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Phagocytosis is the process by which cells engulf some solid particles to form internal vesicles known as phagosomes. Phagocytosis is in fact a specific form of endocytosis involving the vesicular interiorization of particles. Phagocytosis is essentially a defensive reaction against infection and invasion of the body by foreign substances and, in the immune system, phagocytosis is a major mechanism used to remove pathogens and/or cell debris. For these reasons, phagocytosis in vertebrates has been recognized as a critical component of the innate and adaptive immune responses to pathogens. Furthermore, more recent studies have revealed that phagocytosis is also crucial for tissue homeostasis and remodeling. Professional phagocytes in teleosts are monocyte/macrophages, granulocytes and dendritic cells. Nevertheless, in recent years phagocytic properties have also been attributed to teleost lymphocytes and thrombocytes. The possible implications of such cells on this important biological process, new factors affecting phagocytosis, evasion of phagocytosis or new forms of phagocytosis will be considered and discussed.

Natural antibodies play crucial roles in pathogen elimination, B-cell survival and homeostasis, and inflammatory and autoimmune diseases. Although estrogens are able to regulate both innate and adaptive immune responses, their role in the production of natural antibodies is unknown. Here, we show that the dietary intake of the synthetic estradiol analog, 17α-ethinylestradiol (EE2), one of the most potent pharmaceutical estrogens and intensively used in human therapeutics as a component of most oral contraceptives, regulates the abundance and proliferation of T and IgM+ B lymphocytes in the teleost fish gilthead seabream (Sparus aurata L.). Furthermore, for the first time in vertebrates, it is shown that estrogen signaling through G protein-coupled estrogen receptor 1 (GPER1) induces the production of polyreactive natural antibodies, which are able to crossreact with unrelated antigens and commensal and pathogenic bacteria. In addition, the serum from fish treated with EE2 or the GPER1 agonist G1 shows higher complement-dependent bactericidal activity than that from non-treated specimens. These results demonstrate that estrogens and GPER1 are the key regulators of natural antibody production and pathogen clearance in fish, paving the way for future studies in other vertebrate classes.

Pollutants have been reported to disrupt the endocrine system of marine animals, which may be exposed through contaminated seawater or through the food chain. Although 17α-ethynylestradiol (EE2), a drug used in hormone therapies, is widely present in the aquatic environment, current knowledge on the sensitivity of marine fish to estrogenic pollutants is limited. We report the effect of the dietary intake of 5 µg EE2/g food on different processes of testicular physiology, ranging from steroidogenesis to pathogen recognition, at both pre-spermatogenesis (pre-SG) and spermatogenesis (SG) reproductive stages, of gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost. A differential effect between pre-SG and SG specimens was detected in the sex steroid serum levels and in the expression profile of some steroidogenic-relevant molecules, vitellogenin, double sex- and mab3-related transcription factor 1 and some hormone receptors. Interestingly, EE2 modified the expression pattern of some immune molecules involved in testicular physiology. These differences probably reflect a developmental adjustment of the sensitivity to EE2 in the gilthead seabream gonad.

Endocrine-immune system interactions have been widely demonstrated in mammals, whereas in fish, these relationships remain unclear. Of the organs that constitute the endocrine system, the pineal gland and its secretory product melatonin act in the synchronization of daily and seasonal rhythms in most vertebrates, including fish. Seasonal differences in immunocompetence and disease prevalence have been well documented in humans. Seasonality also strongly influences the life history of fish by controlling the timing of physiological events, such as reproduction, food intake, locomotor activity, and growth performance. Apart from its synchronizing capabilities, the role of melatonin in physiological processes in fish is not thoroughly understood. The purpose of this review is to summarize current studies on the effects of melatonin on the fish immune system. These studies suggest that melatonin represents an important component of fish endocrine-immune system interactions. The elucidation of the defense mechanisms of fish will facilitate the development of health management tools to support the growing finfish aquaculture industry as well as address questions concerning the origins and evolution of the immune system in vertebrates.

SUMMARY Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-κB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-κB. Similarly, TNFα promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis.

