Permeability properties and occludin expression in a primary cultured model gill epithelium from the stenohaline freshwater goldfish

Techniques for the primary culture of fish gill epithelia on permeable supports have provided ‘reconstructed’ gill models appropriate for the study of gill permeability characteristics in vitro. Models developed thus far have been derived from euryhaline fish species that can tolerate a wide range of environmental salinity. This study reports on procedures for the primary culture of a model gill epithelium derived from goldfish, a stenohaline freshwater (FW) fish that cannot tolerate high environmental salt concentrations. The reconstructed goldfish gill epithelium was cultured on permeable filter inserts and using electron microscopy and immunocytochemical techniques, was determined to be composed exclusively of gill pavement cells. When cultured under symmetrical conditions (i.e. with culture medium bathing both apical and basolateral surfaces), epithelial preparations generated appreciable transepithelial resistance (TER) (e.g. 1,150 ± 46 Ωcm²) within 36–42 h post-seeding in inserts. When apical medium was replaced with FW (asymmetrical conditions to mimic conditions that occur in vivo), epithelia exhibited increased TER and elevated paracellular permeability. Changes in permeability occurred in association with altered occludin-immunoreactive band position by western blot and no change in occludin mRNA abundance. We contend that the goldfish gill model will provide a useful in vitro tool for examining the molecular components of a stenohaline fish gill epithelium that participate in the regulation of gill permeability. The model will allow molecular observations to be made together with assessment of changing physiological properties that relate to permeability. Together, this will allow further insight into mechanisms that regulate gill permeability in fishes.     

The effects of ultraviolet radiation on a freshwater prey fish: physiological stress response,club cell investment,and alarm cue production

Recent anthropogenic activities have caused deleterious effects to the stratospheric ozone layer, resulting in a global increase in the level of ultraviolet radiation (UVR). Understanding the way that organisms respond to such stressors is key to predicting the effects of anthropogenic activities on aquatic ecosystems and the species that inhabit them. The epidermal layer of the skin of fishes is not keratinized and acts as the primary interface between the fish and its environment. The skin of many species of fishes contains large epidermal club cells (ECCs) that are known to release chemicals (alarm cues) serving to warn other fishes of danger. However, the alarm role of the cells is likely secondary to their role in the immune system. Recent research suggests that ECCs in the epidermis may play a role in protecting the fish from damage caused by UVR. In the present study, we examined the effects of in vivo exposure to UVR on fathead minnows (Pimephales promelas), specifically investigating ECC investment, physiological stress responses, and alarm cue production. We found that fish exposed to UVR showed an increase in cortisol levels and a substantive decrease in ECC investment compared to non‐exposed controls. Unexpectedly, our subsequent analysis of the behavioural response of fish to alarm cues revealed no difference in the potency of the cues prepared from the skin of UV‐exposed or non‐exposed minnows. Our results indicate that, although nonlethal, UVR exposure may lead to secondary mortality by altering the fish immune system, although this same exposure may have little influence on chemically‐mediated predator–prey interactions. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 832–841.     

The cutaneous citadel: a holistic view of skin and immunity

Human skin has 4 major functions: endogenous homeostasis (e.g. regulation of body temperature and fluid balance), metabolism (e.g. Vitamin D synthesis), sensory input, and to serve as a barrier to external threats (e.g. infection, mechanical injury, ultraviolet light). It is the latter function which concerns this review, for the skin's remarkable success in protecting the human body from the outside world is a major component of our immune system. The eminent pathologist, Virchow, whose work in the mid 19th century revolutionized many aspects of medical understanding, viewed the skin as an effective but inanimate barrier (1). However, recent technologies have elucidated a highly complex, dynamic interplay between the skin and other members of the immune system.     

The development of an innervated epithelial barrier model using a human corneal cell line and ND7/23 sensory neurons

