Table of Contents

Abstract: Autophagy is an intracellular degradation pathway for long-lived proteins and organelles. This process is activated above basal levels upon cell intrinsic or environmental stress and dysregulation of autophagy has been linked to various human diseases, including those caused by viral infection. Many viruses have evolved strategies to directly interfere with autophagy, presumably to facilitate their replication or to escape immune detection. However, in some cases, modulation of autophagy appears to be a consequence of the virus disturbing the cell’s metabolic signaling networks. Here, we summarize recent advances in research at the interface of autophagy and viral infection, paying special attention to strategies that human tumor viruses have evolved.

Abstract: Autophagy is a process of cellular self-degradation and is a major pathway for elimination of cytoplasmic material by the lysosomes. Autophagy is responsible for the degradation of damaged organelles and protein aggregates and therefore plays a significant role in cellular homeostasis. Despite the initial belief that autophagy is a nonselective bulk process, there is growing evidence during the last years that sequestration and degradation of cellular material by autophagy can be accomplished in a selective and specific manner. Given the role of autophagy and selective autophagy in several disease related processes such as tumorigenesis, neurodegeneration and infections, it is very important to dissect the molecular mechanisms of selective autophagy, in the context of the system and the organism. An excellent genetically tractable model organism to study autophagy is Drosophila, which appears to have a highly conserved autophagic machinery compared with mammals. However, the mechanisms of selective autophagy in Drosophila have been largely unexplored. The aim of this review is to summarize recent discoveries about the selectivity of autophagy in Drosophila.

Abstract: The budding yeast Saccharomyces cerevisiae responds to nutritional stress through the regulated activities of signaling pathways mediating autophagy and other conserved cellular processes. Autophagy has been studied intensely in yeast, where over 30 autophagy-related genes have been identified with defined roles enabling the formation of autophagic vesicles and their subsequent trafficking to the central yeast vacuole. Much less, however, is known regarding the regulatory mechanisms through which autophagy is integrated with other yeast stress responses. Nitrogen limitation initiates autophagy and pseudohyphal growth in yeast, the latter being a fascinating stress response characterized by the formation of multicellular chains or filaments of elongated cells. An increasing body of evidence suggests an interrelationship between processes responsive to nitrogen stress with cAMP-dependent PKA and the TOR kinase complex acting as key regulators of autophagy, pseudohyphal growth, and endocytosis. In this review, we will summarize our current understanding of the regulatory events controlling these processes. In particular, we explore the interplay between autophagy, polarized pseudohyphal growth, and to a lesser extent endocytosis, and posit that the integrated response of these processes in yeast is a critical point for further laboratory experimentation as a model of cellular responses to nitrogen limitation throughout the Eukaryota.

Abstract: Autophagy is an intracellular degradation process responsible for the delivery of cellular material to the lysosomes. One of the key mechanisms for control of autophagy is the modulation of the interaction between the autophagic protein Beclin 1 and the members of the anti-apoptotic Bcl-2 family (e.g., Bcl-2, Bcl-X_L and Mcl-1). This binding is regulated by a variety of proteins and compounds that are able to enhance or inhibit the Bcl-2/Beclin 1 interaction in order to repress or activate autophagy, respectively. In this review we will focus on this interaction and discuss its characteristics, relevance and regulation.

Abstract: Cryopreserved peripheral blood mononuclear cells (PBMC) constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments.

Abstract: Skeletal muscles are the agent of motion and one of the most important tissues responsible for the control of metabolism. The maintenance of muscle homeostasis is finely regulated by the balance between catabolic and anabolic process. Macroautophagy (or autophagy) is a catabolic process that provides the degradation of protein aggregation and damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagy flux is fundamental for the homeostasis of skeletal muscles during physiological situations and in response to stress. Defective as well as excessive autophagy is harmful for muscle health and has a pathogenic role in several forms of muscle diseases. This review will focus on the role of autophagy in muscle homeostasis and diseases.

Abstract: Autophagy is a ubiquitous eukaryotic process that also occurs in trypanosomatid parasites, protist organisms belonging to the supergroup Excavata, distinct from the supergroup Opistokontha that includes mammals and fungi. Half of the known yeast and mammalian AuTophaGy (ATG) proteins were detected in trypanosomatids, although with low sequence conservation. Trypanosomatids such as Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for serious tropical diseases in humans. The parasites are transmitted by insects and, consequently, have a complicated life cycle during which they undergo dramatic morphological and metabolic transformations to adapt to the different environments. Autophagy plays a major role during these transformations. Since inhibition of autophagy affects the transformation, survival and/or virulence of the parasites, the ATGs offer promise for development of drugs against tropical diseases. Furthermore, various trypanocidal drugs have been shown to trigger autophagy-like processes in the parasites. It is inferred that autophagy is used by the parasites in an—not always successful—attempt to cope with the stress caused by the toxic compounds.

