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

The management of unresectable metastatic melanoma is a major clinical challenge because of the lack of reliably effective systemic therapies. Blocking cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) has recently been proposed as a strategy to enhance cell-mediated immune responses to cancer, and clinical trials have demonstrated that anti-CTLA-4 therapy can produce durable outcomes with different response patterns than cytotoxic chemotherapy. We enrolled eight out of 155 patients with advanced melanoma in a multicentre phase II trial that evaluated the activity and tolerability of ipilimumab, a fully human, anti-CTLA-4 monoclonal antibody ( www.clinicaltrials.gov ; NCT00289627; CA184-008). Here we report our experience with three of these patients, who experienced progressive disease after a variety of previous therapies, including prior immunotherapies, and who achieved good outcomes with ipilimumab. One patient had a partial response ongoing at 17+ months on ipilimumab despite failure with four prior therapies, and the other two patients showed durable stable disease, both still ongoing at 17+ and 20+ months, respectively. The patient achieving a partial response experienced no side effects while receiving ipilimumab. The other two patients developed immune-related adverse events (irAEs) including rash (one case; grade 2) and diarrhoea (both cases; grades 1 and 2, respectively); the histopathology of colon biopsy samples from both was suggestive of colitis, with an abundant CD8+ T-cell infiltrate. Nausea, vomiting and acute pancreatitis were also observed in one patient. In addition, immunohistochemical findings of a dense CD8+, TIA1+ and granzyme B+ lymphoid infiltrate within a biopsied lesion provide indirect evidence of functional T-cell activation induced by treatment. These case reports highlight the potential for anti-CTLA-4-based therapy in previously treated patients with advanced melanoma. Moreover, because the patterns of response to ipilimumab differ from chemotherapy, we need to understand how and when patients may respond to treatment so that appropriate clinical decisions can be made.

The intratumoral injection of cytokines, in particular IL2, has shown promise for cutaneous melanoma patients with unresectable disease or continuous recurrence despite surgery. We recently reported that the intralesional injection of L19-IL2, an immunocytokine combining IL2 and the human monoclonal antibody fragment L19, resulted in efficient regional control of disease progression, increased time to distant metastasis and evidence of effect on circulating immune cell populations. We have also shown in preclinical models of cancer a remarkable synergistic effect of the combination of L19-IL2 with L19-TNF, a second clinical-stage immunocytokine, based on the same L19 antibody fused to TNF. Here, we describe the results of a phase II clinical trial based on the intralesional administration of L19-IL2 and L19-TNF in patients with stage IIIC and IVM1a metastatic melanoma, who were not candidate to surgery. In 20 efficacy-evaluable patients, 32 melanoma lesions exhibited complete responses upon intralesional administration of the two products, with mild side effects mainly limited to injection site reactions. Importantly, we observed complete responses in 7/13 (53.8 %) non-injected lesions (4 cutaneous, 3 lymph nodes), indicating a systemic activity of the intralesional immunostimulatory treatment. The intralesional administration of L19-IL2 and L19-TNF represents a simple and effective method for the local control of inoperable melanoma lesions, with a potential to eradicate them or make them suitable for a facile surgical removal of the residual mass.

Ozone is well recognized as a bactericidal agent and its beneficial effect on wound healing could be a consequence of this property. Because ozone itself does not penetrate the cells but immediately reacts with polyunsaturated fatty acids, its effects should be the results of oxidative reaction. For this reason, ozonated oils could be a way to deliver ozone messengers to the skin. This paper evaluated the therapeutic effects of three different grades of ozonated sesame oil in acute cutaneous wounds made in the skin of SKH1 mice. Specifically, wound closure rate, histological parameters, and the level of key proteins such as vascular endothelial growth factors and cyclin D1 have been analyzed in relation to the peroxide level present in the ozonated oil. Treatment with moderately ozonated sesame oil--expressed as peroxide value about 1,500)--has a faster wound closure rate in the first 7 days than treatment with oil containing either lower or higher peroxide value, and even with controls. Moreover, under the same treatment, an earlier and higher response of cells involved in wound repair, a higher angiogenesis, as well as an enhanced vascular endothelial growth factors and cyclin D1 expression were observed. The present study shows the validity of ozonated sesame oil in cutaneous wound healing and emphasizes the importance of the ozonation grade.

Epidemiological studies suggest a correlation between increased airborne particulate matter (PM) and adverse health effects. The mechanisms of PM-health effects are believed to involve oxidative stress and inflammation. To evaluate the ability of PM promoting skin tissue damage, one of the main organs exposed to outdoor pollutants, we analyzed the effect of concentrated ambient particles (CAPs) in a reconstructed human epidermis (RHE) model. RHE tissues were exposed to 25 or 100 µg/ml CAPs for 24 or 48 h. Data showed that RHE seems to be more susceptible to CAPs-induced toxicity after 48 h exposure than after 24 h. We found a local reactive O(2) species (ROS) production increase generated from metals present on the particle, which contributes to lipids oxidation. Furthermore, as a consequence of altered redox status, NFkB nucleus translocation was increase upon CAPs exposure, as well as cyclooxygenase 2 and cytochrome P450 levels, which may be involved in the inflammatory response initiated by PM. CAPs also triggered an apoptotic process in skin. Surprisingly, by transition electron microscopy analysis we showed that CAPs were able to penetrate skin tissues. These findings contribute to the understanding of the cutaneous pathophysiological mechanisms initiated by CAPs exposure, where oxidative stress and inflammation may play predominant roles.

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Obese adults have shorter telomeres than their normal-weight counterparts, while this phenomenon is not present in childhood.