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Temozolomide a recent, oral, second generation alkylating agent is a chemotherapeutic with demonstrated efficacy for the treatment of high-grade gliomas; its efficacy has been demonstrated in both pre-clinical and phase I and II studies. The goal of this study is to determine the activity and safety of temozolomide in improving overall survival (OS), progression-free survival (PFS) and health-related quality of life (HQL) in patient with malignant gliomas.Forty-two patients with newly diagnosed glioblastoma, anaplastic astrocytoma and anaplastic oligodendroglioma were studied. The mean follow-up period was 12 months. The overall response rate (only responsive patient) for all histological groups was 40%, 10 patients (24%) showed a stabilization of disease. The median PFS and OS was respectively 8.35 and 14.1 months; time to progression was 34 week ranging from 21 to 47. In all patients, treatment with temozolomide was associated with improvement of performance status including the patient showing disease progression; Karnofski score improved in all patients by a minimum of 10, with a median of 20 at 6 months. No patient stopped the treatment due to side-effects, no major adverse events were recorded.Conclusion: Temozolomide appears to be an ideal, first-line, single-agent, with a safe profile and demonstrated HQL benefits in patients with high-grade gliomas. [ABSTRACT FROM AUTHOR]

Cell therapies based on pluripotent stem cells (PSC), have opened new therapeutic strategies for neurodegenerative diseases. However, insufficiently differentiated PSC can lead to tumor formation. Ideally, safety switch therapies should selectively kill proliferative transplant cells while preserving post-mitotic neurons. In this study, we evaluated the potential of nucleoside analogs and thymidine kinase-based suicide genes. Among tested thymidine kinase variants, the humanized SR39 (SR39h) variant rendered cells most sensitive to suicide induction. Unexpectedly, post-mitotic neurons with ubiquitous SR39h expression were killed by ganciclovir, but were spared when SR39h was expressed under the control of the cell cycle-dependent Ki67 promoter. The efficacy of six different nucleoside analogs to induce cell death was then evaluated. Penciclovir (PCV) showed the most interesting properties with an efficiency comparable to ganciclovir (GCV), but low toxicity. We tested three nucleoside analogs in vivo: at concentrations of 40 mg/kg/day, PCV and GCV prevented tumor formation, while acyclovir (ACV) did not. In summary, SR39h under the control of a cell cycle-dependent promoter appears most efficient and selective as safety switch for neural transplants. In this setting, PCV and GCV are efficient inducers of cell death. Because of its low toxicity, PCV might become a preferred alternative to GCV.

Rimeporide, a first-in-class sodium/proton exchanger Type 1 inhibitor (NHE-1 inhibitor) is repositioned by EspeRare for patients with Duchenne Muscular Dystrophy (DMD). Historically, NHE-1 inhibitors were developed for cardiac therapeutic interventions. There is considerable overlap in the pathophysiological mechanisms in Congestive Heart Failure (CHF) and in cardiomyopathy in DMD, therefore NHE-1 inhibition could be a promising pharmacological approach to the cardiac dysfunctions observed in DMD. Extensive preclinical data was collected in various animal models including dystrophin-deficient (mdx) mice to characterise Rimeporide's anti-fibrotic and anti-inflammatory properties and there is evidence that NHE-1 inhibitors could play a significant role in modifying DMD cardiac and also skeletal pathologies, as the NHE-1 isoform is ubiquitous. We report here the first study with Rimeporide in DMD patients. This 4-week treatment, open label phase Ib, multiple oral ascending dose study, enrolled 20 ambulant boys with DMD (6-11 years), with outcomes including safety, pharmacokinetic (PK) and pharmacodynamic (PD) biomarkers. Rimeporide was safe and well-tolerated at all doses. PK evaluations showed that Rimeporide was well absorbed orally reaching pharmacological concentrations from the lowest dose, with exposure increasing linearly with dose and with no evidence of accumulation upon repeated dosing. Exploratory PD biomarkers showed positive effect upon a 4-week treatment, supporting its therapeutic potential in patients with DMD, primarily as a cardioprotective treatment, and provide rationale for further efficacy studies., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Idiopathic pulmonary fibrosis (IPF) is a rapid progressive fibro-proliferative disorder with poor prognosis similar to lung cancer. The pathogenesis of IPF is uncertain, but loss of epithelial cells and fibroblast proliferation are thought to be central processes. Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-β signaling, both recognized factors driving lung fibrosis. Differentially spliced BARD1 isoforms, in particular BARD1β, are oncogenic drivers of proliferation in cancers of various origins. We therefore hypothesized that BARD1 and/or its isoforms might play a role in lung fibrosis., Methods: We investigated BARD1 expression as a function of TGF-β in cultured cells, in mice with experimentally induced lung fibrosis, and in lung biopsies from pulmonary fibrosis patients., Results: FL BARD1 and BARD1β were upregulated in response to TGF-β in epithelial cells and fibroblasts in vitro and in vivo. Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1β in fibrotic tissues., Conclusion: Our data suggest that FL BARD1 and BARD1β might be mediators of pleiotropic effects of TGF-β. In particular BARD1β might be a driver of proliferation and of pulmonary fibrosis pathogenesis and progression and represent a target for treatment.

