Due to the radiosensitivity of the lung, toxic endpoints, in the form of radiation pneumonitis and pulmonary fibrosis, are relatively frequent outcomes following radiation treatment of thoracic ...neoplasms. Because of the potential lethal nature of these normal tissue reactions, they not only lead to quality-of-life issues in survivors, but also are deemed dose-limiting and thereby compromise treatment. The mitigation and treatment of lung normal tissue late effects has therefore been the goal of many investigations; however, the complexity of both the organ itself and its response to injury has resulted in little success. Nonetheless, current technology allows us to propose likely targets that are either currently being researched or should be considered in future studies.
Highlights ► We examined if some nanoparticles (NPs) can interfere with an LDH assay. ► Copper NP and a silver NP could interfere with the assay by inactivating LDH. ► Titanium dioxide NPs were found ...to adsorb LDH but only at high doses. ► Thus, the validity of the assay for assessing NP toxicity depends on NP types.
We present a real-time, high-throughput, and cost-effective method of detecting apoptosis in vitro using a previously developed reagent that detects caspase activation by fluorescence. Current ...methods of assessing apoptosis fail to account for the dimension of time, and thus are limited in data yielded per sample. This reagent allows real-time detection of apoptosis, but until now has been restricted to a costly automated detection system. Here, we describe apoptosis detection with the Essen Bioscience IncuCyte® Caspase-3/7 Reagent using a multimode microplate reader, a common instrument in biological laboratories, which may be used prior to or in lieu of the automated system. This modified microplate reader apoptosis assay was validated against the established automated system, and was shown to detect a strong dose-response relationship (automated system r2 = 0.9968, microplate reader r2 = 0.9924). We also propose a quick and reliable method of quantifying cell density by Hoechst 33342 nuclear staining in microplates (r2 = 0.8812 between Hoechst signal and cell density). We assert that the dimension of time should not be overlooked, and that the method presented here is an accessible strategy for many researchers due to low startup cost and precise detection of apoptosis in real time.
•Popular methods of apoptosis detection yield a single reading per sample•Accounting for kinetics of the apoptotic response provides more robust and precise data outputs•Current real time methods of apoptosis detection are either expensive or have low sample capacity•A high-throughput microplate reader based assay is shown here to accurately detect apoptosis in real time
Lung exposure to radiation induces an injury response that includes the release of cytokines and chemotactic mediators; these signals recruit immune cells to execute inflammatory and wound-healing ...processes. However, radiation alters the pulmonary microenvironment, dysregulating the immune responses and preventing a return to homeostasis. Importantly, dysregulation is observed as a chronic inflammation, which can progress into pneumonitis and promote pulmonary fibrosis; inflammatory monocytes, which are bone marrow derived and express CCR2, have been shown to migrate into the lung after radiation exposure. Although the extent to which recruited inflammatory monocytes contribute to radiation-induced pulmonary fibrosis has not been fully investigated, we hypothesize that its pathogenesis is reliant on this population. The CC chemokine ligand, CCL2, is a chemotactic mediator responsible for trafficking of CCR2+ inflammatory cells into the lung. Therefore, the contribution of this mediator to fibrosis development was analyzed. Interleukin (IL)-1β, a potent pro-inflammatory cytokine expressed during the radiation response, and its receptor, IL-1R1, were also evaluated. To this end, CCR2–/–, IL-1β–/– and IL-1R1–/– chimeric mice were generated and exposed to 12.5 Gy thoracic radiation, and their response was compared to wild-type (C57BL/6) syngeneic controls. Fibrotic foci were observed in the periphery of the lungs of C57 syngeneic mice and CCR2–/– recipient mice that received C57 bone marrow (C57 > CCR2–/–) by 16 and 12 weeks after irradiation, respectively. In contrast, in the mice that had received bone marrow lacking CCR2 (CCR2–/– > C57 and CCR2–/– syngeneic mice), no pulmonary fibrosis was observed at 22 weeks postirradiation. This observation correlated with decreased numbers of infiltrating and interstitial macrophages compared to controls, as well as reduced proportions of pro-inflammatory Ly6C+ macrophages observed at 12–18 weeks postirradiation, suggesting that CCR2+ macrophages contribute to radiation-induced pulmonary fibrosis. Interestingly, reduced proportions of CD206+ lung macrophages were also present at these time points in CCR2–/– chimeric mice, regardless of donor bone marrow type, suggesting that the phenotype of resident subsets may be influenced by CCR2. Furthermore, chimeras, in which either IL-1β was ablated from infiltrating cells or IL-1R1 from lung tissues, were also protected from fibrosis development, correlating with attenuated CCL2 production; these data suggest that IL-1β may influence chemotactic signaling after irradiation. Overall, our data suggest that CCR2+ infiltrating monocyte-derived macrophages may play a critical role in the development of radiation-induced pulmonary fibrosis.