Distributed cyber-physical systems (DCPS) are pervasive in areas such as aeronautics and ground transportation systems, including the case of distributed hybrid systems. DCPS design and verification is quite challenging because of asynchronous communication, network delays, and clock skews. Furthermore, their model checking verification typically becomes unfeasible due to the huge state space explosion caused by the system's concurrency. The PALS ("physically asynchronous, logically synchronous") methodology has been proposed to reduce the design and verification of a DCPS to the much simpler task of designing and verifying its underlying synchronous version. The original PALS methodology assumes a single logical period, but Multirate PALS extends it to deal with multirate DCPS in which components may operate with different logical periods. This paper shows how Multirate PALS can be applied to formally verify a nontrivial multirate DCPS. We use Real-Time Maude to formally specify a multirate distributed hybrid system consisting of an airplane maneuvered by a pilot who turns the airplane according to a specified angle through a distributed control system. Our formal analysis revealed that the original design was ineffective in achieving a smooth turning maneuver, and led to a redesign of the system that satisfies the desired correctness properties. This shows that the Multirate PALS methodology is not only effective for formal DCPS verification, but can also be used effectively in the DCPS design process, even before properties are verified.

TNFα is a pleiotropic pro-inflammatory cytokine with a key role in the activation of the immune system to fight viral infections. Despite its antiviral role, a few viruses might utilize the host produced TNFα to their benefit. Some recent reports have shown that anti-TNFα therapies could be utilized to treat certain viral infections. However, the underlying mechanisms by which TNFα can favor virus replication have not been identified. Here, a rhabdoviral infection model in zebrafish allowed us to identify the mechanism of action by which Tnfa has a deleterious role for the host to combat certain viral infections. Our results demonstrate that Tnfa signals through its receptor Tnfr2 to enhance viral replication. Mechanistically, Tnfa does not affect viral adhesion and delivery from endosomes to the cytosol. In addition, the host interferon response was also unaffected by Tnfa levels. However, Tnfa blocks the host autophagic response, which is required for viral clearance. This mechanism of action provides new therapeutic targets for the treatment of SVCV-infected fish, and advances our understanding of the previously enigmatic deleterious role of TNFα in certain viral infections.

Biocides are chemical substances that can deter or kill the microorganisms responsible for biofouling. The rapid expansion of the aquaculture industry is having a significant impact on the marine ecosystems. As the industry expands, it requires the use of more drugs, disinfectants and antifoulant compounds (biocides) to eliminate the microorganisms in the aquaculture facilities. The use of biocides in the aquatic environment, however, has proved to be harmful as it has toxic effects on the marine environment. Organic booster biocides were recently introduced as alternatives to the organotin compounds found in antifouling products after restrictions were imposed on the use of tributyltin (TBT). The replacement products are generally based on copper metal oxides and organic biocides. The biocides that are most commonly used in antifouling paints include chlorothalonil, dichlofluanid, DCOIT (4,5-dichloro-2-n-octyl-4-isothiazolin-3-one, Sea-nine 211®), Diuron, Irgarol 1051, TCMS pyridine (2,3,3,6-tetrachloro-4-methylsulfonyl pyridine), zinc pyrithione and Zineb. There are two types of risks associated with the use of biocides in aquaculture: (i) predators and humans may ingest the fish and shellfish that have accumulated in these contaminants and (ii) the development of antibiotic resistance in bacteria. This paper provides an overview of the effects of antifouling (AF) biocides on aquatic organisms. It also provides some insights into the effects and risks of these compounds on non-target organisms.

The PALS architecture reduces distributed, real-time asynchronous system design to the design of a synchronous system under reasonable requirements. Assuming logical synchrony leads to fewer system behaviors and provides a conceptually simpler paradigm for engineering purposes. One of the current limitations of the framework is that from a set of independent "synchronous machines", one must compose the entire synchronous system by hand, which is tedious and error-prone. We use Maude's meta-level to automatically generate a synchronous composition from user-provided component machines and a description of how the machines communicate with each other. We then use the new capabilities to verify the correctness of a distributed topology control protocol for wireless networks in the presence of nodes that may fail.

