Two X-Linked Chronic Granulomatous Disease Patients with Unusual NADPH Oxidase Properties

Background

Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two X-linked CGD patients with mutations in gp91^phox that alter the regions in this membrane-bound NADPH oxidase component involved in docking of the cytosolic component p47^phox.

Materials and Methods

Hydrogen peroxide and superoxide generation, bactericidal activity, and NADPH oxidase protein expression by the patients?? neutrophils were measured, and genetic analysis was performed.

Results

We report two patients, each with a novel missense mutation in CYBB, the gene that encodes gp91^phox. Surprisingly, neutrophils from these patients showed total absence of superoxide production, although they retained 13?C30% of the hydrogen peroxide production capability. We speculate that this is due to direct electron transfer from flavin adenine dinucleotide (FAD) in gp91^phox to oxygen, leading to inefficient hydrogen peroxide formation instead of efficient superoxide production.

Conclusions

X-linked CGD patients with mutations that alter the gp91^phox protein in regions involved in docking of the cytosolic NADPH oxidase component p47^phox may have higher than expected hydrogen peroxide generation capability.     

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.     

Prenatal Diagnosis of X‐Linked Chronic Granulomatous Disease by Percutaneous Umbilical Blood Sampling

In this study, we investigated how to prenatally diagnose X‐linked chronic granulomatous disease (X‐CGD) effectively and accurately. Percutaneous umbilical blood sampling was conducted in the 22nd week of pregnancy. NADPH oxidase activity and gp91^phox protein expression of neutrophils were analysed using flow cytometry. Direct sequencing was used to detect CYBB gene mutations. Umbilical blood was obtained from seven foetuses whose mothers were X‐CGD carriers. Six foetuses, whose mothers needed prenatal diagnosis because of other diseases, were used as control. The neutrophils in all 13 foetuses showed lower hydrogen peroxide generation (stimulation index < 100) and gp91^phox protein expression. Among the seven foetuses whose mothers were X‐CGD carriers, four foetuses (Family 2, 4, 5 and 7) had CYBB gene mutations and showed very low hydrogen peroxide generation (stimulation index < 10) and no gp91^phox expression. The other three foetuses (Family 1, 3 and 6) had no CYBB gene mutations and showed stimulation index of 20–50 and partial or normal gp91^phox protein expression (no difference with controls). Two of the three mothers (Family 1 and 3) have delivered healthy infants with normal hydrogen peroxide generation and expression of gp91^phox in neutrophils. Combined with direct sequencing, dihydrorhodamine oxidation analysis and gp91^phox protein detection is an effective and accurate method for prenatal screening for X‐CGD.     

Baculovirus mediated expression of human phagocytic cell oxidase cytochrome b558 in sf9 insect cells

Chronic granulomatous disease (CGD) results from deficient production of components of the phagocyte NADPH oxidase. Most commonly affected is cytochrome b₅₅₈, a heterodimer composed of a 22-kDa protein (p22^phox noncovalently bound to a 91-kDa transmembrane glycoprotein (gp91^phox). CGD phagocytes lack both p22^phox and gp91^phox peptides when either gene is affected, suggesting that both peptides must be produced for individual subunit stability. Both genes have been cloned, but eukaryotic expression of recombinant gp91^phox has not been reported. To investigate the stability and interaction of cytochrome b₅₅₈ subunits, we introduced p22^phox and gp91^phox cDNA into recombinant baculoviruses. Recombinant gp91^phox (rgp91^phox) and p22^phox (rp22^phox) were detected individually and together in the same cells by in situ immunofluorescence and by SDS-PAGE immunoblotting of membranes from sf9 cells infected with baculovirus constructs. Formation of rp22^phox/ rgp91^phox complexes was demonstrated by coprecipitation using subunit-specific antibodies. This study demonstrates for the first time that cDNA encoding either subunit is capable of initiating production of stable recombinant cytochrome b₅₅₈ subunits in eukaryotic cells.     

