Reproducibility is a current concern for everyone involved in the conduct and publication of biomedical research. Recent attempts testing reproducibility, particularly the reproducibility project in cancer biology published in elife (https://elifesciences.org/collections/9b1e83d1/reproducibility-project-cancer-biology), have exposed major difficulties in repeating published preclinical experimental work. It is thought that some of these difficulties relate to uncertainty about the provenance of tools, lack of clarity in methodology and use of inappropriate approaches for analysis; the latter particularly related to untoward manipulation of images. In the past, some of these so-called untoward practices were considered the 'norm'; however, today, the landscape is different. The expectations, not only of the readers of the published scientific word but also of the publishers and funders of research, have changed. This collective group now expects that any published data should be reproducible; but for this to be possible, experimental detail, confirmation of selectivity and quality of reagents/ tools, analytical and statistical methods used need to be described adequately. Two powerful methodologies often used to support researchers' findings allow the detection of changes in protein expression, that is, immunoblotting (widely known as Western blotting) and immunohistochemistry. Undeniably, as a result of unintentional mistakes (often related to lack of antibody specificity; Baker, 2015), but, in some cases, deliberate alterations and questionable interpretations of results, the use of these two methods has led to many high profile retractions. Indeed, such images have driven the retractions that have occurred in BJP over the last two years.Today, immunoblotting and immunohistochemistry serve as primary methodologies for the detection and quantification of molecular signalling pathways and identification of therapeutic targets. This necessitates clear guidance for the application of these techniques, the need for controls (both positive and negative) and the most appropriate methods for quantification. Indeed, this need has spawned a number of initiatives to support researchers in assessing the validity of antibody resources including antibodypedia (Bjorling and Uhlen, 2008) and the resources available within 'The Human Protein Atlas' (Thul et al., 2017). The aim of this article is to outline the rationale for, and the expectations of, the BJP with respect to work published in the Journal that includes immunoblotting or immunohistochemical data. In creating these guidelines, our aim is to reduce potential misinterpretations and to maximise the communication and transparency of essential information, particularly with respect to the methodologies employed.We have generated the guidelines below for the benefit of authors, editors and reviewers. While we recognise other recently published guidelines (Uhlen et al., 2016) and indeed we have incorporated some of the advice provided in such reports, we focus, here, on th...
Influenza A virus pandemics and emerging anti-viral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and lung inflammation. We investigated whether the primary enzymatic source of inflammatory cell ROS (reactive oxygen species), Nox2-containing NADPH oxidase, is a novel pharmacological target against the lung inflammation caused by influenza A viruses. Male WT (C57BL/6) and Nox2−/y mice were infected intranasally with low pathogenicity (X-31, H3N2) or higher pathogenicity (PR8, H1N1) influenza A virus. Viral titer, airways inflammation, superoxide and peroxynitrite production, lung histopathology, pro-inflammatory (MCP-1) and antiviral (IL-1β) cytokines/chemokines, CD8+ T cell effector function and alveolar epithelial cell apoptosis were assessed. Infection of Nox2−/y mice with X-31 virus resulted in a significant reduction in viral titers, BALF macrophages, peri-bronchial inflammation, BALF inflammatory cell superoxide and lung tissue peroxynitrite production, MCP-1 levels and alveolar epithelial cell apoptosis when compared to WT control mice. Lung levels of IL-1β were ∼3-fold higher in Nox2−/y mice. The numbers of influenza-specific CD8+DbNP366+ and DbPA224+ T cells in the BALF and spleen were comparable in WT and Nox2−/y mice. In vivo administration of the Nox2 inhibitor apocynin significantly suppressed viral titer, airways inflammation and inflammatory cell superoxide production following infection with X-31 or PR8. In conclusion, these findings indicate that Nox2 inhibitors have therapeutic potential for control of lung inflammation and damage in an influenza strain-independent manner.
The imminent threat of viral epidemics and pandemics dictates a need for therapeutic approaches that target viral pathology irrespective of the infecting strain. Reactive oxygen species are ancient processes that protect plants, fungi and animals against invading pathogens including bacteria. However, in mammals reactive oxygen species production paradoxically promotes virus pathogenicity by mechanisms not yet defined. Here we identify that the primary enzymatic source of reactive oxygen species, NOX2 oxidase, is activated by single stranded RNA and DNA viruses in endocytic compartments resulting in endosomal hydrogen peroxide generation, which suppresses antiviral and humoral signaling networks via modification of a unique, highly conserved cysteine residue (Cys98) on Toll-like receptor-7. Accordingly, targeted inhibition of endosomal reactive oxygen species production abrogates influenza A virus pathogenicity. We conclude that endosomal reactive oxygen species promote fundamental molecular mechanisms of viral pathogenicity, and the specific targeting of this pathogenic process with endosomal-targeted reactive oxygen species inhibitors has implications for the treatment of viral disease.