Radiation models, such as whole thorax lung irradiation (WTLI) or partial-body irradiation (PBI) with bone-marrow sparing, have shown that affected lung tissue displays a continual progression of ...injury, often for months after the initial insult. Undoubtably, a variety of resident and infiltrating cell types either contribute to or fail to resolve this type of progressive injury, which in lung tissue, often develops into lethal and irreversible radiation-induced pulmonary fibrosis (RIPF), indicating a failure of the lung to return to a homeostatic state. Resident pulmonary epithelium, which are present at the time of irradiation and persist long after the initial insult, play a key role in the maintenance of homeostatic conditions in the lung and have often been described as contributing to the progression of radiation-induced lung injury (RILI). In this study, we took an unbiased approach through RNA sequencing to determine the in vivo response of the lung epithelium in the progression of RIPF. In our methodology, we isolated CD326+ epithelium from the lungs of 12.5 Gy WTLI C57BL/6J female mice (aged 8-10 weeks and sacrificed at regular intervals) and compared irradiated and non-irradiated CD326+ cells and whole lung tissue. We subsequently verified our findings by qPCR and immunohistochemistry. Transcripts associated with epithelial regulation of immune responses and fibroblast activation were significantly reduced in irradiated animals at 4 weeks postirradiation. Additionally, alveolar type-2 epithelial cells (AEC2) appeared to be significantly reduced in number at 4 weeks and thereafter based on the diminished expression of pro-surfactant protein C (pro-SPC). This change is associated with a reduction of Cd200 and cyclooxygenase 2 (COX2), which are expressed within the CD326 populations of cells and function to suppress macrophage and fibroblast activation under steady-state conditions, respectively. These data indicate that either preventing epithelial cell loss that occurs after irradiation or replacing important mediators of immune and fibroblast activity produced by the epithelium are potentially important strategies for preventing or treating this unique injury.
Purpose: Thoracic irradiation injures lung parenchyma, triggering inflammation and immune cell activation, leading to pneumonitis and fibrosis. Macrophage polarization contributes to these processes. ...Since IL-4 promotes pro-fibrotic macrophage activation, its role in radiation-induced lung injury was investigated.
Materials and methods: Lung macrophage subpopulations were characterized from 3-26 weeks following exposure of WT and IL-4−/− mice to 0 or 12.5 Gray single dose thoracic irradiation.
Results: Loss of IL-4 did not prevent fibrosis, but blunted macrophage accumulation within the parenchyma. At 3 weeks following exposure, cell numbers and expression of F4/80 and CD206, an alternative activation marker, decreased in alveolar macrophages but increased in infiltrating macrophages in WT mice. Loss of IL-4 impaired recovery of these markers in alveolar macrophages and blunted expansion of these populations in infiltrating macrophages. CD206+ cells were evident in fibrotic regions of WT mice only, however Arg-1+ cells increased in fibrotic regions in IL-4−/− mice only. Radiation-induced proinflammatory Ly6C expression was more apparent in alveolar and interstitial macrophages from IL-4−/− mice.
Conclusions: IL-4 loss did not prevent alternative macrophage activation and fibrosis in irradiated mice. Instead, a role is indicated for IL-4 in maintenance of macrophage populations in the lung following high single dose thoracic irradiation.
