Altered vascular activation due to deficiency of the NADPH oxidase component p22phox

BackgroundReactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play important roles in vascular activation. The p22^phox subunit is necessary for the activity of NADPH oxidase complexes utilizing Nox1, Nox2, Nox3, and Nox4 catalytic subunits.MethodsWe assessed p22^phox-deficient mice and human tissue for altered vascular activation.ResultsMice deficient in p22^phox were smaller than their wild-type littermates but showed no alteration in basal blood pressure. The wild-type littermates were relatively resistant to forming intimal hyperplasia following carotid ligation, and the intimal hyperplasia that developed was not altered by p22^phox deficiency. However, at the site of carotid artery ligation, the p22^phox-deficient mice showed significantly less vascular elastic fiber loss compared with their wild-type littermates. This preservation of elastic fibers was associated with a reduced matrix metallopeptidase (MMP) 12/tissue inhibitor of metalloproteinase (TIMP) 1 expression ratio. A similar decrease in the relative MMP12/TIMP1 expression ratio occurred in human coronary artery smooth muscle cells upon knockdown of the hydrogen peroxide responsive kinase CK1αLS. In the ligated carotid arteries, the p22^phox-deficient mice showed reduced expression of heterogeneous nuclear ribonucleoprotein C (hnRNP-C), suggesting reduced activity of CK1αLS. In a lung biopsy from a human patient with p22^phox deficiency, there was also reduced vascular hnRNP-C expression.ConclusionsThese findings indicate that NADPH oxidase complexes modulate aspects of vascular activation including vascular elastic fiber loss, the MMP12/TIMP1 expression ratio, and the expression of hnRNP-C. Furthermore, these findings suggest that the effects of NADPH oxidase on vascular activation are mediated in part by protein kinase CK1αLS.     

Gp91<Superscript>phox</Superscript> (NOX2) in classically activated microglia exacerbates traumatic brain injury

Background  

We hypothesized that gp91^phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O₂ ^-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91^phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91^phox knockout mice (gp91^phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91^phox generation.     

Three common polymorphisms in the CYBA gene form a haplotype associated with decreased ROS generation

NOX enzymes are reactive oxygen species (ROS)‐generating NADPH oxidases. Several members of the NOX family depend on the p22^phox subunit, encoded by the CYBA gene. CYBA is highly polymorphic, and has been widely studied as a potential risk factor for various diseases, with conflicting results. In the present study, we used Epstein‐Barr (EBV)‐transformed B‐lymphocytes from 50 healthy unrelated individuals to analyze their CYBA mRNA sequence and NOX2‐dependent ROS generation. Seven single‐nucleotide polymorphisms (SNPs) were identified (five previously described, two novel). The combination of these SNPs yielded 11 distinct haplotypes, which could be grouped into seven haplogroups (A–G). Haplogroup C (c.214T>C, c.521T>C, and c.^*24G>A) showed a significantly lower ROS generation, as compared to the most frequent haplogroup, A. CYBA variants from the seven haplogroups were transduced into p22^phox‐deficient B‐lymphocytes. The haplogroup C variant showed significantly lower ROS production. c.214T>C and c.521T>C lead to nonsynonymous codon changes, while c.^*24G>A lies within the 3′UTR. Using a luciferase/3′UTR construct, we showed that the ^*24A allele led to decreased reporter gene activity. These results help to unravel the complex nature of how genetic variations in CYBA influence NOX2 activity, and indicate that haplotypes, rather than individual SNPs, define the effect on ROS generation. Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Vasoactive intestinal peptide dampens formyl-peptide-induced ROS production and inflammation by targeting a MAPK-p47phox phosphorylation pathway in monocytes

Reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase (NOX2) are required for microbial clearance; however, when produced in excess they exacerbate inflammatory response and injure surrounding tissues. NOX2 is a multicomponent enzyme composed of membrane-associated cytochrome b588 and cytosolic components p47^phox, p67^phox, p40^phox, and rac1/2. We investigated whether vasoactive intestinal peptide (VIP), an endogenous immune-modulatory peptide, could affect ROS production by NOX2 in primary human phagocytes. VIP did not modulate basal ROS production by phagocytes, but it inhibited monocyte and not neutrophil ROS production in response to the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). The action of VIP was essentially mediated by high-affinity G-protein coupled receptors VPAC1 as its specific agonist, [ALA^11,22,28]VIP, mimicked VIP-inhibitory effect, whereas the specific VPAC1 antagonist, PG97-269, blunted VIP action. Further, we showed that VIP inhibited fMLF-induced phosphorylation of ERK1/2 (extracellular signal–regulated kinase 1/2), p38MAPK (p38 mitogen-activated protein kinase) pathways, and phosphorylation of p47^phox on Ser345 residue. Also, VIP exerted an anti-inflammatory effect in a model of carrageenan-induced inflammation in rats. We thus found that VIP exerts anti-inflammatory effects by inhibiting the “MAPK–p47^phox phosphorylation–NOX2 activation” axis. These data suggest that VIP acts as a natural anti-inflammatory agent of the mucosal system and its analogs could be novel anti-inflammatory molecules.     

The effect of gender and genetic polymorphisms on matrix metalloprotease (MMP) and tissue inhibitor (TIMP) plasma levels in different infectious and non‐infectious conditions

Matrix metalloproteases (MMPs) are increased in different infections due to their role in controlling immune responses and are regulated by tissue inhibitors (TIMPs). Different MMP promoter single nucleotide polymorphisms (SNPs) induce changes in MMP genes, mRNA and protein expression. Gender might also modify MMP plasma levels. In order to determine the weight of these variables on MMP secretion we studied MMP‐1, ‐2, ‐3, ‐8, ‐9, ‐10, ‐13 and TIMP‐1, ‐2, ‐4 plasma levels in 90 patients with severe bacterial sepsis, 102 with anti‐retroviral (ARV)‐treated HIV monoinfection, 111 with ARV‐treated HIV–hepatitis C virus (HCV) co‐infection and 86 non‐infected controls (45 stroke and 41 trauma patients). MMP‐1(‐1607 1G/2G), MMP‐3(‐1612 5A/6A), MMP‐8(‐799C/T), MMP‐9(‐1562 C/T) and MMP‐13(‐77A/G) SNPs were genotyped. MMP‐3 plasma levels were significantly higher in men than in women in each diagnostic group, and MMP‐3 SNP allele 6A carriers also had higher levels than allele 5A carriers, an effect that was magnified by sepsis. Independent predictors of higher MMP‐3 levels were male gender (P = 0·0001), MMP‐3(‐1612 5A/6A) SNP (P = 0·001), higher levels of TIMP‐4 (P = 0·004) and MMP‐8 (P = 0·006) and lower levels of MMP‐1 (P = 0·03) by multivariate analysis. No strong associations with gender or SNPs were observed for other MMPs or TIMPs. In conclusion, male gender and MMP‐3(‐1612 5A/6A) 6A allele carriage increased MMP‐3 plasma levels significantly, especially in patients with severe bacterial sepsis. This confounding gender effect needs to be addressed when evaluating MMP‐3 plasma levels in any infectious or non‐infectious condition.     

Emerging role of oxidative stress in metabolic syndrome and cardiovascular diseases: important role of Rac/NADPH oxidase

'Oxidative stress' is a term defining states of elevated reactive oxygen species (ROS) levels. Normally, ROS control several physiological processes, such as host defence, biosynthesis of hormones, fertilization and cellular signalling. However, oxidative stress has been involved in different pathologies, including metabolic syndrome and numerous cardiovascular diseases. A major source of ROS involved in both metabolic syndrome and cardiovascular pathophysiology is the NADPH oxidase (NOX) family of enzymes. NOX is a multi‐component enzyme complex that consists of membrane‐bound cytochrome b‐558, which is a heterodimer of gp91phox and p22phox, cytosolic regulatory subunits p47phox and p67phox, and the small GTP‐binding protein Rac1. Rac1 plays many important biological functions in cells, but perhaps the most unique function of Rac1 is its ability to bind and activate the NOX complex. Furthermore, Rac1 has been reported to be a key regulator of oxidative stress through its co‐regulatory effects on both nitric oxide (NO) synthase and NOX. Therefore, the main goal of this review is to give a brief outline about the important role of the Rac1–NOX axis in the pathophysiology of both metabolic syndrome and cardiovascular disease. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Study of matrix metalloproteinases and their tissue inhibitors in ductal in situ carcinomas of the breast

