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 flux 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, 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. 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 target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that 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 assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

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. 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.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms Recent reviews have described the range of assays that have been used for this purpose(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi) Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes This set of guidelines is 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 verify an autophagic response

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-4sx3t3he7d.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Although it was thought that apoptotic cells, when rapidly phagocytosed, underwent a silent death that did not trigger an immune response, in recent years a new concept of immunogenic cell death (ICD) has emerged. The immunogenic characteristics of ICD are mainly mediated by damage-associated molecular patterns (DAMPs), which include surface-exposed calreticulin (CRT), secreted ATP and released high mobility group protein B1 (HMGB1). Most DAMPs can be recognized by pattern recognition receptors (PRRs). In this Review, we discuss the role of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in regulating the immunogenicity of dying cancer cells and the effect of therapy-resistant cancer microevolution on ICD.

Immunogenic cell death and DAMPs in cancer therapy.

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 (4th edition)

We have devised an ex vivo culture system which generates large numbers of eosinophils at high purity (>90%) from unselected mouse bone marrow progenitors. In response to 4 days of culture with recombinant mouse FLT3-L and recombinant mouse stem cell factor followed by recombinant mouse IL-5 alone thereafter, the resulting bone marrow-derived eosinophils (bmEos) express immunoreactive major basic protein, Siglec F, IL-5R alpha-chain, and transcripts encoding mouse eosinophil peroxidase, CCR3, the IL-3/IL-5/GM-CSF receptor common beta-chain, and the transcription factor GATA-1. BmEos are functionally competent: they undergo chemotaxis toward mouse eotaxin-1 and produce characteristic cytokines, including IFN-gamma, IL-4, MIP-1alpha, and IL-6. The rodent pathogen pneumonia virus of mice replicates in bmEos and elevated levels of IL-6 are detected in supernatants of bmEos cultures in response to active infection. Finally, differentiating bmEos are readily transfected with lentiviral vectors, suggesting a means for rapid production of genetically manipulated cells.

/pdf/functionally-competent-eosinophils-differentiated-ex-vivo-in-44q1s50d3l.pdf

Functionally Competent Eosinophils Differentiated Ex Vivo in High Purity from Normal Mouse Bone Marrow

Human Platelets Exhibit Chemotaxis using Functional N-Formyl Peptide Receptors

https://www.exphem.org/article/S0301-472X(03)00390-4/pdf

Platelets deliver costimulatory signals to antigen-presenting cells: a potential bridge between injury and immune activation.

/pdf/photoconversion-of-lysotracker-red-to-a-green-fluorescent-40w6c04kh5.pdf

Photoconversion of Lysotracker Red to a green fluorescent molecule.

Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS in humans, results in a progressive depletion of CD4+ T lymphocytes that culminates in fatal immunodeficiency. Although it is clear that HIV is responsible for massive early destruction of memory CD4+ T lymphocytes (7), the subsequent disease course is prolonged and variable, suggesting additional mechanisms in the chronic stages of the disease. The long asymptomatic period of infection is associated with increased immune activation particularly of the remaining memory CD4+ T cells, increased rates of spontaneous apoptosis, abnormalities in CD4+ T-cell function (13, 14, 20, 21, 24), and destruction of the lymphoid architecture (69). The degree of T-cell activation has value in predicting HIV infection survival time (13, 24). This has led many to suggest that the mechanisms underlying AIDS are more complex than a simple model of slow virus-induced CD4+ T-cell death (6, 18, 28, 29).

