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

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Imatinib (IM) is considered the gold standard for chronic myeloid leukemia (CML) treatment, although resistance is emerging as a significant problem. The proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) play an important role in cell proliferation, survival, and resistance to glucocorticoid-mediated cell death. Several transcription factors such as NF-KB and AP-1 are activated in response to physiopathological increases and modulation of intracellular calcium levels. Our previous study demonstrated that lymphocytes from CML patients showed dysregulated calcium homeostasis and oxidative stress. Alteration in ionized calcium concentration in the cytosol has been implicated in the initiation of secretion, contraction, and cell proliferation. In this study, we hypothesized that IL-6, IL-8, NF-kB, AP-1, and intracellular calcium may be used as selective and prognostic factors to address the follow-up in CML patients treated with imatinib. Our results demonstrated a significant down-regulation in IL-6 and IL-8 release as well as NF-kB and AP-1 activation in lymphomonocytes from Imatinib-treated patients, compared to samples from untreated patients. In parallel, IM treatment, in vivo and in vitro, were able to modulate the intracellular calcium concentration of peripheral blood mononuclear cells of CML patients by acting at the level of InsP(3) receptor in the endoplasmic reticulum and at the level of the purinergic receptors on plasma membrane. The results of this study show that measurements of NF-kB, AP-1, IL-6, IL-8, and intracellular calcium in CML patients treated with Imatinib may give important information to the hematologist on diagnostic criteria and are highly predictive in patients with newly diagnosed CML.

Ochratoxin A (OTA) is a powerful mycotoxin found in various foods and feedstuff, responsible for subchronic and chronic toxicity, such as nephrotoxicity, hepatotoxicity, teratogenicity, and immunotoxicity to both humans and several animal species. The severity of the liver damage caused depends on both dose and duration of exposure. Several studies have suggested that oxidative stress might contribute to increasing the hepatotoxicity of OTA, and several antioxidants, including curcumin (CURC), have been tested to counteract the toxic hepatic action of OTA in various classes of animals. Therefore, the present study was designed to evaluate the protective effect of CURC, a bioactive compound with different therapeutic properties on hepatic injuries caused by OTA in rat animal models. CURC effects were examined in Sprague Dawley rats treated with CURC (100 mg/kg), alone or in combination with OTA (0.5 mg/kg), by gavage daily for 14 days. At the end of the experiment, rats treated with OTA showed alterations in biochemical parameters and oxidative stress in the liver. CURC dosing significantly attenuated oxidative stress and lipid peroxidation versus the OTA group. Furthermore, liver histological tests showed that CURC reduced the multifocal lymphoplasmacellular hepatitis, the periportal fibrosis, and the necrosis observed in the OTA group. This study provides evidence that CURC can preserve OTA-induced oxidative damage in the liver of rats.

The reported data indicate that effective DOPET protection from CsA-induced oxidative stress is associated with a mild effect on histological damages and does not affect the altered glomerular function and the hypertension, thus indicating that kidney injury by CsA is only in part dependent on oxidative stress.