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.

/pdf/nitric-oxide-and-peroxynitrite-in-health-and-disease-3uuuqgfdcz.pdf

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.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

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

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.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

The transcription factor NF-κB regulates multiple aspects of innate and adaptive immune functions and serves as a pivotal mediator of inflammatory responses. NF-κB induces the expression of various pro-inflammatory genes, including those encoding cytokines and chemokines, and also participates in inflammasome regulation. In addition, NF-κB plays a critical role in regulating the survival, activation and differentiation of innate immune cells and inflammatory T cells. Consequently, deregulated NF-κB activation contributes to the pathogenic processes of various inflammatory diseases. In this review, we will discuss the activation and function of NF-κB in association with inflammatory diseases and highlight the development of therapeutic strategies based on NF-κB inhibition.

/pdf/nf-kb-signaling-in-inflammation-r4c2bxufoi.pdf

NF-κB signaling in inflammation

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

https://biblio.ugent.be/publication/8555786/file/8555788.pdf

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Adenoviral delivery of a constitutively active retinoblastoma mutant inhibits neointima formation in a human explant model for vein graft disease

Intimal thickening in arteries is considered as a site of predilection for atherosclerosis. We investigated whether oral application of the nitric oxide (NO) donors SPM-5185 (N-nitratopivaloyl-S-(N'-acetylalanyl)-cysteine ethylester, 10 mg/kg body weight/b.i.d.) and molsidomine (pro-drug of 3-morpholino-sydnonimine (SIN-1), 10 mg/kg body weight/day) can retard intimal thickening and changes in vascular reactivity induced by a silicone collar positioned around the carotid artery of rabbits. Intimal thickening was significantly inhibited by SPM-5185 (cross-sectional area 18 +/- 6 vs. 44 +/- 10 x 10(-3) mm2; P < 0.05), but not by molsidomine (28 +/- 6 vs. 35 +/- 9 x 10(-3) mm2), which is a donor of both NO and superoxide anions. In organ chamber studies collaring was associated with a decreased sensitivity to acetylcholine (ACh). SPM-5185 evoked a tendency towards normalization of the pD2 of ACh in collared arteries. We also investigated whether chronic nitric oxide (NO) treatment affected vascular reactivity and fatty streak development in the rabbit aorta. During 16 weeks rabbits received 150 g/day of a standard diet, or diets with 0.3% cholesterol, with 0.02% molsidomine (10 mg/kg body weight/day) or with the combination. The NO donor enhanced the area of fatty streaks, without affecting hypercholesterolemia. Moreover, it desensitized the smooth muscle cells of the rabbit aorta to vasodilators acting via the cytoplasmic guanylate cyclase and suppressed the capacity of the endothelial cells to release NO in response to muscarinic receptor stimulation. This suggested that chronic exposure to large quantities of NO caused a negative feedback, with selective decreases of both the endothelial capacity to generate NO and the responsiveness to vasodilators operating via cyclic GMP. In conclusion, we demonstrated that exogenous NO can decrease intimal hyperplasia in vivo. However, prolonged in vivo treatment with a donor of NO enhanced atherosclerosis in hypercholesterolemic rabbits.

The effect of chronic treatment with NO donors during intimal thickening and fatty streak formation

The destruction of cartilage is an important characteristic of rheumatoid arthritis (RA). Immune complexes (IC) are usually found in high amounts in RA synovial fluids (SF) and in the superficial layers of RA cartilage. The objective of this study was to investigate if IC have a direct influence on proliferation, survival and production of nitric oxide (NO) of cytokine-activated chondrocytes. Primary bovine chondrocytes were incubated with cytokines (huIL-1alpha, bovIFN-gamma, huTNF-alpha) and IC containing precipitates of peripheral blood (PB) and/or synovial fluid (SF) of 14 RA patients, 5 osteoarthritis (OA) patients and 10 healthy age and sex-matched controls. After 48 h, chondrocyte viability, proliferation, apoptosis, NO production and oxygen radical levels were measured. Staining with May-Grunwald-Giemsa after incubation with IC of RA PB and SF, showed apoptotic chondrocytes with condensation of the nuclei. The proliferation rates of cytokine-activated chondrocytes, incubated with sera and SF IC of RA patients were significantly decreased compared to chondrocytes, incubated with sera and SF IC of OA patients and compared to sera of controls. Quantitative evaluation of apoptotic cells by annexin-V/propidium iodide and TUNEL assays revealed a significant increase after incubation with sera and SF IC of RA patients, compared to control sera and OAs sera and SF. In all TUNEL positive samples, active-caspase-3-positive cells were found. There was a significant increase of chondrocyte NO production, after incubation with SF IC of RA patients, compared to OA SF. These results support the hypothesis that IC, present in serum and SF of RA patients, have a profound influence on chondrocyte growth, NO production and apoptosis, contributing to cartilage destruction in RA.

Synovial fluid and peripheral blood immune complexes of patients with rheumatoid arthritis induce apoptosis in cytokine-activated chondrocytes

The confluence of the immunology and oncology tributaries has precipitated a biomarker turbulence that is novel to the cancer community. On the heels of reversing the dismal outcomes for numerous patients suffering from terminal disease, it has also left behind deep pools of biomarker questions – which ones, when to use them, how to use them and what cells express them – to name just a few. The tumor microenvironment, an arena potentially housing a complex mix of immune infiltrators in the midst of tumor cells, is a wildly heterogeneous terrain, one that severely challenges our single biomarker model. This article examines the recently FDA-approved biomarker assays for PD-1 therapeutics, considers the technical shortcomings of the single biomarker model, scans alternate biomarker technologies and proposes a model that may allow investigators a method to systematically address these complexities.

https://www.tandfonline.com/doi/pdf/10.1080/23808993.2016.1140005

Challenges & Perspectives of Immunotherapy Biomarkers & The HistoOncoImmune™ Methodology

The haemodynamic basis for paradoxical embolization in patients with stroke and decompression sickness has not yet been fully elucidated. Therefore right and left atrial pressures were measured simultaneously with peroperatively placed catheters after coronary artery bypass grafting in 17 patients with sinus rhythm and normal left ventricular function. Recordings were made both during spontaneous breathing and positive pressure ventilation. A cyclic pressure reversal in which right atrial pressure exceeded left atrial pressure was reproducibly recorded. It started on average 215 +/- 5 ms (mean +/- SEM) after the onset of the electrocardiographic P-wave, lasted on average 179 +/- 14 ms and had a maximal amplitude of on average 4.1 +/- 0.3 mmHg. During the expiration phase of spontaneous breathing and inspiration phase of positive pressure ventilation, the onset of the pressure reversal occurred later, its duration was shorter and its amplitude smaller. These observations demonstrate the presence of a cyclic inter-atrial pressure reversal and illustrate the importance of the breathing mode for the time course and amplitude of this reversal.

Influence of the breathing mode on the time course and amplitude of the cyclic inter-atrial pressure reversal in postoperative coronary bypass surgery patients

Mark M. Kockx

Papers

Adenoviral delivery of a constitutively active retinoblastoma mutant inhibits neointima formation in a human explant model for vein graft disease

The effect of chronic treatment with NO donors during intimal thickening and fatty streak formation

Synovial fluid and peripheral blood immune complexes of patients with rheumatoid arthritis induce apoptosis in cytokine-activated chondrocytes

Challenges & Perspectives of Immunotherapy Biomarkers & The HistoOncoImmune™ Methodology

Influence of the breathing mode on the time course and amplitude of the cyclic inter-atrial pressure reversal in postoperative coronary bypass surgery patients