Functional, structural, and molecular remodelling of the goldfish (Carassius auratus) heart under moderate hypoxia.

The goldfish (Carassius auratus) is known for its physiologic ability to survive even long periods of oxygen limitation (hypoxia), adapting the cardiac performance to the requirements of peripheral tissue perfusion. We here investigated the effects of short-term moderate hypoxia on the heart, focusing on ventricular adaptation, in terms of hemodynamics and structural traits. Functional evaluations revealed that animals exposed to 4 days of environmental hypoxia increased the hemodynamic performance evaluated on ex vivo cardiac preparations. This was associated with a thicker and more vascularized ventricular compact layer and a reduced luminal lacunary space. Compared to normoxic animals, ventricular cardiomyocytes of goldfish exposed to hypoxia showed an extended mitochondrial compartment and a modulation of proteins involved in mitochondria dynamics. The enhanced expression of the pro-fission markers DRP1 and OMA1, and the modulation of the short and long forms of OPA1, suggested a hypoxia-related mitochondria fission. Our data propose that under hypoxia, the goldfish heart undergoes a structural remodelling associated with a potentiated cardiac activity. The energy demand for the highly performant myocardium is supported by an increased number of mitochondria, likely occurring through fission events.

Comment on "Exceptional preservation of organs in Devonian placoderms from the Gogo lagerstätte".

Trinajstic et al., (Science, 16 September 2022, p. 1311-1314) describe exceptionally well-preserved organs in fossilized Devonian placoderms to infer the early evolution of the vertebrate heart. We argue that the report has numerous shortcomings and examples of mixed specimen codes. Further, we question whether there indeed is any evidence for a mineralized chambered heart in these placoderms.

Gill structure and neurochemical markers in the African bonytongue (Heterotis niloticus): A preliminary study.

We have conducted a morphological and immunohistochemical study of the gills of juvenile specimens of the obligate air-breathing fish Heterotis niloticus. The study has been performed under normoxic and hypoxic conditions. The gills showed a reduced respiratory surface area by development of an interlamellar cellular mass (ILCM). The ILCM persisted without changes under both normoxia and hypoxia. Neuroepithelial cells (NECs), the major oxygen and hypoxia sensing cell type, were located in the distal end of the gill filaments and along the ILCM edges. These cells expressed 5HT, the neuronal isoform of the nitric oxide synthase (nNOS) and the vesicular acetylcholine transporter (VAChT). Furthermore, NECs appeared associated with nitrergic nerve fibres. The O2 levels did not modify the location, number or the immunohistochemical characteristics of NECs. Pavement cells covering the ILCM were also positive to nNOS and VAChT. The mechanisms of O2 sensing in the gills of Heterotis appears to involve several cell populations, the release of multiple neurotransmitters and a diversity of excitatory, inhibitory and modulatory mechanisms.

Expression of the Antimicrobial Peptide Piscidin 1 and Neuropeptides in Fish Gill and Skin: A Potential Participation in Neuro-Immune Interaction.

Antimicrobial peptides (AMPs) are found widespread in nature and possess antimicrobial and immunomodulatory activities. Due to their multifunctional properties, these peptides are a focus of growing body of interest and have been characterized in several fish species. Due to their similarities in amino-acid composition and amphipathic design, it has been suggested that neuropeptides may be directly involved in the innate immune response against pathogen intruders. In this review, we report the molecular characterization of the fish-specific AMP piscidin1, the production of an antibody raised against this peptide and the immunohistochemical identification of this peptide and enkephalins in the neuroepithelial cells (NECs) in the gill of several teleost fish species living in different habitats. In spite of the abundant literature on Piscidin1, the biological role of this peptide in fish visceral organs remains poorly explored, as well as the role of the neuropeptides in neuroimmune interaction in fish. The NECs, by their role as sensors of hypoxia changes in the external environments, in combination with their endocrine nature and secretion of immunomodulatory substances would influence various types of immune cells that contain piscidin, such as mast cells and eosinophils, both showing interaction with the nervous system. The discovery of piscidins in the gill and skin, their diversity and their role in the regulation of immune response will lead to better selection of these immunomodulatory molecules as drug targets to retain antimicrobial barrier function and for aquaculture therapy in the future.

Immunohistochemical and ultrastructural study of the immune cell system and epithelial surfaces of the respiratory organs in the bimodally breathing African sharptooth catfish (Clarias gariepinus Burchell, 1822).