Aims: To analyse the expression of metalloproteinases (MMPs) and their inhibitors (TIMPs) in ductal carcinoma in situ of the breast (DCIS). Methods and results: An immunohistochemical study was performed in 56 patients with pure DCIS, in 39 with DCIS adjacent to invasive carcinoma (IDC) and 63 patients with T1 IDC, using tissue microarrays and specific antibodies against MMPs and TIMPs. Immunohistochemical results were categorized using a specific software program. The data were analysed by unsupervised hierarchical cluster analysis by each cellular type. IDC showed a higher expression rate of MMP‐7 and TIMP‐1 than pure DCIS, as well as a higher expression rate of MMP‐9 and TIMP‐3 than the DCIS component of mixed cases, whereas pure DCIS showed a higher rate of expression of MMP‐9 and ‐11 and TIMP‐3 than in the DCIS component of mixed cases. Pure DCIS with a periductal inflammatory infiltrate showed significantly higher MMP‐2, ‐14 and TIMP‐1. Dendograms identified two cluster groups with distinct MMP/TIMP expression profiles in neoplastic cells and fibroblastic or mononuclear inflammatory cells surrounding the neoplastic ducts of pure DCIS. Conclusions: The results indicate the distinct variability in MMP/TIMP expression by DCIS, which may be of potential biological and clinical interest in breast cancer.     

Macrophage‐specific NOX2 contributes to the development of lung emphysema through modulation of SIRT1/MMP‐9 pathways

Reactive oxygen species (ROS) participate in the pathogenesis of emphysema. Among ROS‐producing enzymes, NOX NADPH oxidases are thought to be responsible for tissue injury associated with several lung pathologies. To determine whether NOX2 and/or NOX1 participate in the development of emphysema, their expression patterns were first studied by immunohistochemistry in the lungs of emphysematous patients. Subsequently, we investigated their contribution to elastase‐induced emphysema using NOX2‐ and NOX1‐deficient mice. In human lung, NOX2 was mainly detected in macrophages of control and emphysematous lungs, while NOX1 was expressed in alveolar epithelium and bronchial cells. We observed an elevated number of NOX2‐positive cells in human emphysematous lungs, as well as increased NOX2 and NOX1 mRNA expression in mouse lungs following elastase exposure. Elastase‐induced alveolar airspace enlargement and elastin degradation were prevented in NOX2‐deficient mice, but not in NOX1‐deficient mice. This protection was independent of inflammation and correlated with reduced ROS production. Concomitantly, an elevation of sirtuin 1 (SIRT1) level and a decrease of matrix metalloproteinase‐9 (MMP‐9) expression and activity were observed in alveolar macrophages and neutrophils. We addressed the specific role of macrophage‐restricted functional NOX2 in elastase‐induced lung emphysema using Ncf1 mutant mice and Ncf1 macrophage rescue mice (Ncf1 mutant mice with transgenic expression of Ncf1 only in CD68‐positive mononuclear phagocytes; the MN mouse). Compared to WT mice, the lack of functional NOX2 led to decreased elastase‐induced ROS production and protected against emphysema. In contrast, ROS production was restored specifically in macrophages from Ncf1 rescue mice and contributes to emphysema. Taken together, our results demonstrate that NOX2 is involved in the pathogenesis of human emphysema and macrophage‐specific NOX2 participates in elastase‐induced emphysema through the involvement of SIRT1/MMP‐9 pathways in mice. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Epithelial-to-mesenchymal transition in podocytes mediated by activation of NADPH oxidase in hyperhomocysteinemia