Reactive oxygen species (ROS) are key modulators of apoptosis and carcinogenesis. One of the important sources of ROS is NADPH oxidases (NOXs). The isoform NOX5 is highly expressed in lymphoid tissues, but it has not been detected in any common Hodgkin or non-Hodgkin lymphoma cell lines. In diverse, nonlymphoid malignant cells NOX5 exerts an antiapoptotic effect. Apoptosis suppression is the hallmark feature of a rare type of lymphoma, termed anaplastic lymphoma kinase-positive (ALK(+)) anaplastic large-cell lymphoma (ALCL), and a major factor in the therapy resistance and relapse of ALK(+) ALCL tumors. We applied RT-PCR and Western blot analysis to detect NOX5 expression in three ALK(+) ALCL cell lines (Karpas-299, SR-786, SUP-M2). We investigated the role of NOX5 in apoptosis by small-interfering RNA (siRNA)-mediated gene silencing and chemical inhibition of NOX5 using FACS analysis and examining caspase 3 cleavage in Karpas-299 cells. We used immunohistochemistry to detect NOX5 in ALK(+) ALCL pediatric tumors. NOX5 mRNA was uniquely detected in ALK(+) ALCL cells, whereas cell lines of other lymphoma classes were devoid of NOX5. Transfection of NOX5-specific siRNA and chemical inhibition of NOX5 abrogated calcium-induced superoxide production and increased caspase 3-mediated apoptosis in Karpas-299 cells. Immunohistochemistry revealed focal NOX5 reactivity in pediatric ALK(+) ALCL tumor cells. These results indicate that NOX5-derived ROS contribute to apoptosis blockage in ALK(+) ALCL cell lines and suggest NOX5 as a potential pharmaceutical target to enhance apoptosis and thus to suppress tumor progression and prevent relapse in pediatric ALK(+) ALCL patients that resist classical therapeutic approaches., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Inner ear pathologies are associated with major morbidity and loss of life quality in affected patients. In many of these conditions, production of reactive oxygen-species (ROS) is thought to be a key pathological mechanism. While the sources of ROS are complex (including for example mitochondria), there is increasing evidence that activation of NOX enzymes, in particular NOX3, plays a key role. NOX3 is a multi-subunit NADPH oxidase, functionally and structurally closely related to NOX1 and NOX2. In both the vestibular and the cochlear compartments of the inner ear, high levels of NOX3 mRNA are expressed. In NOX3 mutant mice, the vestibular function is perturbed due to a lack of otoconia, while only minor alterations of hearing have been documented. However, there is increasing evidence that activation of NOX3 through drugs, noise and probably also aging, leads to hearing loss. Thus, NOX3 is an interesting target to treat and prevent inner ear pathologies and a few first animal models based on drug - or molecular therapy have been reported. So far however, there are no specific NOX3 inhibitors with a documented penetration into the inner ear. Nevertheless, certain antioxidants and non-specific NOX inhibitors diminish hearing loss in animal models. Development of small molecules inhibitors or molecular strategies against NOX3 could improve specificity and efficiency of redox-targeted treatments. In this review, we will discuss arguments for the involvement of NOX3 in inner ear pathologies and therapeutic approaches to target NOX3 activity.