Acetylcholine (Ach) represents the old neurotransmitter in central and peripheral nervous system. Its muscarinic and nicotinic receptors (mAChRs and nAChRs) constitute an independent cholinergic system that is found in immune cells and play a key role in the regulation of the immune function and cytokine production. Gas exchanging surfaces of the gills and air-breathing organs (ABOs) of the sharptooth catfish Clarias gariepinus were investigated using ultrastructural and confocal immunofluorescence techniques. This study was predominantly focused on the structure of the immune cell types, the expression of their neurotransmitters, including the antimicrobial peptide piscidin 1, and the functional significance of respiratory gas exchange epithelia. A network of immune cells (monocytes, eosinophils, and mast cells) was observed in the gill and the ABO epithelia. Eosinophils containing 5-hydroxytryptamine (5-HT) immunoreactivity were seen in close association with mast cells expressing acetylcholine (Ach), 5-HT, neuronal nitric oxide synthase, and piscidin 1. A rich and dense cholinergic innervation dispersing across the islet capillaries of the gas exchange barrier and the localization of Ach in the squamous pavement cells covering the capillaries were evidenced byVAChT antibodies. We report for the first time that piscidin 1 (Pis 1)-positive mast cells interact with Pis 1-positive nerves found in the epithelia of the respiratory organs. Pis 1 immunoreactivity was also observed in the covering respiratory epithelium of the ABOs and associated with a role in local mucosal immune defense. The above results anticipate future studies on the neuro-immune interactions at mucosal barrier surfaces, like the gill and the skin of fish, areas densely populated by different immune cells and sensory nerves that constantly sense and adapt to tissue-specific environmental challenges.

Neuroepithelial cells (NECs) and mucous cells express a variety of neurotransmitters and neurotransmitter receptors in the gill and respiratory air-sac of the catfish Heteropneustes fossilis (Siluriformes, Heteropneustidae): a possible role in local immune defence.

Heteropneustes fossilis is an air-breathing teleost inhabiting environments with very poor O2 conditions, and so it has evolved to cope with hypoxia. In the gills and respiratory air-sac, the sites for O2 sensing and the response to hypoxia rely on the expression of acetylcholine (Ach) acting via its nicotinic receptor (nAChR). This study examined the expression patterns of neuronal markers and some compounds in the NECs of the gills and respiratory air sac having an immunomodulatory function in mammalian lungs. Mucous cells, epithelial cells and neuroepithelial cells (NECs) were immunopositive to a variety of both neuronal markers (VAChT, nAChR, GABA-B-R1 receptor, GAD679) and the antimicrobial peptide piscidin, an evolutionary conserved humoral component of the mucosal immune system in fish. We speculate that Ach release via nAChR from mucous cells may be modulated by GABA production in the NECs and it is required for the induction of mucus production in both normoxic and hypoxic conditions. The presence of piscidin in mucous cells may act in synergy with the autocrine/paracrine signals of Ach and GABA binding to GABA B R1B receptor that may play a local immunomodulatory function in the mucous epithelia of the gills and the respiratory air sac. The potential role of the NECs in the immunobiological behaviour of the gill/air-sac is at moment a matter of speculation. The extent to which the NECs as such may participate is elusive at this stage and waits investigation.

Expression of acetylcholine, its contribution to regulation of immune function and O2 sensing and phylogenetic interpretations of the African butterfly fish Pantodon buchholzi (Osteoglossiformes, Pantodontidae).

Acetylcholine (Ach) is the main neurotransmitter in the neuronal cholinergic system and also works as a signaling molecule in non-neuronal cells and tissues. The diversity of signaling pathways mediated by Ach provides a basis for understanding the biology of the cholinergic epithelial cells and immune cells in the gill of the species studied. NECs in the gill were not found surprisingly, but specialized cells showing the morphological, histochemical and ultrastructural characteristics of eosinophils were located in the gill filaments and respiratory lamellae. Much remains unknown about the interaction between the nerves and eosinophils that modulate both the release of acetylcholine and its nicotinic and muscarinic receptors including the role of acetylcholine in the mechanisms of O2 chemosensing. In this study we report for the first time the expression of Ach in the pavement cells of the gill lamellae in fish, the mast cells associated with eosinophils and nerve interaction for both immune cell types, in the gill of the extant butterfly fish Pantodon buchholzi. Multiple roles have been hypothesized for Ach and alpha nAChR in the gills. Among these there are the possible involvement of the pavement cells of the gill lamellae as O2 chemosensitive cells, the interaction of Ach positive mast cells with eosinophils and interaction of eosinophils with nerve terminals. This could be related to the use of the vesicular acetylcholine transporter (VAChT) and the alpha 2 subunit of the acetylcholine nicotinic receptor (alpha 2 nAChR). These data demonstrate the presence of Ach multiple sites of neuronal and non-neuronal release and reception within the gill and its ancestral signaling that arose during the evolutionary history of this conservative fish species.

