Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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. For example, 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 versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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 fi eld 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. 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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. 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

Poncet’s disease after the intravesical instillation of Bacillus Calmette–Guérin (BCG): a case report

Abstract Background Poncet’s disease is a rare syndrome characterized by articular impairment in a form of rare tuberculid. One of the theories of its cause involves an autoimmune response induced by the intravesical administration of the Calmette–Guerin Bacillus or the treatment of bladder carcinoma. Furthermore, there may be an appearance of oligoarticular or polyarticular arthritis, beginning 1–3 months after the start of therapy. Few physicians know the disease and the literature related to that syndrome is scarce and restricted to case reports, which contributes to its under diagnosis. Case presentation Female patient, 64 years old, Caucasian, in whom was noticed firstly dark urine, without haematuria or dysuria. Later felt also colic pain in the hypogastric region. Microscopically, the conclusive diagnosis was a high grade non-invasive papillary urothelial carcinoma. Thereupon, the treatment of the tumour began with transurethral resection technique and intravesical instillation of Calmette–Guérin Bacillus as adjuvant treatment. Eight months after the beginning of treatment, the lingering presence of the carcinoma was identified. Nevertheless, arthritis was identified through radiographs, after an increase in the clavicle capitation, right knee and left ankle in bone scintigraphy. Coinciding with the joint manifestations, the patient developed fever and purulent urethral discharge (culture was negative). Therefore, trying to investigate the cause of the arthritis, Purified Protein Derivate was taken, with reactive results. An increase of acute phase reactants was found, with other tests resulting normal: blood chemistry, Complete Blood Count, immunology and serology. Human Leukocyte Antigen typing by polymerase chain reaction revealed the presence of A24/AX, B44, B27, BW4/BW4, DQ7 and DQ5. Consequently, Poncet’s disease was the diagnostic conclusion. The treatment with intravesical Calmette–Guérin Bacillus was immediately discontinued. The patient received corticosteroids associated with etoricoxib and isoniazid for 4 months, achieving disappearance of the inflammatory joint signs in 3 months. After 6 months, no joint pain recurrence or other manifestations suggesting active disease had been seen. Conclusions Therefore, such diagnosis should be considered when confronted with an osteoarticular clinical picture in patients treated with intravesical Calmette–Guérin Bacillus, especially patients with HLA-B27 (+) and B7 (+), as Poncet’s disease is a reactive arthritis