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.)

Nicotine contributes to the onset and progression of several pulmonary diseases. Among the various pathophysiological mechanisms triggered by nicotine, oxidative stress and cell death are reported in several cell types. We found that chronic exposure to nicotine (48h) induced NOX1-dependent oxidative stress and apoptosis in primary pulmonary cells. In murine (MLE-12) and human (BEAS-2B) lung epithelial cell lines, nicotine acted as a sensitizer to cell death and synergistically enhanced apoptosis when cells were concomitantly exposed to hyperoxia. The precise signaling pathway was investigated in MLE-12 cells in which NOX1 was abrogated by a specific inhibitor or stably silenced by shRNA. In the early phase of exposure (1h), nicotine mediated intracellular Ca(2+) fluxes and activation of protein kinase C, which in its turn activated NOX1, leading to cellular and mitochondrial oxidative stress. The latter triggered the intrinsic apoptotic machinery by modulating the expression of Bcl-2 and Bax. Overexpression of Bcl-2 completely prevented nicotine's detrimental effects, suggesting Bcl-2as a downstream key regulator in nicotine/NOX1-induced cell damage. These results suggest that NOX1 is a major contributor to the generation of intracellular oxidative stress induced by nicotine and might be an important molecule to target in nicotine-related lung pathologies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Arginine, a semiessential amino acid implicated in diverse cellular processes, is a substrate for two arginases-Arg1 and Arg2-having different expression patterns and functions. Although appropriately regulated Arg1 expression is critical for immune responses, this has not been documented for Arg2. We show that Arg2 is the dominant enzyme in dendritic cells (DCs) and is repressed by microRNA-155 (miR155) during their maturation. miR155 is known to be strongly induced in various mouse and human DC subsets in response to diverse maturation signals, and miR155-deficient DCs exhibit an impaired ability to induce Ag-specific T cell responses. By means of expression profiling studies, we identified Arg2 mRNA as a novel miR155 target in mouse DCs. Abnormally elevated levels of Arg2 expression and activity were observed in activated miR155-deficient DCs. Conversely, overexpression of miR155 inhibited Arg2 expression. Bioinformatic and functional analyses confirmed that Arg2 mRNA is a direct target of miR155. Finally, in vitro and in vivo functional assays using DCs exhibiting deregulated Arg2 expression indicated that Arg2-mediated arginine depletion in the extracellular milieu impairs T cell proliferation. These results indicate that miR155-induced repression of Arg2 expression is critical for the ability of DCs to drive T cell activation by controlling arginine availability in the extracellular environment., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Reactive oxygen species (ROS) contribute to alveolar cell death in acute respiratory distress syndrome (ARDS) and we previously demonstrated that NOX1-derived ROS contributed to hyperoxia-induced alveolar cell death in mice. The study investigates whether NOX1 expression is modulated in epithelial cells concomitantly to cell death and associated to STAT3 signaling in the exudative phase of ARDS. In addition, the role of STAT3 activation in NOX1-dependent epithelial cell death was confirmed by using a lung epithelial cell line and in mice exposed to hyperoxia. NOX1 expression, cell death and STAT3 staining were evaluated in the lungs of control and ARDS patients by immunohistochemistry. In parallel, a stable NOX1-silenced murine epithelial cell line (MLE12) and NOX1-deficient mice were used to characterize signalling pathways. In the present study, we show that NOX1 is detected in alveolar epithelial cells of ARDS patients in the exudative stage. In addition, increased alveolar epithelial cell death and phosphorylated STAT3 are observed in ARDS patients and associated with NOX1 expression. Phosphorylated STAT3 is also correlated with TUNEL staining. We also confirmed that NOX1-dependent STAT3 activation participates to alveolar epithelial cell death. Silencing and acute inhibition of NOX1 in MLE12 led to decreased cell death and cleaved-caspase 3 induced by hyperoxia. Additionally, hyperoxia-induced STAT3 phosphorylation is dependent on NOX1 expression and associated with cell death in MLE12 and mice. This study demonstrates that NOX1 is involved in human ARDS pathophysiology and is responsible for the damage occurring in alveolar epithelial cells at least in part via STAT3 signalling pathways.