The present study tested the hypothesis that hyperhomocysteinemia (hHcys) induces podocytes to undergo epithelial-to-mesenchymal transition (EMT) through the activation of NADPH oxidase (Nox). It was found that increased homocysteine (Hcys) level suppressed the expression of slit diaphragm-associated proteins, P-cadherin and zonula occludens-1 (ZO-1), in conditionally immortalized mouse podocytes, indicating the loss of their epithelial features. Meanwhile, Hcys remarkably increased the abundance of mesenchymal markers, such as fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA). These phenotype changes in podocytes induced by Hcys were accompanied by enhanced superoxide ( \textO₂ ^·- {\text{O}}_2^{{ \cdot - }} ) production, which was substantially suppressed by inhibition of Nox activity. Functionally, Hcys significantly enhanced the permeability of the podocyte monolayer coupled with increased EMT, and this EMT-related increase in cell permeability could be restored by Nox inhibitors. In mice lacking gp91 ^phox (gp91^−/−), an essential Nox subunit gene, hHcys-enhanced podocyte EMT and consequent glomerular injury were examined. In wild-type (gp91^+/+) mice, hHcys induced by a folate-free diet markedly enhanced expression of mesenchymal markers (FSP-1 and α-SMA) but decreased expression of epithelial markers of podocytes in glomeruli, which were not observed in gp91^−/− mouse glomeruli. Podocyte injury, glomerular sclerotic pathology, and marked albuminuria observed in gp91^+/+ mice with hHcys were all significantly attenuated in gp91^−/− mice. These results suggest that hHcys induces EMT of podocytes through activation of Nox, which represents a novel mechanism of hHcys-associated podocyte injury.     

Hyperpolarized δ‐[1‐13C]gluconolactone as a probe of the pentose phosphate pathway

The pentose phosphate pathway (PPP) is thought to be upregulated in trauma (to produce excess NADPH) and in cancer (to provide ribose for nucleotide biosynthesis), but simple methods for detecting changes in flux through this pathway are not available. MRI of hyperpolarized ¹³C–enriched metabolites offers considerable potential as a rapid, non‐invasive tool for detecting changes in metabolic fluxes. In this study, hyperpolarized δ‐[1‐¹³C]gluconolactone was used as a probe to detect flux through the oxidative portion of the pentose phosphate pathway (PPP_ox) in isolated perfused mouse livers. The appearance of hyperpolarized (HP) H¹³CO₃^? within seconds after exposure of livers to HP‐δ‐[1‐¹³C]gluconolactone demonstrates that this probe rapidly enters hepatocytes, becomes phosphorylated, and enters the PPP_ox pathway to produce HP‐H¹³CO₃^? after three enzyme catalyzed steps (6P–gluconolactonase, 6‐phosphogluconate dehydrogenase, and carbonic anhydrase). Livers perfused with octanoate as their sole energy source show no change in production of H¹³CO₃^? after exposure to low levels of H₂O₂, while livers perfused with glucose and insulin showed a twofold increase in H¹³CO₃^? after exposure to peroxide. This indicates that flux through the PPP_ox is stimulated by H₂O₂ in glucose perfused livers but not in livers perfused with octanoate alone. Subsequent perfusion of livers with non‐polarized [1,2‐¹³C]glucose followed by ¹H NMR analysis of lactate in the perfusate verified that flux through the PPP_ox is indeed low in healthy livers and modestly higher in peroxide damaged livers. We conclude that hyperpolarized δ‐[1‐¹³C]gluconolactone has the potential to serve as a metabolic imaging probe of this important biological pathway.     