The brain appears to be a target of air pollution. This study aimed to further ascertain behavioral and neurobiological mechanisms of our previously observed preference for immediate reward (Allen, ...J. L., Conrad, K., Oberdorster, G., Johnston, C. J., Sleezer, B., and Cory-Slechta, D. A. (2013). Developmental exposure to concentrated ambient particles and preference for immediate reward in mice. Environ. Health Perspect. 121, 32-38), a phenotype consistent with impulsivity, in mice developmentally exposed to inhaled ultrafine particles. It examined the impact of postnatal and/or adult concentrated ambient ultrafine particles (CAPS) or filtered air on another behavior thought to reflect impulsivity, Fixed interval (FI) schedule-controlled performance, and extended the assessment to learning/memory (novel object recognition (NOR)), and locomotor activity to assist in understanding behavioral mechanisms of action. In addition, levels of brain monoamines and amino acids, and markers of glial presence and activation (GFAP, IBA-1) were assessed in mesocorticolimbic brain regions mediating these cognitive functions. This design produced four treatment groups/sex of postnatal/adult exposure: Air/Air, Air/CAPS, CAPS/Air, and CAPS/CAPS. FI performance was adversely influenced by CAPS/Air in males, but by Air/CAPS in females, effects that appeared to reflect corresponding changes in brain mesocorticolimbic dopamine/glutamate systems that mediate FI performance. Both sexes showed impaired short-term memory on the NOR. Mechanistically, cortical and hippocampal changes in amino acids raised the potential for excitotoxicity, and persistent glial activation was seen in frontal cortex and corpus callosum of both sexes. Collectively, neurodevelopment and/or adulthood CAPS can produce enduring and sex-dependent neurotoxicity. Although mechanisms of these effects remain to be fully elucidated, findings suggest that neurodevelopment and/or adulthood air pollution exposure may represent a significant underexplored risk factor for central nervous system diseases/disorders and thus a significant public health threat even beyond current appreciation.
Abstract As materials are produced at smaller scales, the properties that make them especially useful for biological applications such as drug delivery, imaging or sensing applications also render ...them potentially harmful. There has been a reasonable amount of work addressing the interactions of biological fluids at material surfaces that demonstrates the high affinity of protein for particle surfaces and some looking at the role of particle surface chemistry in cellular associations, but mechanisms have been too little addressed outside the context of intended, specific interactions. Here, using cultured endothelium as a model for vascular transport, we demonstrate that the capacity of nanoparticle surfaces to adsorb protein is indicative of their tendency to associate with cells. Quantification of adsorbed protein shows that high binding nanoparticles are maximally coated in seconds to minutes, indicating that proteins on particle surfaces can mediate cell association over much longer time scales. We also remove many of the most abundant proteins from culture media which alters the profile of adsorbed proteins on nanoparticles but does not affect the level of cell association. We therefore conclude that cellular association is not dependent on the identity of adsorbed proteins and therefore unlikely to require specific binding to any particular cellular receptors.
Models of thoracic irradiation have been developed as clinicians and scientists have attempted to decipher the events that led up to the pulmonary toxicity seen in human subjects following radiation ...treatment. The most common model is that of whole thorax irradiation (WTI), applied in a single dose. Mice, particularly the C57BL/6J strain, has been frequently used in these investigations, and has greatly informed our current understanding of the initiation and progression of radiation-induced lung injury (RILI). In this review, we highlight the sequential progression and dynamic nature of RILI, focusing primarily on the vast array of information that has been gleaned from the murine model. Ample evidence indicates a wide array of biological responses that can be seen following irradiation, including DNA damage, oxidative stress, cellular senescence and inflammation, all triggered by the initial exposure to ionizing radiation (IR) and heterogeneously maintained throughout the temporal progression of injury, which manifests as acute pneumonitis and later fibrosis. It appears that the early responses of specific cell types may promote further injury, disrupting the microenvironment and preventing a return to homeostasis, although the exact mechanisms driving these responses remains somewhat unclear. Attempts to either prevent or treat RILI in preclinical models have shown some success by targeting these disparate radiobiological processes. As our understanding of the dynamic cellular responses to radiation improves through the use of such models, so does the likelihood of preventing or treating RILI.
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