Julie Rae

Ischemic Stroke Injury Is Reduced in Mice Lacking a Functional NADPH Oxidase

BACKGROUND AND PURPOSE Free radicals account for a significant proportion of the brain damage that occurs during ischemic stroke. Using mutant mice (X-CGD) with a dysfunctional phagocytic NADPH oxidase, we investigated the role of this superoxide-generating enzyme as a mediator of the reperfusion injury in a mouse model of middle cerebral artery occlusion. METHODS Transient (2 hour) middle cerebral artery occlusion was performed in X-CGD or wild-type litter mates (8- to 10-week-old). After 22 hours of reperfusion, brains were harvested and infarct volume delineated using 2,3,5-triphenyl-tetrazolium chloride. To elucidate the origin of the damaging NADPH oxidase, transient ischemia was also performed in X-CGD or wild-type mice transplanted with wild-type C57 B1/6J or X-CGD bone marrow, respectively. RESULTS The infarct volume induced by transient ischemia was significantly less in X-CGD mice (29.1 +/- 5.6 mm3; n = 13) than wild-type littermates (54.0 +/- 10.6 mm3; n = 10; P < .05). The elimination of a functional NADPH oxidase from either the circulation or the central nervous system, by performing the appropriate bone marrow transplant experiments, did not reduce the infarct size induced by transient ischemia. This suggests that in order to confer protection against transient ischemia and reperfusion, a putative neuronal and circulating NADPH oxidase need to be inactivated. CONCLUSIONS Brain injury was reduced in mice lacking a functional NADPH oxidase in both the central nervous system and peripheral leukocytes, suggesting a pivotal role for the NADPH oxidase in the pathogenesis of ischemia-reperfusion injury in the brain.

Host defense molecule polymorphisms influence the risk for immune-mediated complications in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited disorder of phagocyte function in which defective superoxide production results in deficient microbicidal activity. CGD patients suffer from recurrent, life-threatening infections, and nearly half develop chronic gastrointestinal (GI) complications (colitis, gastric outlet obstruction, or perirectal abscess) and/or autoimmune/rheumatologic disorders (AIDs). To identify genetic modifiers of disease severity, we studied a cohort of 129 CGD patients, in whom seven candidate genes (myeloperoxidase [MPO], mannose binding lectin [MBL], Fcgamma receptors IIa, IIIa, IIIb, TNF-alpha, and IL-1 receptor antagonist), each containing a physiologically relevant polymorphism predicted to influence the host inflammatory response, were selected for analysis. Genotypes of MPO (P = 0.003) and FcgammaRIIIb (P = 0.007) were strongly associated with an increased risk for GI complications, while an FcgammaRIIa (P = 0.05) genotype was suggestive for an association. Patients with all three associated genotypes had the highest risk for GI complications (P < 0.0001). The risk of AIDs was strongly associated with variant alleles of MBL (P = 0.01) and weakly associated with an FcgammaRIIa genotype (P = 0.04). Patients with variant forms of both MBL and FcgammaRIIa had the highest risk of developing an AID (P = 0.003).

Induction of gp91-phox, a component of the phagocyte NADPH oxidase, in microglial cells during central nervous system inflammation.

Gp91-phox is an integral component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex that generates reactive oxygen species (ROS) in activated circulating phagocytes. The authors previously demonstrated that gp91-phox knockout (KO) mice show significant protection from neuronal injury after cerebral ischemia–reperfusion injury, suggesting a pivotal role for this enzyme. Moreover, results from chimeric mice suggested that elimination of gp91-phox from both circulating phagocytes and a putative central nervous system (CNS) source were required to confer neuroprotection. In the current study, the authors demonstrated gp91-phox–specific immunostaining of perivascular cells in the CNS of control rats. However, after transient cerebral ischemia, gp91-phox–positive phagocytes were observed within the core ischemic region and activated microglial cells were positive in the penumbra. Such activated microglial cells were also gp91-phox–positive in the CNS of a chimpanzee with mild meningitis. Finally, in humans, both normal adult CNS tissues and isolated fetal microglial cells expressed gp91-phox mRNA. These microglia also expressed mRNA for the five other known components that comprise the NADPH oxidase complex. These data strongly suggest that microglial cells may contain a functionally active NADPH oxidase capable of generating ROS during CNS inflammation.

Cytochrome b-245 of the neutrophil superoxide-generating system contains two nonidentical hemes. Potentiometric studies of a mutant form of gp91phox.

Analysis of potentiometric titrations of the cytochrome b from a X chronic granulomatous disease patient with an Arg Ser mutation in gp91 indicates that the mutant form of the cytochrome contains two nonidentical hemes with midpoint potentials of E = −220 and E = −300 mV. In the light of this information, reanalysis of redox titrations of wild-type cytochrome b implies that it probably also contains two separate heme centers with midpoint potentials of E = −225 and E = −265 mV. The effect of the Arg Ser substitution is to reduce the midpoint potential of one of the heme centers by approximately 35 mV and suggests possible interaction between Arg and a heme propionate side chain.