The corneal epithelium is a highly innervated tissue and hence in vitro models that mimic the effects of chemicals or radiation (e.g. ultra violet) on this important barrier should include consideration of the potential role of innervation. A sensory neural cell line, ND7/23, was incorporated into a 2D and 3D model of a corneal epithelium, using a human corneal cell line, and effects on barrier integrity were neither adverse nor stimulatory. In the 3D model the nerve cell bodies were separated from the corneal epithelium, via a porous polycarbonate insert membrane. The ND7/23 cells were induced to form neurites and cease division when cultured in the keratinocyte medium employed for the corneal cells. In the absence of calcium, the epithelial barrier function was lost, shown by enhanced fluorescein leakage and relocation of ZO-1 and E-cadherin from the cell membrane. At 60 microM calcium, and above, the corneal cells formed tight junctions, with peripheral membrane location of ZO-1 and E-cadherin. The presence of the ND7/23 cells did not compromise or enhance the time taken to form these junctions, when monitored at 24-h intervals over 72 h. Both male- and female-derived human corneal cell lines showed a similar tight junction functional response to different medium calcium concentrations in the presence or absence of the ND7/23 cells. Once differentiated in keratinocyte medium, patch-clamped ND7/23 cells were capable of producing a whole-cell current when exposed to low pH (5.4), indicative of the presence of active pH-gated ion channels.     

Role of N-acetyl-beta-d-hexosaminidase in cholesteatoma tissue

Cholesteatoma is a destructive disease characterized by the progressive expansion of keratinizing squamous epithelium in the middle ear and mastoid, and chronic inflammatory reaction of the subepithelial connective tissue. N-Acetyl-beta-d-hexosaminidase (HEX) catalyzes the release of terminal non-reducing N-acetyl-d-hexosamine residues acting on glucosides and galactosides in glycoproteins, G(M2)-gangliosides and glycosaminoglycans (GAGs). In this study the activities of HEX were measured in cholesteatoma tissue and in normal skin to demonstrate a possible role of HEX in bone resorption in the area adjacent to cholesteatoma. Cholesteatomas (n = 21) and normal adult retroauricular skin (controls, n = 21), were collected from patients during surgery due to chronic otitis media. In 20 of 21 specimens a significantly higher activity of HEX was observed in cholesteatoma tissue compared with that in normal skin. Mean release of HEX from the activated cells was 68.55 +/- 30.77 nkat/g wet tissue in cholesteatoma and 31.79 +/- 10.02 nkat/g wet tissue in skin specimens. It may explain the process of bone resorption in the area adjacent to cholesteatoma, i.e. ossicles or temporal bone. This study suggests that drugs inhibiting HEX activity, such as iminocyclitols, may be useful in cholesteatoma treatment.     

Research progress on gut health of farmers teleost fish: a viewpoint concerning the intestinal mucosal barrier and the impact of its damage

Reviews in Fish Biology and Fisheries - The intestinal mucosal barrier plays a critical role in the maintenance of host health. In farmed teleost fish, the intestinal epithelium is challenged by a...     

A hassle a day may keep the doctor away: stress and the augmentation of immune function

Stress may be defined as a sequence of events, that begins witha stimulus (stressor), that is recognized by the brain (stressperception), and which results in the activation of physiologicfight/flight/fright systems within the body (stress response).Many evolutionary selection pressures are stressors, and oneof the primary functions of the brain is to perceive stress,warn the body of danger, and enable an organism to respond.We hypothesized that under acute conditions, just as the stressresponse prepares the cardiovascular and musculoskeletal systemsfor fight or flight, it may also prepare the immune system forchallenges (e.g., wounding) which may be imposed by a stressor(e.g., an aggressor). Initial studies showed that acute (2h)stress induced a significant trafficking of immune cells tothe skin. Since the skin is an organism's major protective barrier,we hypothesized that this leukocyte redistribution may serveto enhance skin immunity during acute stress. We tested thishypothesis using the delayed type hypersensitivity (DTH) reaction,which mediates resistance to various infectious agents, as amodel for skin immune function. Acute stress administered immediatelybefore antigen exposure significantly enhanced skin DTH. Adrenalectomy(ADX) eliminated the stress-induced enhancement of DTH whileadministration of physiological doses of corticosterone and/orepinephrine to ADX animals enhanced skin DTH in the absenceof stress. These studies showed that changes in leukocyte distributionand circulating stress hormones are systemic mediators of theimmunoenhancing effects of acute stress. We recently identifiedgamma interferon as a local cytokine mediator of a stress-inducedimmunoenhancement. Our results suggest that during acute stressthe brain sends preparatory warning signals to the immune systemjust as it does to other fight/flight systems of the body.     

