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

De opvattingen over onze oudere vaderlandsche geschiedenis bij de Hollandsche historici der 16e en 17e eeuw

Proefschrift--Leiden, 1917."Lijst der gebruikte auteurs," p. [xi]-xxviii

Two donor-related infections in a heart transplant recipient: One common, the other a tropical surprise

BACKGROUND Infection is the most frequent complication after heart transplantation (HTx) In this report and brief literature review we present a recipient who some 6 weeks post HTx had two donor related infections a "common' primary cytomegalovirus (CMV) infection and simultaneously a highly unusual donor-related Strongyloides stercoralis infection METHODS The parasite was discovered by chance in a skin biopsy CMV was treated with ganciclovir and the strongyloidiasis was cured with two courses of anti-helminthic therapy initially with ivermectine and albendazol and, in response to eosinophilia with ivermectine monotherapy The patient s recovery was further complicated by two successive rejection episodes a relapse of the CMV syndrome and a novel influenza A/H1N1 infection These episodes were treated with steroids ganciclovir and oseltamivir respectively RESULTS It took almost 9 months before a permanent IgG anti-CMV response was seen At 13 months post-HTx coronary angiography showed only slight vessel wall abnormalities At present the patient is back at home and in good condition CONCLUSION Until now only 4 recipient-derived strongyloidiasis cases have been described in post HTx patients all diagnosed by autopsies This is the first report of a donor related Strongyloides infection in a patient after HTx J Heart Lung Transplant 2010 29 1433-7 (C) 2010 International Society for Heart and Lung Transplantation All rights reserve

Inhibition of T Cell Responses in Vitro by an Antibody Against a Novel Lymphocyte Surface Molecule (QCA-1)

We have identified an antigen present on the surface of lymphocytes in the rat which appears to play an important role in the preliminary stages of the immune response. This antigen, which we have called quiescent cell antigen 1 because of its apparent expression only on quiescent cells, is present on the majority of peripheral T and B cells and a small percentage of thymocytes which are located mainly in the medullary region. SDS-PAGE analysis of membrane molecules shows two bands on unreduced gels at approximately 43 and 47 kd. On reduction the bands ran at approximately 46 and 60 kd. When a monoclonal antibody against this antigen (HIS45) is present in an allogeneic mixed leukocyte reaction, it inhibits the proliferation of responding cells completely. When the antibody HIS45 is added to cytotoxic T lymphocyte mediated lysis assays it does not inhibit lysis nor does it affect the specificity of this lysis. Comparison with other antibodies which have been reported to affect lymphocyte function in rats and in other species fail to reveal any which have similar properties