recessive CGD patient shows at least a population of neutrophils with significantly reduced oxidative burst. The X-linked CGD patient does not usually have any neutrophils capable of mounting oxidative burst. o Below is another example of the flow cytometric pattern associated with DHR oxidation by neutrophils to measure oxidative burst. In the laboratory test done at Mayo, the neutrophils are identified based on their size and granularity, using forward and side scatter measurements on the flow cytometer. Left (Normal): The data in the 3 upper panels shows the identification of neutrophils in a whole blood sample along with the absence of any fluorescence in the unstimulated neutrophil population, which is in stark contrast to the lower panels, where the neutrophils on stimulation with PMA mount a strong oxidative burst with a complete right shift in fluorescence. Middle (X-Linked): This slide provides a typical example of flow data for neutrophil oxidative burst from a patient with X-linked CGD. In contrast to the previous slide, where the lower panel demonstrated a right shift of the neutrophils undergoing oxidative burst, there is absolutely no shift with the activated neutrophils indicating a complete lack of oxidative burst as would be expected from patients with the X-linked form of the disease. The 19 year-old male patient whose results are shown here had both the clinical features of CGD as well as a confirmed de novo mutation in the CYBB gene encoding the gp91phox protein by gene sequencing.

Right (Carrier): This slide represents the DHR flow assay data from a 23 year-old female patient with a known family history of X-linked CGD and a personal clinical history of CGD and probably ulcerative colitis with recurrent abscesses and granulomas. The flow results reveal a slightly unusual carrier phenotype with two populations of neutrophils one negative and the other positive for oxidative burst. In general, for carriers, the distribution of the positive and negative populations is 50% each, however, in this patient it was more of 30% to 70% positive to negative neutrophils for oxidative burst. However, the clinical history combined with the flow data clearly indicates a carrier female who also has a clinical phenotype due to skewed lyonization of the X-chromosome. Gene sequencing revealed the presence of a single copy (heterozygous) of the familial X-linked CYBB mutation.

Treatment of CGD
Treatment of CGD is typically managed using a triple regimen: Trimethoprim/sulfamethoxazole is prescibed to prevent bacterial infections Itraconazole is prescribed to prevent a fungal infection

Interferon- is prescribed for the prevention of infection

Interferon- (INF-), a therapy commonly used for treatment of X-linked CGD, enhances transcription of the CYBB gene encoding the subunit of NADPH oxidase (gp91phox). This results in stimulation of production of superoxide by normal neutrophils as well as by CGD neutrophils, especially when a mutation in gp91phox exists that decreases its transcription even though the coding region is intact. Once superoxide production is restored to 10% of normal levels, resistance to infection is greatly improved.

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The host microbicidal mechanisms (a) include the NOX2 (also known as CYBB) NADPH oxidase, the inducible NO synthase (iNOS), iron scavengers and exporters, such as lactoferrin and natural resistance-associated macrophage protein 1 (NRAMP1; also known as SLC11A1), plus antimicrobial peptides and proteins that permeabilize and degrade the bacteria. Bacterial defensive mechanisms (b) include modification of their surface to resist or break down antimicrobial peptides, and expression of enzymes, such as catalase, that convert reactive species to less harmful compounds or prevent recruitment of the protein complexes that synthesize reactive nitrogen species (RNS) or reactive oxygen species (ROS) (see the main text for details). SOD, superoxide dismutase.

Leukocyte recruitment involves their rolling, firm adhesion, lateral migration and transendothelial diapedesis, controlled by chemokines. Whereas soluble chemokines mediate direct leukocyte recruitment, chemokines immobilized on the surface of activated endothelial cells (ECs) through glycosaminoglycan (GAG) trigger leukocyte arrest through G proteincoupled receptors (GPCRs), which activate leukocyte integrins. Celldependent and lesion-stagedependent usage of chemokines and their receptors12 show the robustness and complexity of the chemokine system, providing a signature combination of chemokines in each stage to attract specific leukocyte subsets. The combined action of chemokines in leukocyte recruitment can result in synergistic effects, as for the CCL5-CXCL4 heteromer23. Whether chemokines can also mediate cell egress from atherosclerotic lesions remains unclear27 and could be inherent to the aortic transplantation model studied26, 28. Some chemokines and their receptors are involved in cell homeostasis, such as CX3CL1 and CX3CR1 (ref. 24) and CCL17 (ref. 25) in monocyte and Treg cell homeostasis, respectively. Interference with chemokine functions increasingly gains attention in cardiovascular drug research (blue boxes)7. Blocking chemokines (or their receptors) with small-molecule antagonists or antibodies or modulation of chemokine affinity for receptors or of GAG binding can reduce chemokine signaling. Tailored manipulation of heterophilic chemokine interactions may even be more promising, as it can leave normal physiological and immunological functions intact. SELPLG, selectin P ligand; SELP, selectin P; SELE, selectin E; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1.

FIGURE 12.1. Following injury and release of interleukin-1 (IL-1) and tumor necrosis factor a (TNF-a), the resting state of the endothelium is changed by the appearance of selectins on the cell surface. These selectins bind to the counter-receptor on circulating neutrophils, slowing the polymorphonuclear neutrophils (PMNs) to a rolling adhesion. The release of IL-8 macrophage inflammatory protein and monocyte chemotactic protein results in the activation of integrins on the neutrophil surface. The integrins bind tightly to intracellular adhesion molecules (ICAMS) on the endothelial cell surface. Diapedesis (transendothelial migration) follows, facilitated by plateletendothelial cell adhesion molecules (PECAM-1). Antagonism at any point can reduce the inflammatory process.