We tested whether significant leukocyte infiltration occurs in a mouse model of permanent cerebral ischemia. C57BL6/J male mice underwent either permanent (3 or 24 hours) or transient (1 or 2 hours þ 22-to 23-hour reperfusion) middle cerebral artery occlusion (MCAO). Using flow cytometry, we observed B15,000 leukocytes (CD45 þ high cells) in the ischemic hemisphere as early as 3 hours after permanent MCAO (pMCAO), comprising B40% lymphoid cells and B60% myeloid cells. Neutrophils were the predominant cell type entering the brain, and were increased to B5,000 as early as 3 hours after pMCAO. Several cell types (monocytes, macrophages, B lymphocytes, CD8þ T lymphocytes, and natural killer cells) were also increased at 3 hours to levels sustained for 24 hours, whereas others (CD4 þ T cells, natural killer T cells, and dendritic cells) were unchanged at 3 hours, but were increased by 24 hours after pMCAO. Immunohistochemical analysis revealed that leukocytes typically had entered and widely dispersed throughout the parenchyma of the infarct within 3 hours. Moreover, compared with pMCAO, there were B50% fewer infiltrating leukocytes at 24 hours after transient MCAO (tMCAO), independent of infarct size. Microglial cell numbers were bilaterally increased in both models. These findings indicate that a profound infiltration of inflammatory cells occurs in the brain early after focal ischemia, especially without reperfusion.
Cerebral infarct volume is typically smaller in premenopausal females than in age-matched males after ischemic stroke, but the underlying mechanisms are poorly understood. In this study we provide evidence in mice that this gender difference only occurs when the ischemic brain is reperfused. The limited tissue salvage achieved by reperfusion in male mice is associated with increased expression of proinflammatory proteins, including cyclooxygenase-2 (Cox-2), Nox2, and vascular cell adhesion molecule-1 (VCAM-1), and infiltration of Nox2-containing T lymphocytes into the infarcted brain, whereas such changes are minimal in female mice after ischemia-reperfusion (I-R). Infarct volume after I-R was no greater at 72 h than at 24 h in either gender. Infarct development was Nox2 dependent in male but not in female mice, and Nox2 within the infarct was predominantly localized in T lymphocytes. Stroke resulted in an B15-fold increase in Nox2-dependent superoxide production by circulating, but not spleen-derived, T lymphocytes in male mice, and this was Bsevenfold greater than in female mice. These circulating immune cells may thus represent a major and previously unrecognized source of superoxide in the acutely ischemic and reperfused brain of males (and potentially in postmenopausal females). Our findings provide novel insights into mechanisms that could be therapeutically targeted in acute ischemic stroke patients who receive thrombolysis therapy to induce cerebral reperfusion.
Background and Purpose-Ly6Chi monocytes are generally thought to exert a proinflammatory role in acute tissue injury, although their impact after injuries to the central nervous system is poorly defined. CC chemokine receptor 2 is expressed on Ly6C hi monocytes and plays an essential role in their extravasation and transmigration into the brain after cerebral ischemia. We used a selective CC chemokine receptor 2 antagonist, INCB3344, to assess the effect of Ly6C hi monocytes recruited into the brain early after ischemic stroke. Methods-Male C57Bl/6J mice underwent occlusion of the middle cerebral artery for 1 hour followed by 23 hours of reperfusion. Mice were administered either vehicle (dimethyl sulfoxide/carboxymethylcellulose) or INCB3344 (10, 30 or 100 mg/kg IP) 1 hour before ischemia and at 2 and 6 hours after ischemia. At 24 hours, we assessed functional outcomes, infarct volume, and quantified the immune cells in blood and brain by flow cytometry or immunofluorescence. Gene expression of selected inflammatory markers was assessed by quantitative polymerase chain reaction. Results-Ly6Chi monocytes were increased 3-fold in the blood and 10-fold in the brain after stroke, and these increases were selectively prevented by INCB3344 in a dose-dependent manner. Mice treated with INCB3344 exhibited markedly worse functional outcomes and larger infarct volumes, in association with reduced M2 polarization and increased peroxynitrite production in macrophages, compared with vehicle-treated mice. Conclusions-Our data suggest that Ly6Chi monocytes exert an acute protective effect after ischemic stroke to limit brain injury and functional deficit that involves promotion of M2 macrophage polarization. Furthermore, there is evidence that CCR2/CCL2 mechanisms are important for the migration of neuroblasts from neurogenic regions to damaged regions of brain after cerebral ischemia in mice, suggesting a role in tissue recovery after stroke. 17 Thus, for the development of future therapies to target detrimental elements of the poststroke acute inflammatory response, it is vital to clarify the role of CCR2 + /Ly6C hi monocytes. To our knowledge, no study has examined the effect of a selective CCR2 antagonist in ischemic stroke. INCB3344 is a novel CCR2 antagonist that inhibits the binding of CCL2 to monocytes with high potency and it is highly selective for the CCR2 receptor.18 It effectively reduces monocyte/macrophage accumulation in autoimmune encephalomyelitis in mice and in inflammatory arthritis in rats 18 and in deoxycorticosterone acetate/salt-induced hypertension in mice.19 Its potency and selectivity for CCR2 inhibition have thus been used in this study to evaluate the effect of inhibiting Ly6C hi monocyte migration to the brain early during poststroke inflammation. Materials and Methods AnimalsThis study was approved by the Monash University Animal Ethics Committee (Project MARP/2011/112) and performed in accordance with the National Health and Medical Research Council of Australia guidelines for the care and use ...
CH augments membrane depolarization-induced pulmonary VSM Ca(2+) sensitization and vasoconstriction through EGFR-dependent stimulation of Rac1 and NOX 2.
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