BMP‐2 Regulates the Formation of Oral Sulcus in Mouse Tongue by Altering the Balance Between TIMP‐1 and MMP‐13

The aim of this study is to investigate whether BMP‐2 regulates the oral sulcus formation of mouse embryonic tongue by modifying the expression of TIMP and MMP. The BMP‐2 siRNA induced a 180% increase in the depth of oral sulcus cavity (P < 0.01) by stimulating the invagination of oral sulcus into the mesenchymal tissues consisting of tongue floor, whereas the recombinant BMP‐2 suppressed the process in the organ culture system of mouse embryonic tongue. The BMP‐2 siRNA induced a 60% decrease in the expression of TIMP‐1 mRNA (P < 0.05) and a drastic decline in TIMP‐1 protein was observed around the oral sulcus in the BMP‐2 siRNA treated mandibles. The recombinant BMP‐2 induced a 220% increases in the expression of TIMP‐1 mRNA and the area of the immunostaining for TIMP‐1 around the oral sulcus was larger in the mandibles treated with the recombinant BMP‐2 than the vehicle. The BMP‐2 siRNA induced a 60% increase in the expression of MMP‐13 protein and a marked increase in the staining intensity for MMP‐13 was observed in the epithelial region of the BMP‐2 siRNA treated mandibles. The recombinant BMP‐2 induced a 70% decrease in the expression of MMP‐13 mRNA and the decrease was mainly observed in the tissues around oral sulcus. The expressions of BMP‐2, TIMP‐1, and MMP‐13 were verified in the tissues around in vivo developing oral sulcus at E11, 12, and 13 by immunohistochemistry. These results suggest that BMP‐2 regulates the formation of oral sulcus by altering the balance between TIMP‐1 and MMP‐13. Anat Rec 293:1408–1415, 2010. © 2010 Wiley‐Liss, Inc.     

阿托伐他汀通过抑制PKC的激活对抗高糖诱导的人脐静脉内皮细胞氧化应激反应

目的:探讨阿托伐他汀对高糖诱导的人脐静脉血管内皮细胞(HUVECs)产生氧化应激的影响及其作用机制。方法:体外培养HUVECs,以25 mmol/L葡萄糖干预,模拟糖尿病患者体内环境,通过流式细胞术和共聚焦显微镜检测细胞内的活性氧(ROS)水平,采用Lucigenin分析方法测定还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性,分别应用实时荧光定量PCR和免疫印迹杂交的方法检测 NADPH氧化酶亚基Nox4和Nox2/gp91^phox的表达水平,用免疫印迹杂交方法检测蛋白激酶C(PKC)蛋白的磷酸化水平。结果:(1)在高糖环境(终浓度为25 mmol/L)下,HUVECs内ROS生成显著增加,NADPH氧化酶的活性显著增强,NADPH 氧化酶Nox4和Nox2/gp91^phox亚基的mRNA和蛋白表达水平显著上调;(2)阿托伐他汀可显著抑制高糖诱导的ROS 生成、NADPH氧化酶活性的增强及NADPH 氧化酶Nox4和Nox2/gp91^phox亚基表达水平的增加幅度,且具有浓度依赖性;(3)PKC抑制剂(PKC inhibitor peptide, 20 μmol/L)可显著抑制高糖环境下ROS的生成、NADPH氧化酶活性的增强及NADPH 氧化酶Nox4和Nox2/gp91^phox亚基表达水平的增加幅度;(4)阿托伐他汀可抑制高糖诱导的PKC蛋白的磷酸化。结论:PKC的活化参与了高糖诱导的HUVECs产生的氧化应激反应。阿托伐他汀通过抑制PKC蛋白的活化对抗高糖诱导的内皮细胞产生的氧化应激反应。     

Does NAD(P)H oxidase-derived H<Subscript>2</Subscript>O<Subscript>2</Subscript> participate in hypotonicity-induced insulin release by activating VRAC in β-cells?