Author Correction: Human neutrophils phagocytose and kill Acinetobacter baumannii and A. pittii.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Expression of Acetylcholine- and G protein coupled Muscarinic receptor in the Neuroepithelial cells (NECs) of the obligated air-breathing fish, Arapaima gigas (Arapaimatidae: Teleostei).

The air-breathing specialization has evolved idependently in vertebrates, as many different organs can perfom gas exchange. The largest obligate air-breathing fish from South America Arapaima gigas breathe air using its gas bladder, and its dependence on air breathing increases during its growth. During its development, gill morphology shows a dramatic change, remodeling with a gradual reduction of gill lamellae during the transition from water breathing to air breathing . It has been suggested that in this species the gills remain the main site of O2 and CO2 sensing. Consistent with this, we demonstrate for the first time the occurrence of the neuroepithelial cells (NECs) in the glottis, and in the gill filament epithelia and their distal halves. These cells contain a broader spectrum of neurotransmitters (5-HT, acetylcholine, nNOS), G-protein subunits and the muscarininic receptors that are coupled to G proteins (G-protein coupled receptors). We report also for the first time the presence of G alpha proteins coupled with muscarinic receptors on the NECs, that are thought as receptors that initiate the cardiorespiratory reflexes in aquatic vertebrates. Based on the specific orientation in the epithelia and their closest vicinity to efferent vasculatures, the gill and glottal NECs of A. gigas could be regarded as potential O2 and CO2 sensing receptors. However, future studies are needed to ascertain the neurophysiological characterization of these cells.

Identification of Human Pathological Mitral Chordae Tendineae Using Polarization-sensitive Optical Coherence Tomography.

Defects of the mitral valve complex imply heart malfunction. The chordae tendineae (CTs) are tendinous strands connecting the mitral and tricuspid valve leaflets to the papillary muscles. These CTs are composed of organized, wavy collagen bundles, making them a strongly birefringent material. Disorder of the collagen structure due to different diseases (rheumatic, degenerative) implies the loss or reduction of tissue birefringence able to be characterized with Polarization Sensitive Optical Coherence Tomography (PS-OCT). PS-OCT is used to discriminate healthy from diseased chords, as the latter must be excised and replaced in clinical conventional interventions. PS-OCT allows to quantify birefringence reduction in human CTs affected by degenerative and rheumatic pathologies. This tissue optical property is proposed as a diagnostic marker for the identification of degradation of tendinous chords to guide intraoperative mitral valve surgery.

Human neutrophils phagocytose and kill Acinetobacter baumannii and A. pittii.

Acinetobacter baumannii is a common cause of health care associated infections worldwide. A. pittii is an opportunistic pathogen also frequently isolated from Acinetobacter infections other than those from A. baumannii. Knowledge of Acinetobacter virulence factors and their role in pathogenesis is scarce. Also, there are no detailed published reports on the interactions between A. pittii and human phagocytic cells. Using confocal laser and scanning electron microscopy, immunofluorescence, and live-cell imaging, our study shows that immediately after bacteria-cell contact, neutrophils rapidly and continuously engulf and kill bacteria during at least 4 hours of infection in vitro. After 3 h of infection, neutrophils start to release neutrophil extracellular traps (NETs) against Acinetobacter. DNA in NETs colocalizes well with human histone H3 and with the specific neutrophil elastase. We have observed that human neutrophils use large filopodia as cellular tentacles to sense local environment but also to detect and retain bacteria during phagocytosis. Furthermore, co-cultivation of neutrophils with human differentiated macrophages before infections shows that human neutrophils, but not macrophages, are key immune cells to control Acinetobacter. Although macrophages were largely activated by both bacterial species, they lack the phagocytic activity demonstrated by neutrophils.

Acinetobacter baumannii and A. pittii clinical isolates lack adherence and cytotoxicity to lung epithelial cells in vitro.