Network Analysis Highlights Complex Interactions between Pathogen,Host and Commensal Microbiota

Interactions between bacteria and their host represent a full continuum from pathogenicity to mutualism. From an evolutionary perspective, host-bacteria relationships are no longer considered a two-component system but rather a complex network. In this study, we focused on the relationship between brook charr (Salvelinus fontinalis) and bacterial communities developing on skin mucus. We hypothesized that stressful conditions such as those occurring in aquaculture production induce shifts in the bacterial community of healthy fish, thus allowing pathogens to cause infections. The results showed that fish skin mucus microbiota taxonomical structure is highly specific, its diversity being partly influenced by the surrounding water bacterial community. Two types of taxonomic co-variation patterns emerged across 121 contrasted communities’ samples: one encompassing four genera well known for their probiotic properties, the other harboring five genera mostly associated with pathogen species. The homeostasis of fish bacterial community was extensively disturbed by induction of physiological stress in that both: 1) the abundance of probiotic-like bacteria decreased after stress exposure; and 2) pathogenic bacteria increased following stress exposure. This study provides further insights regarding the role of mutualistic bacteria as a primary host protection barrier.     

New insights into the role of zinc in the respiratory epithelium

Over the past 30 years, many researchers have demonstrated the critical role of zinc (Zn), a group IIb metal, in diverse physiological processes, such as growth and development, maintenance and priming of the immune system, and tissue repair. This review will discuss aspects of Zn physiology and its possible beneficial role in the respiratory epithelium. Here we have detailed the mechanisms by which Zn diversely acts as: (i) an anti-oxidant; (ii) an organelle stabilizer; (iii) an anti-apopototic agent; (iv) an important cofactor for DNA synthesis; (v) a vital component for wound healing; and (vi) an anti-inflammatory agent. This paper will also review studies from the authors' laboratory concerning the first attempts to map Zn in the respiratory epithelium and to elucidate its role in regulating caspase-3 activated apoptosis. We propose that Zn, being a major dietary anti-oxidant has a protective role for the airway epithelium against oxyradicals and other noxious agents. Zn may therefore have important implications for asthma and other inflammatory diseases where the physical barrier is vulnerable and compromised.     

Environmental pollutants and skin cancer

We are increasingly exposed to environmental pollution. Pollutants can be inhaled, ingested or come into contact with the skin depending on the form in which they occur. On metabolization, activation, or accumulation, pollutants can become extremely toxic for the vital organs and this is often related to a strong genotoxic effect. Since the skin acts as a barrier between the organism and the environment, it is frequently directly exposed to pollution. It is very often degraded by polluting agents and acts as an inlet toward other tissues. Numerous studies in man recognize and demonstrate the carcinogenic power of certain pollutants in the digestive and respiratory tracts. The "pollutants" that react most specifically with the skin are: ultraviolet radiation, polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene), volatile organic compounds (e.g., benzene), heavy metals, and ozone. Ultraviolet radiation, a "physical" pollutant, has been described as being the factor responsible for most skin cancers in man. The genotoxicity of UV light is well documented (type of lesion or mutation, etc.) and its carcinogenic effect is clearly demonstratedin vivo in man. A few epidemiological studies describe the carcinogenicity of certain pollutants such as arsenic or lead on the skin. However, most of the evidence for the role of pollutants in skin cancers comes fromin vivo animal studies or fromin vitro studies (e.g., PAHs). In this report, different studies are presented to illustrate the research strategies developed to investigate the mechanism of action of "chemical" pollutants and their potential role in human skin pathology. All the study models and the associated techniques of investigation are tools for a better understanding and thus more efficient prevention of the deleterious effects caused by the environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Protein transport across the lung epithelial barrier

Alveolar lining fluid normally contains proteins of important physiological, antioxidant, and mucosal defense functions [such as albumin, immunoglobulin G (IgG), secretory IgA, transferrin, and ceruloplasmin]. Because concentrations of plasma proteins in alveolar fluid can increase in injured lungs (such as with permeability edema and inflammation), understanding how alveolar epithelium handles protein transport is needed to develop therapeutic measures to restore alveolar homeostasis. This review provides an update on recent findings on protein transport across the alveolar epithelial barrier. The use of primary cultured rat alveolar epithelial cell monolayers (that exhibit phenotypic and morphological traits of in vivo alveolar epithelial type I cells) has shown that albumin and IgG are absorbed via saturable processes at rates greater than those predicted by passive diffusional mechanisms. In contrast, secretory component, the extracellular portion of the polymeric immunoglobulin receptor, is secreted into alveolar fluid. Transcytosis involving caveolae and clathrin-coated pits is likely the main route of alveolar epithelial protein transport, although relative contributions of these internalization steps to overall protein handling of alveolar epithelium remain to be determined. The specific pathways and regulatory mechanisms responsible for translocation of proteins across lung alveolar epithelium and regulation of the cognate receptors (e.g., 60-kDa albumin binding protein and IgG binding FcRn) expressed in alveolar epithelium need to be elucidated.     