Background: Volatile inhaled anesthetics exert a differential protective effect against bronchospasm development after cholinergic stimulation. However, their ability to inhibit the adverse respiratory consequences of an anaphylactic reaction after exposure to an allergen has not been characterized. We therefore compared the abilities of isoflurane, sevoflurane, and desflurane to prevent the lung constriction induced by an allergic reaction in a pediatric model of an anaphylactic reaction., Methods: Low-frequency respiratory input impedance (Zrs) was measured in 4 groups of ovalbumin (OVA)-sensitized 5-week-old rabbit pups anesthetized with midazolam (group IV) and with inhaled isoflurane (group ISO), sevoflurane (group SEVO), or desflurane (group DES) at 1 minimum alveolar concentration. Zrs was measured under baseline conditions and after an anaphylactic lung response provoked by IV allergen injection (OVA 1 mg), during which the changes in airway resistance (Raw), tissue damping (G), and elastance obtained from Zrs were followed for 15 minutes., Results: Allergen provocation generated immediate severe bronchoconstriction, with no statistically significant difference in Raw increase among the groups in the first 3 minutes. Conversely, the inhalation of volatile anesthetics accelerated the recovery from the allergen-induced bronchoconstriction, particularly in group SEVO where the Raw was significantly lower than that in group IV 4 minutes after the allergen challenge. These changes were paralleled by significant elevations in G in all groups, with a significantly more pronounced deterioration in the animals in group DES. The anesthetic regimen did not statistically significantly affect the sustained increases in elastance after OVA injections., Conclusions: Our results reveal the lack of potential of the commonly used volatile anesthetics to inhibit the most severe acute phase of the constrictor response to allergen after anaphylaxis in both the central airway and peripheral lung compartments. Inhalation of volatile anesthetics, particularly sevoflurane, promotes an earlier easing of the bronchospasm; this beneficial profile may be advantageous in children with atopic lung diseases.

Chronic granulomatous disease (CGD), caused by a lack of reactive oxygen species (ROS) generation by the phagocyte NADPH oxidase NOX2, leads to massively increased inflammatory responses. In order to identify the type of phagocyte which requires NOX2 activity to limit inflammation, we investigated mice with a loss of function mutation in the Ncf1 gene coding for the p$47^{\rm{phox}}$ subunit of NOX2 and mice with transgenic rescue of Ncf1 under control of the CD68 promoter. To induce CGD hyperinflammation, different mouse genotypes were injected intradermally with β-glucan. Ncf1 mutant mice showed massive and prolonged hyperinflammation. Hyperinflammatory lesions were characterized by persistent neutrophilic infiltration, along with ulceration and necrosis. In contrast, in CD68 promoter rescue mice inflammation resolved within days, as seen in wild-type animals. Measurements of ROS in rescue mice demonstrated functional NOX2 in mononuclear phagocytes (macrophages and dendritic cells) but not in neutrophils. This absence of NOX2 function was also confirmed in inflammatory tissue neutrophils. Lack of functional NOX2 in mononuclear phagocytes increased the secretion of IL-1β at early time points and of IL-6 and TNFα at later time points. Thus, CGD hyperinflammation is a redox dysregulation in mononuclear phagocytes, demonstrating a cell type-specific anti-inflammatory function of NOX2., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), is characterized by acute inflammation, disruption of the alveolar-capillary barrier, and in the organizing stage by alveolar pneumocytes hyperplasia and extensive lung fibrosis. The cellular and molecular mechanisms leading to the development of ALI/ARDS are not completely understood, but there is evidence that reactive oxygen species (ROS) generated by inflammatory cells as well as epithelial and endothelial cells are responsible for inflammatory response, lung damage, and abnormal repair. Among all ROS-producing enzymes, the members of NADPH oxidases (NOXs), which are widely expressed in different lung cell types, have been shown to participate in cellular processes involved in the maintenance of lung integrity. It is not surprising that change in NOXs' expression and function is involved in the development of ALI/ARDS. In this context, the use of NOX inhibitors could be a possible therapeutic perspective in the management of this syndrome. In this article, we summarize the current knowledge concerning some cellular aspects of NOXs localization and function in the lungs, consider their contribution in the development of ALI/ARDS and discuss the place of NOX inhibitors as potential therapeutical target.

