At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

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Free Radicals in the Physiological Control of Cell Function

The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

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Nitric Oxide and Peroxynitrite in Health and Disease

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.

For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.

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Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Immunological dysregulation is the cause of many non-infectious human diseases such as autoimmunity, allergy and cancer. The gastrointestinal tract is the primary site of interaction between the host immune system and microorganisms, both symbiotic and pathogenic. In this Review we discuss findings indicating that developmental aspects of the adaptive immune system are influenced by bacterial colonization of the gut. We also highlight the molecular pathways that mediate host–symbiont interactions that regulate proper immune function. Finally, we present recent evidence to support that disturbances in the bacterial microbiota result in dysregulation of adaptive immune cells, and this may underlie disorders such as inflammatory bowel disease. This raises the possibility that the mammalian immune system, which seems to be designed to control microorganisms, is in fact controlled by microorganisms.

The gut microbiota shapes intestinal immune responses during health and disease

It is likely that most vertebrate genes are associated with 'HTF islands'--DNA sequences in which CpG is abundant and non-methylated. Highly tissue-specific genes, though, usually lack islands. The contrast between islands and the remainder of the genome may identify sequences that are to be constantly available in the nucleus. DNA methylation appears to be involved in this function, rather than with activation of tissue specific genes.

CpG-rich islands and the function of DNA methylation

Immunogenicity for T cell-independent B-cell response assessed by splenic plaque-forming cell (PFC) response and cell-surface expression measured by laser flow cytometry of various class I H-2 antigens on mouse red blood cells (RBC) were compared. It was found that the order of magnitude of both immunogenicity and cell-surface expression on RBC is H-2Dd ≫ H-2Db > H-2Kd, H-2Kb. Furthermore, H-2d public antigens and H-2Ld antigens were neither immunogenic nor easily demonstrable on RBC. These findings contrasted with poor immunogenicity for PFC response (Nakashima et al. 1982, 1983) and proportionally strong expression of H-2 antigens on lymphoid cells. Immunogenicity and cell-surface expression of H-2Dd antigen on RBC were not shown to be controlled by the action of genes outside H-2D. It was therefore suggested that a number of H-2 antigens, including H-2Kd private, H-2Kb private, and H-2d public specificities are at least functionally defective on RBC. This is possibly due to the structural characteristics of the antigens. Since immunogenicity and cell-surface expression were in parallel, the expression of H-2 antigens on RBC must be dictated by a subset of B cells whose activity was assessed by PFC response. This finding supports the view that the H-2 molecules display a new category of activity which is different from their ability to activate T cells and depends on their expression on RBC.

Aberrancy in immunogenicity and cell-surface expression of H-2 antigens on erythrocytes.

The donor cell type controls anti-hapten (fluorescein isothiocyanate) primary antibody response to hapten-modified syngeneic cells

Phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus thuringiensis, which cleaves phosphatidylinositol or glycosylphosphatidylinositol on the external cell surface to generate a second messenger for intracellular signal transduction (S. Rahman et al., FEBS Lett. 303:193-196, 1992), was found to preferentially promote the generation of alloantigen-specific cytotoxic T lymphocytes in mixed leukocyte culture. PIPLC affected an early stage of cytotoxic T-lymphocyte activation in culture, and there was no evidence of any soluble cellular mediators of this PIPLC action. PIPLC neither enhanced overall cell proliferation nor noticeably promoted interleukin-2 and -4 production in mixed leukocyte culture. The relative population size of Ly-2+ T cells was increased, however, in a late mixed leukocyte culture with PIPLC. In addition, PIPLC enhanced an anti-CD3 monoclonal antibody-induced early increase in [Ca2+]i. These results suggest a new parasite (bacterium)-oriented mechanism for enhancing antigen-driven host cytotoxic T-lymphocyte immunity which does not include promotion of interleukin-2 production.

Promotion of cytotoxic T-cell generation in mixed leukocyte culture by phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis.

Accumulation of macrophages in solid tumor tissue has been recognized clinically by histopathological examination. However, the function and molecular characteristics of these macrophages got elucidated only recently. These macrophages, called tumor-associated macrophages (TAMs), can enhance tumor invasion, migration, and angiogenesis, and human TAMs have been characterized by the expression of CD163 and CD68 on plasma membrane. Similar to alternatively activated macrophages (M2 macrophages), TAMs are known to express arginase-1, chitinase 3-like proteins 3 and 4, and RELMα and also express VGEF. In addition, TAMs can suppress T cell proliferation or induce regulatory T cells by a way relevant to the expression of IL-10, TGFβ, and prostaglandins. These immunosuppressive features of TAMs connect them to the macrophages called myeloid-derived suppressor cells (MDSCs). Therefore, an important aim for TAMs research is to repolarize TAMs from tumor promotive macrophages to tumor suppressive macrophages, meaning to establish new strategies efficient for reeducating TAMs to be cytotoxic in tumors.

Tumor-Associated Macrophages and Cancer Development

The lymphocyte signal transduction, as determined by intracellular free Ca2+ mobilization of concanavalin A-stimulated T lymphocytes and of anti-immunoglobulin mu chain antibody-stimulated B lymphocytes, was suppressed in spleen cells from mice injected with murine P1.HTR mastocytoma-induced ascites and in spleen cells treated with the ascites in vitro. The suppression was observed both at the peak level and in the reactive pattern of Ca2+ influx. In the suppression, the ascites were replaceable with tumor culture supernatants or tumor homogenates. Correspondingly, primary and secondary cytotoxic T lymphocyte (CTL) responses of DBA/2 mice to allogeneic antigen were also significantly suppressed by injection of the syngeneic P1.HTR tumor-derived ascites. This new finding suggested that the mechanism of the tumorous ascites or of the tumor-derived factor-mediated immunosuppression involves at least in part the suppression of the early event of the signal transduction for lymphocyte activation.

Ken-ichi Isobe

Papers

Aberrancy in immunogenicity and cell-surface expression of H-2 antigens on erythrocytes.

The donor cell type controls anti-hapten (fluorescein isothiocyanate) primary antibody response to hapten-modified syngeneic cells

Promotion of cytotoxic T-cell generation in mixed leukocyte culture by phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis.

Tumor-Associated Macrophages and Cancer Development

Suppression of lymphocyte signal transduction by murine mastocytoma ascites.