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

Dengue virus infection causes dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), whose pathogeneses are not clearly understood. Current hypotheses of antibody-dependent enhancement, virus virulence, and IFN-gamma/TNFalpha-mediated immunopathogenesis are insufficient to explain clinical manifestations of DHF/DSS such as thrombocytopenia and hemoconcentration. Dengue virus infection induces transient immune aberrant activation of CD4/CD8 ratio inversion and cytokine overproduction, and infection of endothelial cells and hepatocytes causes apoptosis and dysfunction of these cells. The coagulation and fibrinolysis systems are also activated after dengue virus infection. We propose a new hypothesis for the immunopathogenesis for dengue virus infection. The aberrant immune responses not only impair the immune response to clear the virus, but also result in overproduction of cytokines that affect monocytes, endothelial cells, and hepatocytes. Platelets are destroyed by crossreactive anti-platelet autoantibodies. Dengue-virus-induced vasculopathy and coagulopathy must be involved in the pathogenesis of hemorrhage, and the unbalance between coagulation and fibrinolysis activation increases the likelihood of severe hemorrhage in DHF/DSS. Hemostasis is maintained unless the dysregulation of coagulation and fibrinolysis persists. The overproduced IL-6 might play a crucial role in the enhanced production of anti-platelet or anti-endothelial cell autoantibodies, elevated levels of tPA, as well as a deficiency in coagulation. Capillary leakage is triggered by the dengue virus itself or by antibodies to its antigens. This immunopathogenesis of DHF/DSS can account for specific characteristics of clinical, pathologic, and epidemiological observations in dengue virus infection.

Ground glass hepatocyte (GGH) represents a histological hallmark of chronic hepatitis B virus infection and contains surface antigens in the endoplasmic reticulum (ER). Several types of GGHs are recognized at different hepatitis B virus replicative stages. The recent identification of pre-S mutants from GGHs encourages us to investigate whether different GGHs may harbor specific mutants and exhibit differential biological activities. In this study, we applied laser capture microdissection to isolate specific GGHs from a total of 50 samples on eight resected liver specimens. The surface genes in two major types of GGHs were analyzed. Type I GGHs expressed an inclusion-like pattern of hepatitis B surface antigens and harbored mutants with deletions over pre-S1 region, whereas type II GGHs, distributed in clusters and emerged at late replicative phase, contained mutants with deletions over pre-S2 region that defines a cytotoxic T lymphocyte (CTL) immune epitope, and may represent an immune escape mutant. Transfection of pre-S mutants in Huh7 revealed decreased syntheses of middle and small S proteins with accumulation of large surface antigen in ER, which in turn led to the activation of ER stress response with differential activities for different mutants. This study therefore demonstrates that different GGHs may contain specific mutants and exhibit differential biological activities.

A C/EBP-like transcription factor, AGP/EBP, that binds to three distinct motifs in the 5'-flanking region of al-acid glycoprotein gene (AGP) has been identified. Here we report the cloning and properties of cDNA corresponding to mouse AGP/EBP. AGP/EBP and C/EBP share 87% amino acid sequence homology in the "leucine zipper" and its associated DNA-binding domains, while their sequences outside these domains and the sizes of their mRNAs are different. Unlike the limited expression of C/EBP in tissues and cells, AGP/EBP appears to be ubiquitously expressed in tissues like lung, spleen, kidney, heart, testis, and liver and cell lines like p388D1, 129P (hepatoma cell line of C3H/HeJ), FO (mouse myeloma), and L929. Antibody against cloned and expressed AGP/EBP which was raised in rabbits could recognize AGP/EBP from nuclear extract of a number of cells and tissues. On the basis of our findings about the structural relationship and the similarity of motif recognition, we propose that a family of C/EBP-like transcription factors exists. a,-Acid glycoprotein (AGP) is a liver-derived plasma glycoprotein which increases severalfold during acute-phase reaction (3-5, 37). AGP is transcriptionally regulated in rat, mouse, and human hepatocytes by interleukin-1 (IL-1) (31,34,36), hepatocyte-stimulating factor (beta interferon subtype 2 and IL-6) (6,13,16,24), and glucocorticoids (3,4,7,22,23,40). In the rat AGP gene, the hormonal stimulation by IL-1 and IL-6 is mediated by the distal regulatory element located at -5300 to -5150 (34), and the glucocorticoidresponsive element has been limited to positions -120 to -42 (7). The corresponding enhancer elements in the human and mouse AGP genes have not yet been defined. Unlike many glucocorticoid-responsive genes, such as those encoding the mouse mammary tumor virus (38, 40), human metallothionein (21), and tyrosine aminotransferase (33), the induction of AGP RNA by glucocorticoids is diminished in cells treated with protein synthesis inhibitor (e.g., cycloheximide) (3,22,23). It has been demonstrated that the rat AGP 5'-flanking region contains a DNA sequence (-121 to -107), exhibiting a high degree of homology to the glucocorticoid-responsive element consensus sequence 5'-GGTACAN3TGTTCT-3', which serves to specifically bind purified rat glucocorticoid receptor in vitro (22). A 15-bp oligonucleotide representing the rat AGP glucocorticoidresponsive element (5'-GGAACATTTTGTGCA-3') confers glucocorticoid responsiveness on a heterologous promoter; such regulation is not diminished by concurrent inhibition of protein synthesis. However, inclusion of the AGP sequences immediately downstream of the AGP glucocorticoid-responsive element (-106 to -42) renders the hormonal induction sensitive to inhibition of protein synthesis (22). DNase I footprinting with nuclear extracts prepared from HTC hepatoma cells indicates the presence of DNA-protein interactions spanning the region from -110 to -68 of the rat AGP gene (22).Three functional AGP genes (Agp-1, Agp-2, and Agp-3) have been isolated f...

Abstract. In this study dengue virus (DV) was found to infect primary endothelial cells derived from human umbilical cord veins (HUVEC) and alter their cytokine production. Dengue virus infection of HUVEC was confirmed by an increase in plaque-forming units in the culture supernatant and by immunofluorescence assay. HUVEC produced large amounts of interleukin (IL)-6 and IL-8 but not IL-1␤ after DV infection. Both the replication of DV and the production of IL-6 and IL-8 by HUVEC after DV infection were inhibited by ribavirin, an antiviral synthetic guanosine analogue. Additionally, increased serum levels of IL-6 and IL-8 were observed in patients with dengue hemorrhagic fever but not dengue fever. Therefore, our results suggest that endothelial cells can be a target for DV infection, and that DV-induced IL-6 and IL-8 production by endothelial cells may contribute to the pathogenesis of dengue hemorrhagic fever.