The molecular and genetic basis of Acinetobacter baumannii and Acinetobacter pittii virulence remains poorly understood, and there is still lack of knowledge in host cell response to these bacteria. In this study, we have used eleven clinical Acinetobacter strains (A. baumannii n = 5; A. pittii n = 6) to unravel bacterial adherence, invasion and cytotoxicity to human lung epithelial cells. Our results showed that adherence to epithelial cells by Acinetobacter strains is scarce and cellular invasion was not truly detected. In addition, all Acinetobacter strains failed to induce any cytotoxic effect on A549 cells.

Expression patterns and quantitative assessment of neurochemical markers in the lung of the gray bichir, Polypterus senegalus (Cuvier, 1829).

Anatomical and functional studies of the autonomic innervation and the putative oxygen receptors-the neuroepithelial (NEC)-like cells of the bichirs are lacking. The present paper describes the distribution of both NEC-like cells and the polymorphous granular cells (PGCs) that populate the mucociliated epithelium of the lung in the air breathing fish Polypterus senegalus. By using confocal immunohistochemistry we determined the coexpression of specific neurochemical markers. Colocalization studies showed that 5HT is coexpressed with calbindin and nNOS in the NEC-like cells and PGCs, and choline acetyltransferase (ChAT) is coexpressed with nNOS in both the two types of cells. Distribution of neurotransmitters (5HT, NO) and neurochemical marker ChAT is also investigated in the lung muscle. The role of these transmitters may be the autonomic control of circulation and respiration. However, the importance of these signals for the respiratory responses in the species studied is still not known. The present study also shows for the first time the simultaneous occurrence of piscidin 1 and 5HT in the PGCs. The function of these cells being equivalent to ones found in fish gill subepithelial parenchyma, is still not known. Due to the importance of piscidin 1 in local immune defense, more research is useful to understand a possible interaction of PGCs with immune response in the bichir lung.

Biofilm Formation and Quorum-Sensing-Molecule Production by Clinical Isolates of Serratia liquefaciens.

Serratia spp. are opportunistic human pathogens responsible for an increasing number of nosocomial infections. However, little is known about the virulence factors and regulatory circuits that may enhance the establishment and long-term survival of Serratia liquefaciens in the hospital environment. In this study, two reporter strains, Chromobacterium violaceum CV026 and VIR24, and high-resolution triple-quadrupole liquid chromatography-mass spectrometry (LC-MS) were used to detect and to quantify N-acyl-homoserine lactone (AHL) quorum-sensing signals in 20 S. liquefaciens strains isolated from clinical samples. Only four of the strains produced sufficient amounts of AHLs to activate the sensors. Investigation of two of the positive strains by high-performance liquid chromatography (HPLC)-MS confirmed the presence of significant amounts of short-acyl-chain AHLs (N-butyryl-l-homoserine lactone [C4-HSL] and N-hexanoyl-l-homoserine lactone [C6-HSL]) in both strains, which exhibited a complex and strain-specific signal profile that included minor amounts of other short-acyl-chain AHLs (N-octanoyl-l-homoserine lactone [C8-HSL] and N-3-oxohexanoyl-l-homoserine lactone [OC6-HSL]) and long-acyl-chain (C10, C12, and C14) AHLs. No correlation between biofilm formation and the production of large amounts of AHLs could be established. Fimbria-like structures were observed by transmission electron microscopy, and the presence of the type 1 fimbrial adhesin gene fimH in all strains was confirmed by PCR. The ability of S. liquefaciens to adhere to abiotic surfaces and to form biofilms likely contributes to its persistence in the hospital environment, increasing the probability of causing nosocomial infections. Therefore, a better understanding of the adherence properties of this species will provide greater insights into the diseases it causes.

New aspects in the biology of Photobacterium damselae subsp. piscicida: pili, motility and adherence to solid surfaces.