NADPH oxidase inhibition improves neurological outcomes in surgically-induced brain injury

Neurosurgical procedures can result in brain injury by various means including direct trauma, hemorrhage, retractor stretch, and electrocautery. This surgically-induced brain injury (SBI) can cause post-operative complications such as brain edema. By creating a mouse model of SBI, we tested whether NADPH oxidase, an important reactive oxygen species producing enzyme, is involved in SBI using transgenic mice lacking gp91^phox subunit of NADPH oxidase (gp91^phox KO) and apocynin, a specific inhibitor of NADPH oxidase. Neurological function and brain edema were evaluated at 24 h post-SBI in gp91^phox KO and wild-type littermates grouped into SBI and sham-surgery groups. Alternatively, mice were grouped into vehicle- and apocynin-treated (5 mg/kg, i.p. 30 min before SBI) groups. Oxidative stress indicated by lipid peroxidation (LPO) was measured at 3 and 24 h post-SBI. The gp91^phox KO mice, but not the apocynin-treated mice showed significantly improved neurological scores. Brain edema was observed in both gp91^phox KO and wild-type groups after SBI; however, there was no significant difference between these two groups. Brain edema was also not affected by apocynin-pretreatment. LPO levels were significantly higher in SBI group in both gp91^phox KO and wild-type groups as compared to sham group. A trend, although without statistical significance, was noted towards attenuation of LPO in the gp91^phox KO animals as compared to wild-type group. LPO levels were significantly attenuated at 3 h post-SBI by apocynin-pretreatment but not at 24 h post-SBI. These results suggest that chronic and acute inhibition of NADPH oxidase activity does not reduce brain edema after SBI. Long-term inhibition of NADPH oxidase, however improves neurological functions after SBI.     

In vitro effects of Helicobacter pylori-induced infection in gastric epithelial AGS cells on microglia-mediated toxicity in neuroblastoma SH-SY5Y cells

Objective:  To investigate the in vitro effects of H. pylori-conditioned medium (HCM) from gastric epithelial AGS cell cultures on microglia and neuronal cells. Material:  H. pylori, human gastric epithelial AGS cells, microglia-like BV-2 cells and human neuroblastoma SH-SY5Y cells. Treatment:  Treated AGS cells with H. pylori at ratios from 1:100 to 1:900 for 24 h. Cultured BV-2 cells and SH-SY5Y cells were treated with HCM from AGS cell cultures. Methods:  Cell viability was measured by a quantitative colorimetric assay with MTT. Nitric oxide (NO) was determined by using Griess reagent. IL-8 was measured by an enzyme-linked immunosorbent assay. Protein expressions were revealed by western blot analysis. Results:  H. pylori increased IL-8, NO, COX-2 and gp91^phox in AGS cell cultures. When BV-2 cells were cocultured with AGS cells, HCM increased COX-2, gp91^phox, iNOS and NO of BV-2 cells. HCM also enhanced the degradation of IκBα in BV-2 cells. HCM up-regulated expression of nNOS, COX-2, and gp91^phox of SH-SY5Y cells co-cultured with BV-2 cells. Particularly, the decrease of cell viability of SH-SY5Y induced by HCM was dependent on the presence of BV-2 cells. Conclusions:  H. pylori-induced infection induces microglia-mediated inflammation and neurotoxicity. The present results suggest that microglia play a critical role in HCM-induced toxicity of neuronal SH-SY5Y cells. Received 15 April 2008; returned for revision 10 May 2008; received from final revision 14 September 2008; accepted by M. Katori 18 September 2008     

Regulatory T cell features in chronic granulomatous disease

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in any of the genes encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, responsible for the production of reactive oxygen species (ROS). CGD is marked by invasive bacterial and fungal infections and by autoinflammation/autoimmunity, of which the exact pathophysiology remains elusive. Contributing factors include decreased neutrophil apoptosis, impaired apoptotic neutrophil clearance, increased proinflammatory protein expression and reduced ROS-mediated inflammasome dampening. We have explored a fundamentally different potential mechanism: it has been reported that macrophage-mediated induction of regulatory T cells (T_regs) depends on ROS production. We have investigated whether numerical or functional deficiencies exist in T_regs of CGD patients. As the prevalence of autoinflammation/autoimmunity differs between CGD subtypes, we have also investigated T_regs from gp91^phox-, p47^phox- and p40^phox-deficient CGD patients separately. Results show that T_reg numbers and suppressive capacities are not different in CGD patients compared to healthy controls, with the exception that in gp91^phox-deficiency effector T_reg (eT_reg) numbers are decreased. Expression of T_reg markers CD25, inducible T cell co-stimulator (ICOS), Helios, cytotoxic T lymphocyte antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR) did not provide any clue for differences in T_reg functionality or activation state. No correlation was seen between eT_reg numbers and patients' clinical phenotype. To conclude, the only difference between T_regs from CGD patients and healthy controls is a decrease in circulating eT_regs in gp91^phox-deficiency. In terms of autoinflammation/autoimmunity, this group is the most affected. However, upon culture, patient-derived T_regs showed a normal phenotype and normal functional suppressor activity. No other findings pointed towards a role for T_regs in CGD-related autoinflammation/autoimmunity.     