Chronic granulomatous disease caused by a deficiency in p47phox mimicking Crohn’s disease

Abstract We describe 2 cases of autosomal recessive chronic granulomatous disease (CGD) in 2 sisters presenting with a picture consistent with inflammatory bowel disease. The index case is a 10-year-old girl with a history of refractory Crohns colitis treated with aggressive immunosuppressive therapy whose course subsequently was complicated by central nervous system aspergillosis. Additional evaluation showed a diagnosis of CGD, an underlying immunodeficiency in which phagocytes fail to produce microbicidal reactive oxygen intermediates because of inherited defects in the reduced form of nicotinamide-adenine phosphate dinucleotide (NADPH) oxidase. The diagnosis of a typically X-linked inherited disease in our female patient suggested that she had 1 of the 3 less common autosomal recessive forms of the disease. This was confirmed by studies showing the absence of the p47 phox subunit of NADPH oxidase in her neutrophils and the presence of a homozygous dinucleotide deletion in the neutrophil cytosolic factor 1 gene that encodes p47 phox . Additional analyses of members of the patients immediate family showed the same homozygous mutation in 2 siblings, 1 of whom also developed chronic colitis consistent with a diagnosis of Crohns disease. These 2 cases emphasize the importance of high clinical suspicion for an alternative diagnosis of immune deficiency in the setting of presumed inflammatory bowel disease and opportunistic infection.

Long polymerase chain reaction-based fluorescence in situ hybridization analysis of female carriers of X-linked chronic granulomatous disease deletions.

Chronic granulomatous disease (CGD) is a rare inherited disorder in which antimicrobial activity of phagocytes is impaired due to the lack of reactive oxygen species, or oxidative burst, produced by NADPH oxidase. The X-linked form of CGD, representing approximately 70% of all cases, is caused by mutations in the cytochrome b beta subunit (CYBB) gene, which maps to chromosome Xp21.1. CYBB encodes the gp91-phox protein, a necessary component in the NADPH oxidase pathway. A wide variety of mutations have been identified in X-linked CGD patients, all of which lead to deletion of the functional protein and no oxidative burst activity. The mutations vary from single nucleotide substitutions to deletions of the entire gene. In this article, we report a mutation detection method for probands of female relatives at risk for carrier status of large deletions of the CYBB gene. Through fluorescent in situ hybridization of metaphase chromosomes, we were able to consistently distinguish carriers from noncarriers using polymerase chain reaction-derived, labeled DNA specific for exons 2 to 13 of the CYBB region at Xp21.1.

Pre-clinical and translational pharmacology of a human interleukin-22 IgG fusion protein for potential treatment of infectious or inflammatory diseases

Graphical abstract Figure. No Caption available. ABSTRACT Interleukin (IL)‐22 plays protective roles in infections and in inflammatory diseases that have been linked to its meditation of innate immunity via multiple mechanisms. IL‐22 binds specifically to its heterodimeric receptor, which is expressed on a variety of epithelial tissues. UTTR1147A is a recombinant fusion protein that links the human cytokine IL‐22 with the Fc portion of human immunoglobulin (Ig) G4. Here, we report extensive in vitro and in vivo nonclinical studies that were conducted to characterize the pharmacological activity of UTTR1147A. The in vitro activity and potency of UTTR1147A were analyzed using primary human hepatocytes and human colonic epithelial cell lines. Assessment of in vivo efficacy was performed in a mouse colitis model and by measuring relevant pharmacodynamic biomarkers, including antimicrobial peptides REG3A/&bgr;, serum amyloid protein A (SAA) and lipopolysaccharide binding protein (LBP). The pharmacokinetic and pharmacodynamic characteristics of UTTR1147A were assessed in healthy mice, rats and cynomolgus monkeys. UTTR1147A induced STAT3 activation through binding to IL‐22 receptor expressed in primary human hepatocytes and human colon cell lines. In both, activation occurred in a concentration‐dependent manner with similar potencies. In the mouse colitis model, murine IL‐22Fc‐ (muIL‐22Fc) treated groups at doses of 1.25 &mgr;g and above had statistically lower average histologic colitis scores compared to the control treated group. Administration of muIL‐22Fc or UTTR1147A was associated with a dose‐dependent induction of PD markers REG3&bgr; and SAA in rodents as well as REG3A, SAA and LBP in cynomolgus monkeys. The combined data confirm pharmacological activity of IL‐22Fc and support potential regenerative and protective mechanisms in epithelial tissues.