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

Toxoplasma gondii is an important protozoan pathogen of humans that can cause encephalitis in immunocompromised individuals such as those with AIDS. This encephalitis is due to reactivation of latent infection in T. gondii-seropositive patients. Latent organisms survive within tissue cysts, which are specialized parasitophorous vacuoles containing bradyzoites. The cyst wall of this structure is produced by modification of the parasitophorous vacuole by the parasite and is important in cyst survival. The components of the cyst wall have been poorly characterized. By using immunofluorescence and immunoelectron microscopy, we have identified a monoclonal antibody (MAb 93.18) that reacts with the cyst wall. This antibody recognizes a 116-kDa glycoprotein, which we have termed CST1, containing sugar residues that bind Dolichos biflorans lectin (DBA). CST1 is distinct from T. gondii antigen labeled with succinyl Triticum vulgare lectin (S-WGA) and represents the major DBA-binding component in T. gondii. The carbohydrate components of the tissue cyst, such as CST1, are probably important in both providing stability and facilitating persistence in its host. As is seen in the carbohydrate capsules of fungi, glycoproteins in the T. gondii cyst wall may protect cysts from the immune response of the host. Further characterization of the formation of the cyst wall and its components should lead to insights into the mechanism of tissue cyst persistence and may suggest novel therapeutic approaches to eliminate tissue cysts of this organism.

Toxoplasma gondii is an important pathogen in the central nervous system, where it causes a severe and often fatal encephalitis in patients with AIDS. Cytokines play an important role in the regulation of T. gondii replication in the central nervous system (5, 10, 11). Gamma interferon (IFN-␥) has been shown to be the main cytokine preventing reactivation of Toxoplasma encephalitis in the brain (17,18). Several studies have demonstrated that IFN-␥ can control the growth of T. gondii in the brain via the activation of microglia (3, 4). The anti-Toxoplasma activity in microglia is via a nitric oxide (NO)-mediated mechanism (7). IFN-␥ can also activate astrocytes to inhibit the growth of T. gondii (8). The mechanism of IFN-␥-mediated anti-Toxoplasma activity in murine astrocytes has been found to be independent of the mechanisms previously demonstrated in other cells, e.g., mechanisms involving NO, tryptophan starvation, reactive oxygen intermediates, and iron deprivation (9).IFN-␥ is thought to exert its effects largely by activation of IFN-␥-responsive genes, of which over 200 have been identified (2). For most of these genes, their contributions in mediating the effects of IFN-␥ are unknown. One recently identified IFN-␥-regulated gene is IGTP (19). It is representative of a family of at least six genes encoding 47-to 48-kDa proteins that contain a GTP-binding sequence and which are expressed at high levels in immune and nonimmune cells after exposure to IFN-␥. Several of these proteins, including IGTP, localize to the endoplasmic reticulum (ER) of cells, suggesting that they may be involved in the processing or trafficking of immunologically relevant proteins, such as antigens or cytokines (20).Recently it has been found that IGTP-deficient (⌬IGTP) mice display a loss of host resistance to acute infection with T. gondii (21). In this study, we investigated the potential involvement of IGTP in IFN-␥ inhibition of T. gondii in murine astrocytes using primary astrocytes cultivated from IGTP-deficient mice. MATERIALS AND METHODSPrimary astrocyte culture. Murine astrocytes from C57BL/6 ϫ SV129 mice or syngeneic mice, deficient in IGTP (21), were cultivated from the brains of neonatal (less-than-24-h-old) mice. Murine pups were sacrificed, the brains were removed from the cranium, the forebrains dissected, and the meninges were removed. The tissue was minced and incubated in 0.25% trypsin for 5 min at 37°C. After 5 min, the trypsin was inactivated with a solution containing DNase and soybean trypsinase inhibitors, and the tissue was further disrupted by trituration in a 20-ml pipette. The dissociated cells were filtered through a 74-mpore-size Nitex mesh, centrifuged at 200 ϫ g, and suspended in growth medium (GIBCO-BRL, Gaithersburg, Md.) supplemented with 20% fetal bovine serum (FBS) (GIBCO-BRL), 5% glucose, and 1% penicillin and streptomycin (GIBCO-BRL) per ml. The growth medium was changed every 3 days. After 7 days in vitro, a confluent layer of 1 ϫ 10 4 to 2 ϫ 10 4 cells/cm was reached. By this method, cells we...

Challenge with the intracellular protozoan parasite Toxoplasma gondii induces a potent CD8؉ T-cell response that is required for resistance to infection, but many questions remain about the factors that regulate the presentation of major histocompatibility complex class I (MHC-I)-restricted parasite antigens and about the role of professional and nonprofessional accessory cells. In order to address these issues, transgenic parasites expressing ovalbumin (OVA), reagents that track OVA/MHC-I presentation, and OVA-specific CD8 ؉ T cells were exploited to compare the abilities of different infected cell types to stimulate CD8؉ T cells and to define the factors that contribute to antigen processing. These studies reveal that a variety of infected cell types, including hematopoietic and nonhematopoietic cells, are capable of activating an OVA-specific CD8 ؉ T-cell hybridoma, and that this phenomenon is dependent on the transporter associated with antigen processing and requires live T. gondii. Several experimental approaches indicate that T-cell activation is a consequence of direct presentation by infected host cells rather than cross-presentation. Surprisingly, nonprofessional antigen-presenting cells (APCs) were at least as efficient as dendritic cells at activating this MHC-I-restricted response. Studies to assess whether these cells are involved in initiation of the CD8 ؉ T-cell response to T. gondii in vivo show that chimeric mice expressing MHC-I only in nonhematopoietic compartments are able to activate OVA-specific CD8 ؉ T cells upon challenge. These findings associate nonprofessional APCs with the initial activation of CD8 ؉ T cells during toxoplasmosis.