Synthesis of two potential NK<Subscript>1</Subscript>-receptor ligands using [1-<Superscript>11</Superscript>C]ethyl iodide and [1-<Superscript>11</Superscript>C]propyl iodide and initial PET-imaging

Background

The previously validated NK₁-receptor ligand [O-methyl-¹¹C]GR205171 binds with a high affinity to the NK₁-receptor and displays a slow dissociation from the receptor. Hence, it cannot be used in vivo for detecting concentration changes in substance P, the endogenous ligand for the NK₁-receptor. A radioligand used for monitoring these changes has to enable displacement by the endogenous ligand and thus bind reversibly to the receptor. Small changes in the structure of a receptor ligand can lead to changes in binding characteristics and also in the ability to penetrate the blood-brain barrier. The aim of this study was to use carbon-11 labelled ethyl and propyl iodide with high specific radioactivity in the synthesis of two new and potentially reversible NK₁-receptor ligands with chemical structures based on [O-methyl-¹¹C]GR205171.

Methods

[1-¹¹C]Ethyl and [1-¹¹C]propyl iodide with specific radioactivities of 90 GBq/μmol and 270 GBq/μmol, respectively, were used in the synthesis of [O-methyl-¹¹C]GR205171 analogues by alkylation of O-desmethyl GR205171. The brain uptake of the obtained (2S,3S)-N-(1-(2- [1-¹¹C]ethoxy-5-(3-(trifluoromethyl)-4H-1,2,4-triazol-4-yl)phenyl)ethyl)-2-phenylpiperidin-3-amine (I) and (2S,3S)-2-phenyl-N-(1-(2- [1-¹¹C]propoxy-5-(3-(trifluoromethyl)-4H-1,2,4-triazol-4-yl)phenyl)ethyl)piperidin-3-amine (II) was studied with PET in guinea pigs and rhesus monkeys and compared to the uptake of [O-methyl-¹¹C]GR205171.

Results

All ligands had similar uptake distribution in the guinea pig brain. The PET-studies in rhesus monkeys showed that (II) had no specific binding in striatum. Ligand (I) had moderate specific binding compared to the [O-methyl-¹¹C]GR205171. The ethyl analogue (I) displayed reversible binding characteristics contrary to the slow dissociation rate shown by [O-methyl-¹¹C]GR205171.

Conclusion

The propyl-analogue (II) cannot be used for detecting changes in NK₁-ligand levels, while further studies should be performed with the ethyl-analogue (I).

     

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.     

Reactive oxygen and nitrogen species in sepsis-induced hepatic microvascular dysfunction

Objective and design

Hepatic microvascular dysfunction is a critical event in the development of liver failure during sepsis. Activated blood cells and reactive oxygen and nitrogen species (RONS) have been implicated in the pathogenesis of sepsis.

Methods

Intravital-videomicroscopy was used to determine whether RONS contribute to the recruitment of leukocytes/platelets in the hepatic microvasculature during sepsis. Six hours following cecal-ligation and puncture (CLP), disturbances of the hepatic microvasculature were assessed in WT-mice (C57Bl/6 J; n = 8), in mice lacking gp91^phox(n = 5), overexpressing superoxide-dismutase (SOD, n = 8), in WT-mice treated with a NOS-inhibitor (l-NAME, n = 5), lacking nNOS, eNOS or iNOS (n = 5 each), treated with the NO-donor DetaNO (n = 5), in WT-mice treated with gadolinium-chloride (GdCl₂, n = 5) and compared to a group of WT-mice following a sham operation (n = 8). Six hours post-CLP, the adhesion of leukocytes and platelets in terminal hepatic venules (THV) and sinusoids was quantified.

Results

In WT-mice, CLP elicited increases in the number of adherent leukocytes and platelets. Similar responses to CLP were noted in mice overexpressing SOD or lacking either eNOS or gp91^phox. The blood-cell recruitment was significantly blunted in septic iNOS-knockout mice and this response was reversed by pre-treatment with DetaNO.

Conclusion

These findings suggest that iNOS-derived NO is a determinant of the pro-inflammatory phenotype assumed by the hepatic microvasculature during sepsis.     

Quantitative differences in lipid raft components between murine CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells

Background  

Lipid rafts have been shown to play a role in T cell maturation, activation as well as in the formation of immunological synapses in CD4⁺ helper and CD8⁺ cytotoxic T cells. However, the differential expression of lipid raft components between CD4⁺ and CD8⁺ T cells is still poorly defined. To examine this question, we analyzed the expression of GM1 in T cells from young and aged mice as well as the expression of the glycosylphosphatidylinositol (GPI)-linked protein Thy-1 and cholesterol in murine CD4⁺ and CD8⁺ T cell subpopulations.     

