| 1. |
- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)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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| 2. |
- Chen, Lanlin, et al.
(author)
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A nephritogenic peptide induces intermolecular epitope spreading on collagen IV in experimental autoimmune glomerulonephritis
- 2006
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In: Journal of the American Society of Nephrology. - 1046-6673. ; 17:11, s. 3076-3081
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Journal article (peer-reviewed)abstract
- This group previously identified a peptide p13 of alpha 3(IV)NC1 domain of type IV collagen that induces experimental autoimmune glomerulonephritis (EAG) in rats with generation of antibodies to sites on alpha 3(IV)NC1 external to the peptide as a result of intramolecular epitope spreading. It was hypothesized that intermolecular epitope spreading to other collagen IV chains also was induced. Rats were immunized with nephritogenic peptide that was derived from the amino terminal part of rat alpha 3(IV)NC1 domain, and serum and kidney eluate were examined for antibodies to both native and recombinant NC1 domains of collagen IV. Peptide induced EAG with proteinuria and decreased renal function and glomerular basement membrane IgG deposits. Sera from these rats were examined by ELISA, which revealed reactivity not only to immunizing peptide but also to human and rat alpha 3(IV)NC1 and to human alpha 4(IV)NC1 domains. Kidney eluate that was depleted of alpha 3(IV)NC1 antibodies still reacted to alpha 4(IV)NC1, and alpha 3(IV)NC1 column-bound antibody reacted with alpha 3(IV)NC1. There was minimal reactivity to other collagen chains. Eluate that was adsorbed to NC1 hexamer from rat glonterular basement membrane lost all reactivity to glomerular constituents, and the eluted antibodies reacted to alpha 3(IV)NC1 and alpha 4(IV)NC1 domains. These studies show that a T cell epitope of alpha 3(IV)NC1 induces EAG, intramolecular epitope spreading along alpha 3(IV)NC1, and intermolecular epitope spreading to alpha 4(IV)NC1 domain with minimal or no reactivity to other collagen chains or glomerular constituents. This is the first demonstration in EAG of intermolecular epitope spreading and identification of the spread epitopes.
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| 3. |
- Bolton, W Kline, et al.
(author)
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Molecular mapping of the Goodpasture's epitope for glomerulonephritis
- 2005
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In: Transactions of the American Clinical and Climatological Association. - 0065-7778. ; 116, s. 229-238
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Journal article (peer-reviewed)abstract
- Goodpasture's syndrome is an autoimmune disease characterized by pulmonary hemorrhage, glomerulonephritis, and antiglomerular basement membrane (GBM) antibodies. We have studied a rat model with chimeric proteins (CPs) consisting of portions of the nephritogenic non-collagenous domain of alpha3 type IV collagen (alpha3(IV)NC1) and non-nephritogenic alpha1(IV)NC1. CPs with aminoterminal alpha3 that contains the major epitope for Goodpasture antibody binding induced EAG. We next immunized with D3, an alpha1(IV)NC1 CP with 69AA of alpha3(IV)NC1 (binds Goodpasture sera), D4, the D3 construct shortened by 4 AA (nonbinding), P9 and P10, single AA mutants (nonbinding) and S2, an alpha1(IV)NC1 with nine AA of alpha3(IV)NC1 (binding). GBM, S2 and D3 induced EAG. GBM immunized rats had intense IgG deposits but S2 and D3 rats had minimal deposits. A 13 mer rat peptide encompassing the aminoterminal site induced EAG sans antibody, while peptides not encompassing the region failed to induce GN. Asparagine at position 19 rather than isoleucine was essential for disease induction. These studies define critical limited AA sequences of alpha3(IV)NC1 associated with glomerulonephritis without antibody, and demonstrate that this region contains a T-cell epitope responsible for induction of glomerulonephritis.
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