Unlabelled: The pathogenesis of pulmonary fibrosis is linked to oxidative stress, possibly generated by the reactive oxygen species (ROS) generating NADPH oxidase NOX4. Epithelial cell death is a crucial early step in the development of the disease, followed only later by the fibrotic stage. We demonstrate that in lungs of patients with idiopathic lung fibrosis, there is strong expression of NOX4 in hyperplastic alveolar type II cells., Aim: To study a possible causative role of NOX4 in the death of alveolar cells, we have generated NOX4-deficient mice., Results: Three weeks after administration of bleomycin, wild-type (WT) mice developed massive fibrosis, whereas NOX4-deficient mice displayed almost normal lung histology, and only little Smad2 phosphorylation and accumulation of myofibroblasts. However, the protective effects of NOX4 deficiency preceded the fibrotic stage. Indeed, at day 7 after bleomycin, lungs of WT mice showed massive increase in epithelial cell apoptosis and inflammation. In NOX4-deficient mice, no increase in apoptosis was observed, whereas inflammation was comparable to WT. In vitro, NOX4-deficient primary alveolar epithelial cells exposed to transforming growth factor-β(1) did not generate ROS and were protected from apoptosis. Acute treatment with the NOX inhibitors also blunted transforming growth factor-β(1)-induced apoptosis., Conclusion: ROS generation by NOX4 is a key player in epithelial cell death leading to pulmonary fibrosis.

Reactive oxygen species, ROS, are regulators of endothelial cell migration, proliferation and survival, events critically involved in angiogenesis. Different isoforms of ROS-generating NOX enzymes are expressed in the vasculature and provide distinct signaling cues through differential localization and activation. We show that mice deficient in NOX1, but not NOX2 or NOX4, have impaired angiogenesis. NOX1 expression and activity is increased in primary mouse and human endothelial cells upon angiogenic stimulation. NOX1 silencing decreases endothelial cell migration and tube-like structure formation, through the inhibition of PPARα, a regulator of NF-κB. Administration of a novel NOX-specific inhibitor reduced angiogenesis and tumor growth in vivo in a PPARα dependent manner. In conclusion, vascular NOX1 is a critical mediator of angiogenesis and an attractive target for anti-angiogenic therapies.

Bcl-2 is an anti-apoptotic molecule preventing oxidative stress damage and cell death. We have previously shown that Bcl-2 is able to prevent hyperoxia-induced cell death when overexpressed in a murine fibrosarcoma cell line L929. We hypothesized that its specific overexpression in pulmonary epithelial type II cells could prevent hyperoxia-induced lung injury by protecting the epithelial side of the alveolo-capillary barrier. In the present work, we first showed that in vitro Bcl-2 can rescue murine pulmonary epithelial cells (MLE12) from oxygen-induced cell apoptosis, as shown by analysis of LDH release, annexin V/propidium staining, and caspase-3 activity. We then generated transgenic mice overexpressing specifically Bcl-2 in lung epithelial type II cells under surfactant protein C (SP-C) promoter (Tg-Bcl-2) and exposed them to hyperoxia. Bcl-2 did not hinder hyperoxia-induced mitochondria and DNA oxidative damage of type II cell in vivo. Accordingly, lung damage was identical in both Tg-Bcl-2 and littermate mice strains, as measured by lung weight, bronchoalveolar lavage, and protein content. Nevertheless, we observed a significant lower number of TUNEL-positive cells in type II cells isolated from Tg-Bcl-2 mice exposed to hyperoxia compared with cells isolated from littermate mice. In summary, these results show that although Bcl-2 overexpression is able to prevent hyperoxia-induced cell death at single cell level in vitro and ex vivo, it is not sufficient to prevent cell death of parenchymal cells and to protect the lung from acute damage in mice.

