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- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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- Mohajerani, Pouyan, et al.
(författare)
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FMT-PCCT: Hybrid Fluorescence Molecular Tomography-X-Ray Phase-Contrast CT Imaging of Mouse Models
- 2014
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Ingår i: IEEE Transactions on Medical Imaging. - 1558-254X. ; 33:7, s. 1434-1446
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Tidskriftsartikel (refereegranskat)abstract
- The implementation of hybrid fluorescencemolecular tomography (FMT) and X-ray computed tomography (CT) has been shown to be a necessary development, not only for combining anatomical with functional and molecular contrast, but also for generating optical images of high accuracy. FMT affords highly sensitive 3-D imaging of fluorescence bio-distribution, but in stand-alone form it offers images of low resolution. It was shown that FMT accuracy significantly improves by considering anatomical priors from CT. Conversely, CT generally suffers from low soft tissue contrast. Therefore utilization of CT data as prior information in FMT inversion is challenging when different internal organs are not clearly differentiated. Instead, we combined herein FMT with emerging X-ray phase-contrast CT (PCCT). PCCT relies on phase shift differences in tissue to achieve soft tissue contrast superior to conventional CT. We demonstrate for the first time FMT-PCCT imaging of different animal models, where FMT and PCCT scans were performed in vivo and ex vivo, respectively. The results show that FMT-PCCT expands the potential of FMT in imaging lesions with otherwise low or no CT contrast, while retaining the cost benefits of CT and simplicity of hybrid device realizations. The results point to the most accurate FMT performance to date.
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- Volarevic, Vladislav, et al.
(författare)
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Gal-3 regulates the capacity of dendritic cells to promote NKT-cell-induced liver injury
- 2015
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Ingår i: European Journal of Immunology. - : Wiley. - 1521-4141 .- 0014-2980. ; 45:2, s. 531-543
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Tidskriftsartikel (refereegranskat)abstract
- Galectin-3 (Gal-3), an endogenous lectin, exhibits ro- and anti-inflammatory effects in various disease conditions. In order to explore the role of Gal-3 in KT-cell-dependent pathology, we induced hepatitis in C57BL/6WT and al-3-deficient mice by using specific ligand for KT cells: alpha-galactosylceramide, glycolipid Ag presented by CD1d. The injection of alpha-galactosylceramide significantly enhanced expression of Gal-3 in liver NKT and dendritic cells (DCs). Genetic deletion or selective inhibition of Gal-3 (induced by Gal-3-inhibitor TD139) abrogated the susceptibility to NKT-cell-dependent hepatitis. Blood levels of pro-inflammatory cytokines (TNF-alpha-, IFN-gamma, IL-12) and their production by liver DCs and NKT cells were also downregulated. Genetic deletion or selective inhibition of Gal-3 alleviated influx of inflammatory CD11c(+) CD11b(+) DCs in the liver and favored tolerogenic phenotype and IL-10 production of liver NKT and DCs. Deletion of Gal-3 attenuated the capacity of DCs to support liver damage in the passive transfer experiments and to produce pro-inflammatory cytokines in vitro. Gal-3-deficient DCs failed to optimally stimulate production of pro-inflammatory cytokines in NKT cells, in vitro and in vivo. In conclusion, Gal-3 regulates the capacity of DCs to support NKT-cell-mediated liver injury, playing an important pro-inflammatory role in acute liver injury.
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