Abstract: Autophagy is an evolutionarily conserved process that contributes to maintain cell homeostasis. Although it is strongly regulated by many extracellular factors, induction of autophagy is mainly produced by starvation of nutrients. In mammalian cells, the regulation of autophagy by amino acids, and also by the hormone insulin, has been extensively investigated, but knowledge about the effects of other autophagy regulators, including another nutrient, glucose, is more limited. Here we will focus on the signalling pathways by which environmental glucose directly, i.e., independently of insulin and glucagon, regulates autophagy in mammalian cells, but we will also briefly mention some data in yeast. Although glucose deprivation mainly induces autophagy via AMPK activation and the subsequent inhibition of mTORC1, we will also comment other signalling pathways, as well as evidences indicating that, under certain conditions, autophagy can be activated by glucose. A better understanding on how glucose regulates autophagy not only will expand our basic knowledge of this important cell process, but it will be also relevant to understand common human disorders, such as cancer and diabetes, in which glucose levels play an important role.

Abstract: Autophagy is an intracellular degradative process with a number of roles, one of which can be the protection of eukaryotic cells from invading microbes. Microtubule-associated protein light-chain 3 (LC3) is a key autophagy-related protein that is recruited to the double-membrane autophagosome responsible for sequestering material intended for delivery to lysosomes. GFP-LC3 is widely used as a marker of autophagosome formation as denoted by the formation of green puncta when viewed by fluorescence microscopy. Recently, it has been demonstrated that LC3 can be recruited to other membranes including single-membrane phagosomes, in a process termed LC3-associated phagocytosis (LAP). Thus, the observation of green puncta in cells can no longer, by itself, be taken as evidence of autophagy. This review will clarify those features of LAP which serve to distinguish it from autophagy and that make connections with host autophagic responses in terms of infection by microbial pathogens. More specifically, it will refer to concurrent studies of the mechanism by which LAP is triggered in comparison to autophagy.

Abstract: T cell monitoring is increasingly performed using cryopreserved PBMC. It has been suggested that resting of PBMC after thawing, that is, culturing them overnight in test medium, produces higher antigen-induced spot counts in ELISPOT assays. To evaluate the importance of overnight resting, we systematically tested cryopreserved PBMC from 25 healthy donors. CEF peptides (comprising CMV, EBV and flu antigens) were used to stimulate CD8 cells and mumps antigen to stimulate CD4 cells. The data show that resting significantly increased antigen-elicited T cell responses only for CEF high responder PBMC. The maximal gain observed was doubling of spot counts. For CEF low responders, and for mumps responders of either low- or high reactivity levels, resting had no statistically significant effect on the observed spot counts. Therefore, resting is not a generally applicable approach to improve ELISPOT assay performance, but can be recommended only for clinical subject cohorts and antigens for which it has a proven benefit. Because resting invariably leads to losing about half of the PBMC available for testing, and because doubling the PBMC numbers plated into the assay reliably doubles the antigen-induced spot counts, we suggest the latter approach as a simple and reliable alternative to resting for enhancing the performance of ELISPOT assays. Our data imply that resting is not required if PBMC were cryopreserved and thawed under conditions that minimize apoptosis of the cells. Therefore, this study should draw attention to the need to optimize freezing and thawing conditions for successful T cell work.

Abstract: Autophagy is an evolutionarily conserved catabolic process by which cells degrade their own components through the lysosomal machinery. In physiological conditions, the mechanism is tightly regulated and contributes to maintain a balance between synthesis and degradation in cells undergoing intense metabolic activities. Autophagy is associated with major tissue remodeling processes occurring through the embryonic, fetal and early postnatal periods of vertebrates. Here we survey current information implicating autophagy in cellular death, proliferation or differentiation in developing vertebrates. In developing systems, activation of the autophagic machinery could promote different outcomes depending on the cellular context. Autophagy is thus an extraordinary tool for the developing organs and tissues.

Abstract: Macroautophagy is a non-selective, bulk degradation process conserved in eukaryotes. Response to starvation stress and/or regulation of nutrient breakdown/utilization is the major intracellular function of macroautophagy. Recent studies have revealed requirement for autophagy in diverse functions such as nutrient homeostasis, organelle degradation and programmed cell death in filamentous fungal pathogens, for proper morphogenesis and differentiation during critical steps of infection. In this review, we aim to summarize the physiological functions of autophagy in fungal virulence, with an emphasis on nutrient homeostasis in opportunistic human fungal pathogens and in the rice-blast fungus, Magnaporthe oryzae. We briefly summarize the role of autophagy on the host side: for resistance to, or subversion by, the pathogens.