Rationale: Hyperoxia-induced acute lung injury has been used for many years as a model of oxidative stress mimicking clinical acute lung injury and the acute respiratory distress syndrome. Excess quantities of reactive oxygen species (ROS) are responsible for oxidative stress-induced lung injury. ROS are produced by mitochondrial chain transport, but also by NADPH oxidase (NOX) family members. Although NOX1 and NOX2 are expressed in the lungs, their precise function has not been determined until now., Objectives: To determine whether NOX1 and NOX2 contribute in vivo to hyperoxia-induced acute lung injury., Methods: Wild-type and NOX1- and NOX2-deficient mice, as well as primary lung epithelial and endothelial cells, were exposed to room air or 100% O(2) for 72 hours., Measurements and Main Results: Lung injury was significantly prevented in NOX1-deficient mice, but not in NOX2-deficient mice. Hyperoxia-dependent ROS production was strongly reduced in lung sections, in isolated epithelial type II cells, and lung endothelial cells from NOX1-deficient mice. Concomitantly, lung cell death in situ and in primary cells was markedly decreased in NOX1-deficient mice. In wild-type mice, hyperoxia led to phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), two mitogen-activated protein kinases involved in cell death signaling, and to caspase-3 activation. In NOX1-deficient mice, JNK phosphorylation was blunted, and ERK phosphorylation and caspase-3 activation were decreased., Conclusions: NOX1 is an important contributor to ROS production and cell death of the alveolocapillary barrier during hyperoxia and is an upstream actor in oxidative stress-induced acute lung injury involving JNK and ERK pathways in mice.

Adipocyte differentiation is a complex process regulated among other factors by insulin and the production of reactive oxygen species (ROS). NOX4 is a ROS generating NADPH oxidase enzyme mediating insulin's action in 3T3L1 adipocytes. In the present paper we show that NOX4 is expressed at high levels both in white and brown preadipocytes and that differentiation into adipocytes results in a decrease in their NOX4 mRNA content. These in vitro results were confirmed in vivo by demonstrating that in intact adipose tissue the majority of NOX4 expressing cells are localized within the preadipocyte containing stromal/vascular fraction, rather than in the portion consisting of mature adipocytes. In line with these observations, quantification of NOX4 mRNA in fat derived from different rodent models of insulin resistance indicated that alteration in NOX4 expression reflects changes in the ratio of adipocyte/interstitial fractions. In conclusion, we reveal that decreased NOX4 mRNA content is a hallmark of adipocyte differentiation and that NOX4 expression measured in whole adipose tissue is not an unequivocal indicator of intact or impaired insulin action.

Apoplexy of a pituitary adenoma is a rare and under-diagnosed clinical occurrence. It results from either infarction or haemorrhage into an adenoma of the pituitary gland. Its clinical presentation more often includes rapid development of impaired consciousness, severe headache, visual disturbance and variable association of oculomotor nerve palsy. Meningeal irritation signs are considered very rare and usually not reported as presenting symptoms. A 33-year-old male suffered a pituitary macroadenoma apoplexy, clinically indistinguishable from an infectious meningitis at presentation. Three days after surgery, the patient developed a left ophthalmoplegia due to 3(rd) nerve palsy, which fully resolved within 2 months. A right pterional craniotomy was performed during which complete tumour removal was achieved. In conclusion the authors believe that, despite many reports in the literature, encouraging conservative management in pituitary apoplexy by administering intravenous steroids, surgery should be undertaken in order to avoid eventual visual field defects, relieve pituitary gland compression and prevent a possible recurrent apoplectic episode or tumor re-growth.

Bcl-2 is an antiapoptotic molecule that prevents oxidative stress damage and cell death. We investigated the possible protective mechanisms mediated by Bcl-2 during hyperoxia-induced cell death in L929 cells. In these cells, hyperoxia promoted apoptosis without DNA fragmentation. Overexpression of Bcl-2 significantly protected cells from oxygen-induced apoptosis, as shown by measurement of lactate dehydrogenase release, quantification of apoptotic nuclei, and detection of Annexin-V-positive cells. Bcl-2 partially prevented mitochondrial damage and interfered with the mitochondrial proapoptotic signaling pathway: it reduced Bax translocation to mitochondria, decreased the release of cytochrome c, and inhibited caspase 3 activation. However, treatment with the caspase inhibitor Z-VAD.fmk failed to rescue the cells from death, indicating that protection provided by Bcl-2 was due not only to caspase inhibition. Bcl-2 also prevented the release of mitochondrial apoptotic inducing factor, a mediator of caspase-independent apoptosis, correlating with the absence of oligonucleosomal DNA fragmentation. In addition, Bcl-2-overexpressing cells showed significantly higher intracellular amounts of glutathione after 72 h of oxygen exposure. In conclusion, our results demonstrate that the overexpression of Bcl-2 is able to prevent hyperoxia-induced cell death, by affecting mitochondria-dependent apoptotic pathways and increasing intracellular antioxidant compounds.