Expansion of CD11b<Superscript>+</Superscript>Ly6G<Superscript>high</Superscript> and CD11b<Superscript>+</Superscript>CD49d<Superscript>+</Superscript> myeloid cells with suppressive potential in mice with chronic inflammation and light-at-night-induced circadian disruption

Objective

Myeloid-derived suppressor cells (MDSCs) are important negative regulators of immune processes in cancer and other pathological conditions. We suggested that MDSCs play a key role in pathogenesis of chronic inflammation, which precedes and, to a certain extent, induces carcinogenesis. The present study aimed at investigation of MDSCs arising during chronic inflammation and light-at-night (LN)-induced stress, which is shown to accelerate chronic diseases.

Subjects

67 CD-1 mice and in vitro MDSC cultures.

Treatment

Adjuvant arthritis was induced by a subdermal injection of complete Freund’s adjuvant. LN was induced by illumination of 750 lx at night.

Methods

Flow cytometry for evaluation of cell phenotypes and MTT standard test for cell proliferation were used.

Results

Increased levels of splenic CD11b⁺Ly6G^high and CD11b⁺CD49d⁺ myeloid cells possessing suppressive potential in mice with adjuvant arthritis are shown. LN amplifies the process of CD11b⁺Ly6G^high expansion in mice with adjuvant arthritis. Expression of CD62L and CD195 is elevated on the myeloid cells during exposure to LN.

Conclusions

Our study raises the possibility that CD11b⁺Ly6G^high and CD11b⁺CD49d⁺ MDSCs play an important role in the induction of immunosuppressive environment typical for chronic inflammation. Also, LN can affect immune responses during chronic inflammation through recruitment of MDSCs from the bone marrow.

     

Tubular epithelial cells have the capacity to transdifferentiate into CD68-positive macrophage-like cells by oxidative stress

Objective:  The present study was intended to assess transdifferentiation from tubular epithelial cells to macrophage- like cells. Methods:  Puromycin aminonucleoside nephrotic rats were sacrificed at days 4, 8, 24 and 112. We immunohistochemically evaluated CD68, CD163, and cytokeratin AE1/AE3, known as markers for macrophages and tubular epithelial cells. Nitrotyrosine, gp91^phox and Rac 1 expressions was also analyzed. CD68 expression in cultured murine proximal tubular epithelial cells (mProx) stimulated by crude and pure BSA was examined by flow cytometry and immunofluorescence. Results:  The tubular CD68-positive cells were observed on day 112. Immunoelectronmicroscopy revealed that some CD68-positive cells showed brush borders on the cell membrane and some of cytokeratin-positive tubular cells also expressed CD163 in mirror sections. The tubular CD68-positive cells were also positive for nitrotyrosine, gp91 ^phox and Rac 1. They contained lipid in their cytoplasm. Crude BSA, containing free fatty acid, induced CD68 expression in a dose- and time-dependent manner in mProx, but not pure BSA. The surface expression of CD68 was increased by high dose and long term stimulation with crude BSA as shown by immunofluorescence. Conclusions:  We confirmed that tubular epithelial cells have the capacity to transdifferentiate to CD68-positive macrophage-like cells, which may be linked to oxidative stress. Received 10 September 2006; returned for revision 4 November 2007; received from final revision 21 July 2008; accepted by M. Katori 8 August 2008     

Human thymic stromal lymphopoietin enhances expression of CD80 in human CD14<Superscript>+</Superscript> monocytes/macrophages

Objective  

Human thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine, promotes inflammatory T helper type 2 cell (Th2) differentiation of naive CD4⁺ T cells. TSLP is highly produced in keratinocytes of patients with atopic dermatitis and bronchial epithelia of patients with asthma and was thought to be a master switch for allergic inflammation. We sought to examine the effect of TSLP in human monocytes/macrophages.     

Reduced CD27 Expression on Antigen-Specific CD4<Superscript>+</Superscript> T Cells Correlates with Persistent Active Tuberculosis

Objective  

CD27, a member of the tumor necrosis factor receptor family, has important role in generation of T cell immunity. In this study, association of CD27 expression on mycobacterial antigen-specific CD4⁺ T cells with pulmonary tuberculosis (TB) was investigated.     

