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

Reactive oxygen/nitrogen species contribute substantially to the antileukemia effect of APO866, a NAD lowering agent.

APO866 is a small molecule drug that specifically inhibits nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis from the natural precursor nicotinamide. Although, the antitumor activity of APO866 on various types of cancer models has been reported, information regarding mechanisms by which APO866 exerts its cytotoxic effects is not well defined. Here we show that APO866 induces a strong, time-dependent increase in highly reactive ROS, nitric oxide, cytosolic/mitochondrial superoxide anions and hydrogen peroxide. We provide evidence that APO866-mediated ROS production is modulated by PARP1 and triggers cell death through mitochondria depolarization and ATP loss. Genetic or pharmacologic inhibition of PARP1 prevented hydrogen peroxide accumulation, caspase activation, mitochondria depolarization, ATP loss and abrogates APO866-induced cell death, suggesting that the integrity of PARP1 status is required for cell death. Conversely, PARP1 activating drugs enhanced the anti-leukemia activity of APO866 Collectively, our studies show that APO866 induces ROS/RNS productions, which mediate its anti-leukemia effect. These results support testing new combinatorial strategies to enhance the antitumor activities of APO866

Autoimmune syndrome after neonatal induction of tolerance to alloantigens: analysis of the specificity and of the cellular and genetic origin of autoantibodies.

BALB/c mice neonatally injected with 10(8) semiallogeneic (C57BL/6 x BALB/c)F1 spleen cells become tolerant to the H-2b alloantigens, but also develop a wide range of autoimmune manifestations characteristic of systemic lupus erythematosus (SLE). Indeed, in these mice, the presence of a hypergammaglobulinaemia, autoantibodies--including anti-ssDNA, anti-platelet, thymocytotoxic and rheumatoid factor antibodies--circulating immune complexes, cryoglobulins as well as renal glomerular deposition of immunoglobulins have been observed. In this study, we have shown that the allogenic effect and B cell chimaerism which characterize these F1 cell-injected mice is associated with the expression of a large spectrum of autoantibodies, including anti-ssDNA and anti-cytoskeleton antibodies, and that these autoantibodies are not multispecific. We took advantage of the fact that, in this model, autoantibodies are exclusively produced by F1 donor B cells to inject newborn BALB/c mice with F1 Xid spleen cells lacking the CD5+ B cell subset. Injection of 2 x 10(8) F1 Xid spleen cells triggers the production of anti-ssDNA as well as anti-BrMRBC antibodies, and these mice developed tissue lesions. Finally, analysis of the VH gene family expressed by monoclonal autoantibodies derived from F1 cell-injected mice showed that they used the 2 largest families J558 and 7183. These results suggest that the allogenic effect and B cell chimerism which characterize the neonatal induction of tolerance to MHC alloantigens is associated with the selective triggering of autoreactive B cells producing monospecific IgG autoantibodies. They also imply that upon stimulation by persisting alloreactive CD4+ T cells, either CD5- B cells are able to produce autoantibodies or autoantibody-producing CD5+ B cells can differentiate from Xid spleen cells.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

L’hématologie au temps du COVID-19 [Hematology in the time of COVID-19]

The COVID-19 pandemic impacts the hematology practice. Intensive chemotherapies for high-grade lymphomas and acute leukemias, multiple myeloma treatments and most hematopoietic stem cell transplantations should be performed as usual. Low-grade lymphomas should only be treated when strictly indicated, maintenance can be postponed. Other myeloid neoplasia and their therapies cause imunosupression; dose adjustment is recommended but no brisk stopping. Sickle cell anemia patients are highly succeptible to severe COVID-19 course. Thrombocytopenia and procoagulant state are associated with severe courses of COVID-19, requiring an individualized therapy. No data indicate a risk of SARS-CoV-2 transmission through blood product transfusion

Gut microbiota severely hampers the efficacy of NAD-lowering therapy in leukemia.

Most cancer cells have high need for nicotinamide adenine dinucleotide (NAD <sup>+</sup> ) to sustain their survival. This led to the development of inhibitors of nicotinamide (NAM) phosphoribosyltransferase (NAMPT), the rate-limiting NAD <sup>+</sup> biosynthesis enzyme from NAM. Such inhibitors kill cancer cells in preclinical studies but failed in clinical ones. To identify parameters that could negatively affect the therapeutic efficacy of NAMPT inhibitors and propose therapeutic strategies to circumvent such failure, we performed metabolomics analyses in tumor environment and explored the effect of the interaction between microbiota and cancer cells. Here we show that tumor environment enriched in vitamin B3 (NAM) or nicotinic acid (NA) significantly lowers the anti-tumor efficacy of APO866, a prototypic NAMPT inhibitor. Additionally, bacteria (from the gut, or in the medium) can convert NAM into NA and thus fuel an alternative NAD synthesis pathway through NA. This leads to the rescue from NAD depletion, prevents reactive oxygen species production, preserves mitochondrial integrity, blunts ATP depletion, and protects cancer cells from death.Our data in an in vivo preclinical model reveal that antibiotic therapy down-modulating gut microbiota can restore the anti-cancer efficacy of APO866. Alternatively, NAphosphoribosyltransferase inhibition may restore anti-cancer activity of NAMPT inhibitors in the presence of gut microbiota and of NAM in the diet