Vascular endothelial growth factor (VEGF) is the most potent stimulatory factor of angiogenesis. Its expression is induced by reactive oxygen species (ROS) in hypoxic conditions and by insulin in normoxic cells. Both ROS and insulin can activate mitogen-activated protein kinases (MAPKs) and induce the transcriptional factor Sp1, components that are essential for VEGF gene expression. The aim of this study was to investigate the role of ROS producing NADPH oxidase enzymes (NOX-es) in insulin-regulated VEGF gene activation. To achieve this goal we chose HepG2 cells as our model system as these cells express the NADPH oxidase isoform NOX3 and respond to insulin stimulation with enhanced ROS production and mRNA transcription and production of VEGF. We demonstrate that in control cells insulin stimulation leads to H2O2 generation, a biphasic activation of p42/44 MAPK and the induction of both Sp1 and HIF-1alpha. Transfection of NOX3-specific siRNA abrogates H2O2 production and inhibits exclusively the second phase of p42/44 MAPK phosphorylation and Sp1 DNA binding and thus prevents upregulation of VEGF-A mRNA expression. In conclusion, our results demonstrate that NOX3, a ROS generating NADPH oxidase, plays an integral role in insulin-induced p42/44 MAPK signal transmission and VEGF-A production.

Recently the debate over the management of cervical spondylotic myelopathy (CSM) has regained interest; more specifically whether treatment should be operative versus non-operative, raising the question about the real effectiveness of surgery in influencing the natural history of this pathology and about the choice of the most appropriate approach (anterior vs. posterior). The authors report a retrospective review of 70 consecutive patients who underwent elective anterior cervical corpectomy and fusion with iliac crest autograft or titanium mesh and placement of an anterior cervical plate for the treatment of CSM. The patients underwent pre-and postoperative evaluation, including history, and physical and neurological examination. Patients were also evaluated pre-and postoperatively using a modified version of the Japanese Orthopedics Association Scale (mJOA), which provides a fine semi-quantitative graded evaluation of overall function. Upon discharge home, patients were followed for an average of 42 months (range, 12-63 months). Following an anterior cervical decompression of the spinal cord, 94.2% of patients improved their functional status and 5.8% were unchanged; the mean preoperative mJOA score of all patients was 12.2, the postoperative was 15.4 and the amelioration was also documented by neurophysiological studies which showed an increase in amplitude and decrease in latency of somatosensory evoked potentials and motor evoked potential in 47 patients (67%). Older age and longer duration of preoperative symptoms both were not associated with a lower postoperative mJOA score (p < 0.47, p < 0.29, respectively). Single versus multiple level decompression was not predictive of a lower postoperative mJOA score (p < 0.18). Preoperative spinal cord low signal intensity changes on T1-weighted MRI were related to a lower postoperative mJOA score (p < 0.05), whereas spinal cord high-signal intensity changes on T2-weighted MRI were related to a higher postoperative mJOA score (p < 0.01); finally a lower preoperative mJOA score was highly predictive of a lower postoperative mJOA score (p < 0.0005). Anterior cervical corpectomy and fusion for CSM appears to be an effective procedure with a more favorable neurological improvement when compared to posterior decompressive laminectomy, minimally invasive procedures or non-surgical treatment. It is also a safe procedure even in the elderly population, with low morbidity and the potential for permanent spinal cord decompression and excellent bone stability.