Simian immunodeficiency virus (SIV) originating in sooty mangabey monkeys is perhaps the most relevant model of human AIDS currently available. This lineage includes the virus most commonly used in the field, SIVmac239, which arose following the introduction of SIVsm into rhesus macaques housed in U.S. primate centers (12, 71). However, equally relevant models have been developed by using SIVsm strains from captive sooty mangabeys, including the closely related SIVsmE660 and molecularly cloned SIVsmE543-3 strains derived in our laboratory (1, 31, 34, 35, 42, 63). SIVsm/mac and HIV are remarkably similar in terms of biology and pathogenesis in vivo. Both target CD4+ cells, including T cells and macrophages, and use the chemokine coreceptor CCR5, resulting in direct destruction of memory CD4+ T cells at mucosal sites (7, 48, 50, 60, 66). SIV infection of macaques covers a continuum in terms of rate of disease progression, similar to HIV infection of humans (5, 19, 31, 33, 34, 38, 49, 62, 73, 79). The level at which the virus stabilizes following primary infection is predictive of the rate of disease progression in both SIV and HIV infections (33, 53, 76). Both are associated with intense immune activation during the chronic phase of disease (13, 24, 48, 70, 72), and the pathological manifestations of AIDS are similar (5, 47, 51, 71). End stage disease is characterized by terminal blood and tissue CD4+ T-cell depletion, infections with a variety of opportunistic pathogens (5, 47, 51, 71), and the development of virus-induced encephalitis (3, 17, 26, 57, 61, 62, 68, 81, 82).

Despite remarkable similarities between simian AIDS and human AIDS, there are a number of differences that must be considered. First, the disease course of SIV infection of macaques is compressed relative to that of HIV infection, with a median survival time of 1 to 3 years versus 10 years in untreated human AIDS. Second, SIVsm/mac virus isolates use CCR5 as their primary coreceptor; the emergence of CXCR4-using isolates observed in many human AIDS patients is only rarely observed in SIV infection (45). However, since isolates recovered early in infection with HIV preferentially use CCR5 as a coreceptor, the pathogenesis of SIV and that of HIV appear more similar to one another than to that of CXCR4-using SIV-HIV isolates (39, 60). Third, while rare individuals infected with HIV-1 rapidly develop disease (22, 23, 54, 80), the frequency of an accelerated disease course is much higher in SIV-infected macaques. About 10 to 25% of rhesus macaques inoculated with pathogenic strains of SIVmac/sm fail to mount measurable immune responses and develop disease in less than 6 months (5, 16, 34, 71, 79). Finally, the pathology of simian AIDS includes descriptions of SIV-induced disease in which multinucleated giant cells (MNGC) in the lymphoid tissues, brain, and lungs are a prominent feature (5, 47, 51). With the exception of the brains of HIV-infected patients with encephalitis, MNGC are rarely observed in tissues of humans with AIDS. In our studies, the presence of MNGC was associated with extremely high viremia and transient immune responses, both signs of rapid progression (31, 37, 46). Retrospective studies have also shown that SIV encephalitis (SIVE) is highly correlated with rapid disease progression (3, 61, 62, 77). We speculated that rapid progression of SIV infection of macaques might constitute a unique pathological syndrome, distinct from the classical descriptions of SIV-induced AIDS. The goal of the present study was to compare pathology, virus distribution, and target cells by in situ hybridization (ISH) and confocal microscopy in terminal tissues of conventional progressors (CP) and rapid progressors (RP) infected with SIVsmE660 or SIVsmE543-3.

Unique Pathology in Simian Immunodeficiency Virus-Infected Rapid Progressor Macaques Is Consistent with a Pathogenesis Distinct from That of Classical AIDS

Meggan Czapiga

Papers

Functionally Competent Eosinophils Differentiated Ex Vivo in High Purity from Normal Mouse Bone Marrow

Human Platelets Exhibit Chemotaxis using Functional N-Formyl Peptide Receptors

Platelets deliver costimulatory signals to antigen-presenting cells: a potential bridge between injury and immune activation.

Photoconversion of Lysotracker Red to a green fluorescent molecule.

Unique Pathology in Simian Immunodeficiency Virus-Infected Rapid Progressor Macaques Is Consistent with a Pathogenesis Distinct from That of Classical AIDS