We describe for the first time the presence of pilus-like structures on the surface of Photobacterium damselae subsp. piscicida (Phdp). The hint to this discovery was the ability of one strain to hemagglutinate human erythrocytes. Further analysis of several Phdp strains ultrastructure by electron microscopy revealed the presence of long, thin fibers, similar to pili of other Gram-negative bacteria. These appendages were also observed and photographed by scanning, transmission electron microscopy and immunofluorescence. Although this fish pathogen has been described as non-motile, all strains tested exhibit twitching motility, a flagella-independent type IV-dependent form of bacterial translocation over surfaces. As far as we are aware, the movement of Phdp bacteria on semi-solid or solid surfaces has not been described previously. Moreover, we speculate that Phdp twitching motility may be involved in biofilm formation. Microscopic examination of Phdp biofilms by microscopy revealed that Phdp biofilm architecture display extensive cellular chaining and also bacterial mortality during biofilm formation in vitro. Based on our results, standardized analyses of Phdp surface appendages, biofilms, motility and their impact on Phdp survival, ecology and pathobiology are now feasible.

Induction of proinflammatory cytokines in human lung epithelial cells during Rhodococcus equi infection.

Rhodococcus equi is an opportunistic human pathogen associated with immunosuppressed people. While the interaction of R. equi with macrophages has been comprehensively studied, little is known about its interactions with non-phagocytic cells. Here, we characterized the entry process of this bacterium into human lung epithelial cells. The invasion is inhibited by nocodazole and wortmannin, suggesting that the phosphatidylinositol 3-kinase pathway and microtubule cytoskeleton are important for invasion. Pre-incubation of R. equi with a rabbit anti-R. equi polyclonal antiserum resulted in a dramatic reduction in invasion. Also, the invasion process as studied by immunofluorescence and scanning electron microscopy indicates that R. equi make initial contact with the microvilli of the A549 cells, and at the structural level, the entry process was observed to occur via a zipper-like mechanism. Infected lung epithelial cells upregulate the expression of cytokines IL-8 and IL-6 upon infection. The production of these pro-inflammatory cytokines was significantly enhanced in culture supernatants from cells infected with non-mucoid plasmid-less strains when compared with cells infected with mucoid strains. These results demonstrate that human airway epithelial cells produce pro-inflammatory mediators against R. equi isolates.

Interaction of macrophages with a cytotoxic Serratia liquefaciens human isolate.

Macrophages play key roles in host defense by recognizing, engulfing, and killing microorganisms. Understanding the response of macrophages to pathogens may provide insights into host defenses and the tactics used by pathogens to circumvent these defenses. In the present study, we investigated the interaction between a clinical isolate of Serratia liquefaciens and macrophages. S. liquefaciens strain HUMV-3250 triggers a fast and potent cytotoxic effect upon infection. This process requires the presence of live bacteria, adherence, and protein synthesis but not phagocytosis/bacterial internalization. Moreover, cytotoxicity assays, analysis of DNA integrity, immunofluorescence, and confocal, scanning, and time-lapse microscopy revealed that macrophage viability decreased rapidly with time upon challenge, and depends on the MOI used. Treatment of macrophages with caspase-1 inhibitors, or with specific inhibitors of phagocytosis, did not alter the infection outcome. Moreover, human macrophages exhibited similar cytotoxic changes after infection with this strain. Macrophages responded to this cytotoxic strain with a robust pattern of pro-inflammatory gene expression. However, phagocytosis attempts to engulf live bacteria were unsuccessful, and the phagocytes were unable to kill the bacteria. We conclude that macrophage cell death occurs rapidly as a result of necrotic events after close contact with S. liquefaciens. These results likely have important implications for understanding Serratia pathogenesis and host response to infection.

[Congenital heart disease, heterotaxia and laterality]. / Malformaciones cardíacas, heterotaxia y lateralidad.

Congenital heart disease occurs in about 0,8% of all newborns. Many cardiac malformations occur among relatives and have a polymorphic presentation. The origin of most congenital heart disease is thought to be multifactorial, implying both anomalous expression of genes and the influence of epigenetic factors. However, in a small number of cases, the origin of congenital heart disease has been directly related to chromosomal anomalies or to defects in a single gene. Curiously, defects in a single gene can explain a polymorphic presentation if the anomalous gene controls a basic embryonic process that affects different organs in time and space. Some of these genes appear to control the establishment of laterality. The establishment of the left-right asymmetry starts at the Hensen node. Here, the initial embryonic symmetry is broken by cascades of gene activation that confer specific properties on the left and right sides of the embryo. Although there are variations between species, some basic patterns of gene expression (Nodal, Pitx2) appear to be maintained along the phylogenetic scale. Anomalous expression of these genes induces the heterotaxia syndrome, which usually courses with congenital heart disease. The development of heart malformations is illustrated with the mouse mutant iv/iv, which is a model for the heterotaxia syndrome and the associated congenital heart disease.