NAD(P)H oxidase (NOX)-derived H₂O₂ was recently proposed to act, in several cells, as the signal mediating the activation of volume-regulated anion channels (VRAC) under a variety of physiological conditions. The present study aims at investigating whether a similar situation prevails in insulin-secreting BRIN-BD11 and rat β-cells. Exogenous H₂O₂ (100 to 200 μM) at basal glucose concentration (1.1 to 2.8 mM) stimulated insulin secretion. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous H₂O₂. In patch clamp experiments, exogenous H₂O₂ was observed to stimulate NPPB-sensitive anion channel activity, which induced cell membrane depolarization. Exposure of the BRIN-BD11 cells to a hypotonic medium caused a detectable increase in intracellular level of reactive oxygen species (ROS) that was abolished by diphenyleneiodonium chloride (DPI), a universal NOX inhibitor. NOX inhibitors such as DPI and plumbagin nearly totally inhibited insulin release provoked by exposure of the BRIN-BD11 cells to a hypotonic medium. Preincubation with two other drugs also abolished hypotonicity-induced insulin release and reduced basal insulin output: 1) N-acetyl-L-cysteine (NAC), a glutathione precursor that serves as general antioxidant and 2) betulinic acid a compound that almost totally abolished NOX4 expression. As NPPB, each of these inhibitors (DPI, plumbagin, preincubation with NAC or betulinic acid) strongly reduced the volume regulatory decrease observed following a hypotonic shock, providing an independent proof that VRAC activation is mediated by H₂O₂. Taken together, these data suggest that NOX-derived H₂O₂ plays a key role in the insulin secretory response of BRIN-BD11 and native β-cells to extracellular hypotonicity.     

Oxidative Stress Produced with Cell Migration Increases Synthetic Phenotype of Vascular Smooth Muscle Cells

Phenotypic modulation of vascular smooth muscle cells (VSMC) and reactive oxygen species (ROS) is important in vascular pathogenesis. Understanding how these factors relate to cell migration can improve design of therapeutic interventions to control vascular disease. We compared the proliferation, protein content and migration of cultured aortic VSMC from wild type (WT) versus transgenic mice (Tg^p22phox), in which overexpression of p22phox was targeted to VSMC. Also, we compared H₂O₂ generation and expression of specific phenotypic markers of non-migrating with migrating WT versus Tg^p22phox VSMC in an in vitro wound scratch model. Enhanced H₂O₂ production in Tg^p22phox versus WT VSMC (p < 0.005) significantly correlated with increased protein content, proliferation, and migration. VSMC migrating across the wound edge produced more H₂O₂ than non-migrating VSMC (p < 0.05). The expression of synthetic phenotypic markers, tropomyosin 4 and myosin heavy chain embryonic (SMemb), was enhanced significantly, while the expression of contractile marker, smooth muscle α-actin, was reduced significantly in migrating versus non-migrating cells, and also in Tg^p22phox versus WT (p < 0.005) VSMC. These results are consistent with increased production of ROS accelerating the switch from the contractile to the synthetic phenotype, characterized by increases in proliferation, migration, and expression of TM4 and SMemb and decreased α-actin.     

Impaired host defence against Mycobacterium avium in mice with chronic granulomatous disease

Patients with chronic granulomatous disease (CGD), an inherited disorder of phagocytic cells, often contract recurrent life‐threatening bacterial and fungal infections. CGD is considered to arise from a functional defect of the O₂‐generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. To determine whether or not NADPH oxidase is crucial to the host defence against Mycobacterium avium, we investigated the response against M. avium using CGD model mice (gp91‐phox^‐) of C57BL/6 strain. A tracheal injection of 1 × 10⁷ colony‐forming units (CFU)/head of M. avium strain FN into the CGD mice resulted in a pulmonary infection, while also increasing the mortality rate. In contrast, normal C57BL/6 mice injected with same dose of the organisms did not develop severe pulmonary infection and were able to survive through 2 months of observation. The macrophages obtained from the CGD mice were observed to have a higher burden of the bacterial growth than macrophages from normal C57BL/6 mice. These results suggest that the defect of the NADPH oxidase function impairs the host defence against M. avium infection.     