Abstract: Autophagy is an important cellular program with a “double face” role, since it promotes either cell survival or cell death, also in cancer therapies. Its survival role occurs by recycling cell components during starvation or removing stressed organelles; when damage becomes extensive, autophagy provides another programmed cell death pathway, known as Autophagic Cell Death (ACD). The induction of autophagy is a common outcome in PhotoDynamic Therapy (PDT), a two-step process involving the irradiation of photosensitizer (PS)-loaded cancer cells. Upon tissue oxygen interaction, PS provokes immediate and direct Reactive Oxygen Species (ROS)-induced damage to Endoplasmic Reticulum (ER), mitochondria, plasma membrane, and/or lysosomes. The main biological effects carried out in cancer PDT are direct cytotoxicity to tumor cells, vasculature damage and induction of inflammatory reactions stimulating immunological responses. The question about the role of autophagy in PDT and its putative immunological impact is hotly controversial and largely studied in recent times. This review deals with the induction of autophagy in PDT protocols and its dual role, also considering its interrelationship with apoptosis, the preferential cell death program triggered in the photodynamic process.

Abstract: (Macro)-autophagy is a homeostatic process by which eukaryotic cells dispose of protein aggregates and damaged organelles. Autophagy is also used to degrade micro-organisms that invade intracellularly in a process termed xenophagy. Genome-wide association scans have recently identified autophagy genes as conferring susceptibility to Crohn’s disease (CD), one of the chronic inflammatory bowel diseases, with evidence suggesting that CD arises from a defective innate immune response to enteric bacteria. Here we review the emerging role of autophagy in CD, with particular focus on xenophagy and enteric E. coli strains with an adherent and invasive phenotype that have been consistently isolated from CD patients with ileal disease.

Abstract: Autophagy is a housekeeping survival mechanism with a protective function against stress conditions. However, when stress severity or duration increases, it may promote cell death. Paradoxically, autophagy favors cancer development, since cancer cells could enhance their proliferation potential (thus becoming able to resist anticancer therapy) thanks to the energetic supply provided by organelle degradation typically driven by autophagy following a stepwise pathway. The main actors of the autophagic machinery as well as the features shared with apoptosis will be described. Special attention will be paid to the effects of autophagy manipulation.

Abstract: The intestine lies at the interface between the organism and its environment and responds to infection/inflammation in a multi-leveled manner, potentially leading to chronic inflammatory pathologies and cancer formation. Indeed, the immune response at the intestinal epithelium has been found to be involved in the origin and development of colorectal cancer, which is the third most commonly diagnosed neoplastic disease. Among the mechanisms induced upon inflammation, autophagy appears as a defensive strategy for the clearance of invading microbes and intracellular waste components. Autophagy has also been found to play an important role in colorectal cancer, where it seems to have a pro-survival or pro-death function depending on the stage of the neoplastic process. In this paper we discuss the dual role of autophagy in colorectal cancer and review evidence showing that modulation of autophagy affects the immune response and cancer biology. The study of key players involved in autophagy might contribute to the design of new approaches for colorectal cancer, consisting in combined therapies capable of modifying cancer-specific metabolism rather than simply evoking a generic apoptotic and/or autophagic response, thus enhancing the efficacy of currently used drugs and treatments.

Abstract: All the therapeutic strategies for treating cancers aim at killing the cancer cells via apoptosis (programmed cell death type I). Defective apoptosis endow tumor cells with survival. The cell can respond to such defects with autophagy. Autophagy is a cellular process by which cytoplasmic material is either degraded to maintain homeostasis or recycled for energy and nutrients in starvation. A plethora of evidence has shown that the role of autophagy in tumors is complex. A lot of effort is needed to underline the functional status of autophagy in tumor progression and treatment, and elucidate how to tweak autophagy to treat cancer. Furthermore, during the treatment of cancer, the limitation for the cure rate and survival is the phenomenon of multi drug resistance (MDR). The development of MDR is an intricate process that could be regulated by drug transporters, enzymes, anti-apoptotic genes or DNA repair mechanisms. Reports have shown that autophagy has a dual role in MDR. Furthermore, it has been reported that activation of a death pathway may overcome MDR, thus pointing the importance of other death pathways to regulate tumor cell progression and growth. Therefore, in this review we will discuss the role of autophagy in MDR tumors and a possible link amongst these phenomena.