Orc is a theory of orchestration of services that allows structured programming of distributed and timed computations. Several formal semantics have been proposed for Orc, including a rewriting logic semantics developed by the authors. Orc also has a fully fledged implementation in Java with functional programming features. However, as with descriptions of most distributed languages, there exists a fairly substantial gap between Orc's formal semantics and its implementation, in that: (i) programs in Orc are not easily deployable in a distributed implementation just by using Orc's formal semantics, and (ii) they are not readily formally analyzable at the level of a distributed Orc implementation. In this work, we overcome problems (i) and (ii) for Orc. Specifically, we describe an implementation technique based on rewriting logic and Maude that narrows this gap considerably. The enabling feature of this technique is Maude's support for external objects through TCP sockets. We describe how sockets are used to implement Orc site calls and returns, and to provide real-time timing information to Orc expressions and sites. We then show how Orc programs in the resulting distributed implementation can be formally analyzed at a reasonable level of abstraction by defining an abstract model of time and the socket communication infrastructure, and discuss the assumptions under which the analysis can be deemed correct. Finally, the distributed implementation and the formal analysis methodology are illustrated with a case study.

Distributed embedded systems (DESs) are no longer the exception; they are the rule in many application areas such as avionics, the automotive industry, traffic systems, sensor networks, and medical devices. Formal DES specification and verification is challenging due to state space explosion and the need to support real-time features. This paper reports on an extensive industry-based case study involving a DES product family for a pedestrian and car 4-way traffic intersection in which autonomous devices communicate by asynchronous message passing without a centralized controller. All the safety requirements and a liveness requirement informally specified in the requirements document have been formally verified using Real-Time Maude and its model checking features.

Safety of medical devices and of their interoperation is an unresolved issue causing severe and sometimes deadly accidents for patients with shocking frequency. Formal methods, particularly in support of highly reusable and provably safe patterns which can be instantiated to many device instances can help in this regard. However, this still leaves open the issue of how to pass from their formal specifications in logical time to executable emulations that can interoperate in physical time with other devices and with simulations of patient and/or doctor behaviors. This work presents a specification-based methodology in which virtual emulation environments can be easily developed from formal specifications in Real-Time Maude, and can support interactions with other real devices and with simulation models. This general methodology is explained in detail and is illustrated with two concrete scenarios which are both instances of a common safe formal pattern: one scenario involves the interaction of a provably safe pacemaker with a simulated heart; the other involves the interaction of a safe controller for patient-induced analgesia with a real syringe pump.

TNFα overexpression has been associated with several chronic inflammatory diseases, including psoriasis, lichen planus, rheumatoid arthritis, and inflammatory bowel disease. Paradoxically, numerous studies have reported new-onset psoriasis and lichen planus following TNFα antagonist therapy. Here, we show that genetic inhibition of Tnfa and Tnfr2 in zebrafish results in the mobilization of neutrophils to the skin. Using combinations of fluorescent reporter transgenes, fluorescence microscopy, and flow cytometry, we identified the local production of dual oxidase 1 (Duox1)-derived H₂O₂ by Tnfa- and Tnfr2-deficient keratinocytes as a trigger for the activation of the master inflammation transcription factor NF-κB, which then promotes the induction of genes encoding pro-inflammatory molecules. In addition, pharmacological inhibition of Duox1 completely abrogated skin inflammation, placing Duox1-derived H₂O₂ upstream of this positive feedback inflammatory loop. Strikingly, DUOX1 was drastically induced in the skin lesions of psoriasis and lichen planus patients. These results reveal a crucial role for TNFα/TNFR2 axis in the protection of the skin against DUOX1-mediated oxidative stress and could establish new therapeutic targets for skin inflammatory disorders.

We present an equational decision procedure a la Dijkstra & Scholten for the 'Syllogistic Logic with Complements'. Keywords: Syllogistic Logic with Complements, Decision Procedure, Equational Logic, Rewriting Logic, Maude.