Tread carefully: A functional variant in the human NADPH oxidase 4 (NOX4) is not disease causing

In this study, we report a paediatric patient with a lethal phenotype of respiratory distress, failure to thrive, pancreatic insufficiency, liver dysfunction, hypertrophic cardiomyopathy, bone marrow suppression, humoral and cellular immune deficiency. To identify the genetic basis of this unusual clinical phenotype and potentially make available the option of future prenatal testing, whole exome sequencing (WES) was used followed by functional studies in a bid to confirm pathogenicity. The WES we identified a homozygous novel variant, AK298328; c.9_10insGAG; p.[Glu3dup], in NOX4 in the proband, and parental heterozygosity for the variant (confirmed by Sanger sequencing). NADPH Oxidase 4 NOX4 (OMIM 605261) encodes an enzyme that functions as the catalytic subunit of the NADPH oxidase complex. NOX4 acts as an oxygen sensor, catalysing the reduction of molecular oxygen, mainly to hydrogen peroxide (H₂O₂).However, although, our functional data including 60% reduction in NOX4 protein levels and a 75% reduction in the production of H₂O₂ in patient fibroblast extracts compared to controls was initially considered to be the likely cause of the phenotype in our patient, the potential contribution of the NOX4 variant as the primary cause of the disease was clearly excluded based on following pieces of evidence. First, Sanger sequencing of other family members revealed that two of the grandparents were also homozygous for the NOX4 variant, one of who has fibromuscular dysplasia. Second, re-evaluation of more recent variant databases revealed a high allele frequency for this variant.Our case highlights the need to re-interrogate bioinformatics resources as they are constantly evolving, and is reminiscent of the short-chain acyl-CoA dehydrogenase deficiency (SCADD) story, where a functional defect in fatty acid oxidation has doubtful clinical ramifications.     

BCR‐induced superoxide negatively regulates B‐cell proliferation and T‐cell‐independent type 2 Ab responses

Superoxide and its derivatives have been implicated as secondary messenger molecules that influence signaling cascades in non‐phagocytes. B lymphocytes produce superoxide after BCR ligation. We found that these ROS regulate B‐cell signaling and entry into the cell cycle. B cells from mice deficient in the gp91^phox subunit of the NADPH oxidase complex are unable to generate ROS after BCR ligation. However, after BCR stimulation, more gp91^phox KO B cells enter the G1 stage of the cell cycle and proliferate than WT B cells. BCR ligation leads to a more rapid decrease in p27^Kip1 levels in gp91^phox KO B cells. Gp91^phox KO mice display enhanced T‐cell‐independent type 2, but normal T‐dependent Ab responses. ROS‐dependent regulation of BCR‐induced proliferation may help modulate the size of the humoral response to T‐cell‐independent type 2 Ag immunization.     

Glomerular ultrafiltration in rabbits with superficial glomeruli

The determinants of glomerular ultrafiltration in superficial glomeruli of a strain of English cross-breed rabbits have been studied using micropuncture techniques. Mean arterial blood pressure in the anaesthetised rabbits was 70±2 mmHg. The glomerular filtration rate in the kidney prepared for micropuncture was 4.4±0.4 ml/min, the filtration fraction was 22±1% and renal blood flow was 33±3 ml/min, and these values were comparable to values in conscious rabbits. Glomerular capillary pressure (P _gc) averaged 31±1 mmHg, the single-nephron glomerular filtration rate (SNGFR) averaged 25±2 nl/min, and the mean ultrafiltration pressure (calculated using the whole-kidney filtration fraction) averaged 7±1 mmHg. A net positive pressure at the efferent end of the glomerular capillaries (4.4±0.9 mmHg) indicated that a state of filtration pressure disequilibrium existed, under the experimental conditions of this study, in rabbit glomeruli. The calculated glomerular ultrafiltration coefficient (K _f) was 0.08±0.01 nl s^–1 mmHg^–1. Thus, compared to the Munich-Wistar rat, SNGFR is lower in the rabbit. This reflects the substantially lower glomerular ultrafiltration pressure in the rabbit, although this was offset partially by a higher K_f.