Abstract: Experimental allergic encephalomyelitis (EAE) is mediated by neuroantigen-specific pro-inflammatory T cells of the Th1 and Th17 effector class. Th-17 cells can be clearly defined by expression of IL-17, but not IFN-γ, IL-2 or IL-3. Th1 cells do not express IL-17, but it is unclear presently to what extent they co-express the cytokines canonically assigned to Th1 immunity (i.e., IFN-γ, IL-2 and IL-3) and whether CD4 cells producing these cytokines indeed belong to a single Th1 lineage. It is also unclear to what extent the Th1 response in EAE entails polyfunctional T cells that co-express IFN-γ and IL-2. Therefore, we dissected the Th1 cytokine signature of neuroantigen-specific CD4 cells studying at single cell resolution co-expression of IFN-γ, IL-2 and IL-3 using dual color cytokine ELISPOT analysis. Shortly after immunization, in the draining lymph nodes (dLN), the overall cytokine signature of the neuroantigen-specific CD4 cells was highly type 1-polarized, but IFN-γ, IL-2, and IL-3 were each secreted by different CD4 cells in a mutually exclusive manner. This single cell – single cytokine profile was stable through the course of chronic EAE–polyfunctional CD4 cells co-expressing IL-2 and IFN-γ presented less than 5% of the neuroantigen-specific T cells, even in the inflamed CNS itself. The neuroantigen-specific CD4 cells that expressed IFN-γ, IL-2 and IL-3 in a mutually exclusive manner exhibited similar functional avidities and kinetics of cytokine production, but showed different tissue distributions. These data suggest that Th1 cells do not belong to a single lineage, but different Th1 subpopulations jointly mediate Th1 immunity.

Abstract: In recent years, research on the autophagic process has greatly increased, invading the fields of biology and medicine. Several markers of the autophagic process have been discovered and various strategies have been reported studying this molecular process in different biological systems in both physiological and stress conditions. Furthermore, mechanisms of metalloid- or heavy metal-induced toxicity continue to be of interest given the ubiquitous nature and distribution of these contaminants in the environment where they often play the role of pollutants of numerous organisms. The aim of this review is a critical analysis and correlation of knowledge of autophagic mechanisms studied under stress for the most common arsenic (As) and cadmium (Cd) compounds. In this review we report data obtained in different experimental models for each compound, highlighting similarities and/or differences in the activation of autophagic processes. A more detailed discussion will concern the activation of autophagy in Cd-exposed sea urchin embryo since it is a suitable model system that is very sensitive to environmental stress, and Cd is one of the most studied heavy metal inductors of stress and modulator of different factors such as: protein kinase and phosphatase, caspases, mitochondria, heat shock proteins, metallothioneins, transcription factors, reactive oxygen species, apoptosis and autophagy.

Abstract: Viruses commandeer regulatory pathways of their hosts to optimize their success as cellular parasites. The human tumor viruses, Epstein-Barr Virus (EBV), Kaposi’s Sarcoma Herpesvirus (KSHV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV) all affect autophagy for their own ends. EBV and KSHV regulate it during latent infections, a phase when no progeny virus is produced, while HBV and HCV use autophagy to promote their productive infections. Here we shall compare and contrast how these human tumor viruses regulate autophagy and what they gain by the appropriation of this cellular pathway.

Abstract: Human enamel development of the permanent teeth takes place during childhood and stresses encountered during this period can have lasting effects on the appearance and structural integrity of the enamel. One of the most common examples of this is the development of dental fluorosis after childhood exposure to excess fluoride, an elemental agent used to increase enamel hardness and prevent dental caries. Currently the molecular mechanism responsible for dental fluorosis remains unknown; however, recent work suggests dental fluorosis may be the result of activated stress response pathways in ameloblasts during the development of permanent teeth. Using fluorosis as an example, the role of stress response pathways during enamel maturation is discussed.

Abstract: Virus infections induce heat shock proteins that in turn enhance virus gene expression, a phenomenon that is particularly well characterized for the major inducible 70 kDa heat shock protein (hsp70). However, hsp70 is also readily induced by fever, a phylogenetically conserved response to microbial infections, and when released from cells, hsp70 can stimulate innate immune responses through toll like receptors 2 and 4 (TLR2 and 4). This review examines how the virus-hsp70 relationship can lead to host protective innate antiviral immunity, and the importance of hsp70 dependent stimulation of virus gene expression in this host response. Beginning with the well-characterized measles virus-hsp70 relationship and the mouse model of neuronal infection in brain, we examine data indicating that the innate immune response is not driven by intracellular sensors of pathogen associated molecular patterns, but rather by extracellular ligands signaling through TLR2 and 4. Specifically, we address the relationship between virus gene expression, extracellular release of hsp70 (as a damage associated molecular pattern), and hsp70-mediated induction of antigen presentation and type 1 interferons in uninfected macrophages as a novel axis of antiviral immunity. New data are discussed that examines the more broad relevance of this protective mechanism using vesicular stomatitis virus, and a review of the literature is presented that supports the probable relevance to both RNA and DNA viruses and for infections both within and outside of the central nervous system.