Superoxide production and NADPH oxidase expression in human rheumatoid synovial cells: regulation by interleukin-1β and tumour necrosis factor-α

Objectives to evaluate the rheumatoid synovial cell capacity to produce superoxide anion in response to interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α), and to study the NADPH oxidase involvement in this production. Material and Methods Synovial cells obtained from 7 rheumatoid arthritis (RA), 5 osteoarthritic (OA) patients, and dermal fibroblasts, were stimulated (i) with IL-1β and TNF-α, or (ii) with specific oxidase activators and inhibitors, before studying superoxide production; we also studied NADPH oxidase mRNAs and protein expression, and p47-phox phosphorylation. Results Constitutive superoxide production by RA cells was increased in comparison to OA cells and dermal fibroblasts, and was stimulated by PMA and ionomycin. This production was increased after cytokine treatment of RA synovial cells. Cytokine-induced superoxide production by RA cells was inhibited by iodonium diphenyl or apocynin, suggesting the involvement of NADPH oxidase. RT-PCR and western blot analysis revealed the presence of p47-phox, gp91-phox and Nox4 in RA and OA cells, and in dermal fibroblasts. P47-phox phosphorylation was enhanced after cytokine-treatment in RA and OA cells, suggesting a PKC-mediated up-regulation of NADPH oxidase. Conclusions NADPH oxidase is involved in the superoxide release by RA synovial cells, constitutively and after cytokine up-regulation. These cells express two different homologues (gp91-phox and Nox4). Received 2 August 2005; returned for revision 12 January 2006; returned for final revision 22 May 2006; accepted by J. Di Battista 9 June 2006     

Diabetes, Renal and Cardiovascular Disease in p47 phox−/− Chronic Granulomatous Disease

Chronic granulomatous disease is a rare immunodeficiency due to defects in the phagocyte NADPH oxidase. The X-linked form (gp91 ^phox deficiency) accounts for about 70 % of cases; autosomal recessive p47 ^phox deficiency accounts for about 25 % of cases. We identified a 10 % incidence of diabetes in p47 ^phox deficient CGD, but none in X-linked CGD. Renal and cardiovascular diseases were also higher in p47 ^phox deficiency. p47 ^phox deficient CGD has non-infectious morbidities distinct from those in X-linked CGD.     

Cardiac oxidative stress and remodeling following infarction: role of NADPH oxidase

BackgroundThere is growing recognition that oxidative stress plays a role in the pathogeneses of myocardial repair/remodeling following myocardial infarction (MI). Nicotinamide adenine denucleotide phosphate (NADPH) oxidase is a major source for cardiac reactive oxygen species production. Herein, we studied the importance of NADPH oxidase in development of cardiac oxidative stress and its induced molecular and cellular changes related to myocardial repair/remodeling.MethodsMI was created by coronary artery ligation in C57/BL (wild type) and NADPH oxidase (gp91^phox) knockout mice. Cardiac oxidative stress, inflammatory/fibrogenic responses, apoptosis, and hypertrophy were detected by in situ hybridization, immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), picrosirius red staining, and image analysis, respectively, at different stages post MI.ResultsIn wild-type mice with MI, and compared to sham-operated animals, we observed significantly increased gp91^phox and 3-nitrotyrosine, a marker of oxidative stress, in the infarcted myocardium; accumulated macrophages and myofibroblasts at the infarct site; abundant apoptotic myocytes primarily at border zones on Day 3; and numerous apoptotic inflammatory/myofibroblasts in the later stages. In addition, we detected significantly increased transforming growth factor β1, tissue inhibitor of metalloprotease 2, and type 1 collagen gene expression; continuously increasing collagen volume in the infarcted myocardium; and hypertrophy in noninfarcted myocardium. Compared to wild-type mice with MI, we did not observe significant difference in infarct size/thickness, cardiac hypertrophy, myocyte apoptosis, inflammatory/fibrogenic responses, as well as cardiac oxidative stress in gp91^phox knockout mice.ConclusionOur findings indicate that during NADPH oxidase deficiency, superoxide production can be compensated by other sources, which leads to cardiac oxidative stress and its related molecular/cellular events in the infarcted heart.     

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。     

KRN/I-A<Superscript>g7</Superscript> Mouse Arthritis Is Independent of Complement C3

Background  

KRN/I-A^g7 (KxB/N) is a mouse model of inflammatory arthritis, which resembles human rheumatoid arthritis. Arthritis in these animals is caused by autoreactivity to a ubiquitously expressed autoantigen, glucose-6 phosphate isomerase. Tolerance is broken at both the T cell and B cell level. The sera from KRN/I-A^g7 mice can induce mouse arthritis in healthy mice. Complement components of the alternative complement pathway, including C3, have been shown to be required in induction of mouse arthritis by serum transfer.