Objective: To introduce the possibility of volume-rendered helical computed tomographic (CT) angiographic data sets by use of Medtronic StealthStation Treon surgical navigation technology (Medtronic Surgical Navigation Technologies, Louisville, CO) and to evaluate the clinical usefulness of the method in planning and performing surgical treatment of intracranial aneurysms., Methods: Between November 2002 and July 2003, we studied 15 patients with suspected intracranial aneurysms. All patients but two received conventional digital subtraction angiography, which failed to provide the requested information. Helical CT angiography was performed in all patients, and data sets were transferred to the StealthStation system across an electronic network to be automatically postprocessed by use of three-dimensional (3-D) volume rendering. The 3-D volume-rendered images were accurately analyzed to obtain more complete information about the aneurysm and to provide accurate treatment planning. In all patients, the 3-D volume-rendered model was displayed on the screen of the StealthStation system for the duration of the surgical procedure and compared with the intraoperative image., Results: Data sets from CT angiography were automatically postprocessed by the StealthStation in seconds with excellent results, providing us, before and during surgery, with additional information not always available on traditional digital subtraction angiographic investigation. Because of the very short time necessary to complete this process (<5 min to obtain 3-D volume-rendered images), it was possible to perform emergency clipping of the aneurysms in two patients who had been admitted in very compromised neurological conditions. In 12 patients, integrated digital subtraction angiography and automated 3-D volume-rendered images allowed an accurate presurgical evaluation. Furthermore, in all patients on whom surgery was performed, aneurysms were found in the exact location and with the same anatomic features as depicted by the 3-D volume-rendered models., Conclusion: Reports in the literature indicate that information gathered by CT angiography with volume rendering shows a significant impact on aneurysm management. The StealthStation system upgraded with the adequate algorithm seems to provide a time- and cost-effective method of performing automated 3-D volume rendering of CT angiography and provides an interesting alternative to the available investigation modalities in case of emergency.

The natural polyamines are multifunctional constituents of all eucaryotic cells. The objective of this work was to compare aspects of polyamine metabolism in two related cell lines with the idea to investigate whether metabolic differences can be attributed to functional differences of the cells. The human colon carcinoma-derived cell lines SW480 and SW620 were chosen as models. SW480 cells were isolated from the primary tumour, SW620 cells from a lymph node of the same patient. SW620 cells grow faster, and the key regulatory enzymes of polyamine biosynthesis (ODC and AdoMetDC) are more active in the metastatic cells. Moreover, their ability to accumulate polyamines from the environment is more important than of SW480 cells. Likewise polyamine concentrations were markedly higher in SW620 cells, although they are much smaller than SW480 cells, and have a particularly small cytoplasmic space. Both cell lines show a striking diminution of ODC and AdoMetDC activities and changes in the polyamine patterns at the transition from exponential to non-exponential growth--most probably as a consequence of high cell density. Depletion of putrescine and spermidine due to inactivation of ODC by DFMO causes accumulation of cells in G1, and a proportional decrease of S-phase cells in both cell lines. Based on morphologic and other criteria SW480 and SW620 cells were typified as poorly differentiated. In agreement with their low grade of differentiation they exhibit a low alkaline phosphatase activity. However, the time-dependent decrease of alkaline phosphatase is not typical of differentiation patterns of other adenocarcinoma-derived cell lines or of normal enterocytes. The high capacity of de novo polyamine biosynthesis and of polyamine uptake is presumably a prerequisite for the rapid growth and invasiveness. The fact that these properties were more accentuated in the case of SW620 cells and paralleled enhanced metastatic properties indicate relationships between basic parameters of polyamine metabolism and malignancy.

Geraniol, a natural component of plant essential oils, has antiproliferative effects on human colon cancer cells. To obtain more insight into its mechanism of action, we studied its effect on the resting membrane potential and on the expression of proteins involved in cell signaling pathways. Since geraniol is a well known inhibitor of mevalonate metabolism, the effect of mevalonate supplementation on geraniol-triggered growth inhibition was also determined. Geraniol (400 microM) induced membrane depolarization with a decrease of membrane resistance due to local perforation of the cell membrane. Incubation of Caco-2 cells with geraniol (400 microM) for 6 h caused a 60% reduction of protein kinase C (PKC) activity. After 16 h of incubation, geraniol decreased by 50% the amount of active forms of p44/p42 extracellular signal-regulated protein kinases (ERK). Mevalonate supplementation did not reverse inhibition of cell growth by geraniol. These results indicate that the antiproliferative effect of geraniol on Caco-2 cells was not related to a limitation of the mevalonate pool but was directly linked to the perturbation of cell membrane function leading to the reduction of PKC activity and to the decreased expression of p44/p42 ERK active forms.