Apolipoprotein AI could be a significant determinant of epithelial integrity in rainbow trout gill cell cultures: a study in functional proteomics

The freshwater fish gill forms a barrier against an external hypotonic environment. By culturing rainbow trout gill cells on permeable supports, as intact epithelia, this study investigates barrier property mechanisms. Under symmetrical conditions the apical and basolateral epithelial surfaces contact cell culture media. Replacing apical media with water, to generate asymmetrical conditions (i.e. the situation encountered by the freshwater gill), rapidly increases transepithelial resistance (TER). Proteomic analysis revealed that this is associated with enhanced expression of pre-apolipoprotein AI (pre-apoAI). To test the physiological relevance, gill cells were treated with a dose of 50 microg ml(-1) human apolipoprotein (apoAI). This was found to elevate TER in those epithelia which displayed a lower TER prior to apoAI treatment. These results demonstrate the action of apoAI and provide evidence that the rainbow trout gill may be a site of apoAI synthesis. TER does not differentiate between the trans-cellular (via the cell membrane) and para-cellular (via intercellular tight junctions) pathways. However, despite the apoAI-induced changes in TER, para-cellular permeability (measured by polyethylene glycol efflux) remained unaltered suggesting apoAI specifically reduces trans-cellular permeability. This investigation combines proteomics with functional measurements to show how a proteome change may be associated with freshwater gill function.     

The composition and characteristics of skin and muscle collagens from a freshwater catfish grown in biologically treated tannery effuent water

The skin of catfish Gariepinus spp. reared in tannery effuent water (ETP) had an increased collagen content, decreased acid solubility and high carbohydrate association as compared with skin of normal fish. The skin collagen in either fish was composed of three distinct α chains with mobilities different from that of vertebrate type I α chains and two of these three chains were invariably present in the muscle collagen. The amino acid composition of ETP fish skin and muscle collagens were similar but were characterized by higher degrees of proline and lysine hydroxylation, indicating higher stability. Both the skin and muscle collagens had significantly high denaturation temperatures. Electron microscopy of in vitro reconstituted fibrils of skin and muscle collagens of ETP fish displayed defined periodicities of around 64 nm. This demonstrates that the polymorphism in skin collagen chains is not confined to marine fish. There was a close similarity in the composition of skin and muscle collagens and the ETP environment influences the solubility and stability of the skin collagen. It is hypothesized that the third chain may play a role in the anchorage of skin to muscle, as such chain composition is now evident in fish such as hake, cod and catfish where the musculature is strongly attached to the skin.     

Better living through microbial action: the benefits of the mammalian gastrointestinal microbiota on the host

Mammals live in a homeostatic symbiosis with their gastrointestinal microbiota. The mammalian host provides the microbiota with nutrients and a stable environment; whereas the microbiota helps shaping the host's gut mucosa and provides nutritional contributions. Microorganisms start colonizing the gut immediately after birth followed by a succession of populations until a stable, adult microbiota has been established. However, physiological conditions differ substantially among locations in the gut and determine bacterial density and diversity. While Firmicutes and Bacteroidetes dominate the gut microbiota in all mammals, the bacterial genera and species diversity is huge and reflects mammalian phylogeny. The main function of the gastrointestinal epithelium is to absorb nutrients and to retain water and electrolytes, yet at the same time it is an efficient barrier against harmful compounds and microorganisms, and is able to neutralize antagonists coincidentally breaching the barrier. These processes are influenced by the microbiota, which modify epithelial expression of genes involved in nutrient uptake and metabolism, mucosal barrier function, xenobiotic metabolism, enteric nervous system and motility, hormonal and maturational responses, angiogenesis, cytoskeleton and extracellular matrix, signal transduction, and general cellular functions. Whereas such effects are local at the gut epithelium they may eventually have systemic consequences, e.g. on body weight and composition.     

Imaging Mass Spectrometry Visualizes Ceramides and the Pathogenesis of Dorfman-Chanarin Syndrome Due to Ceramide Metabolic Abnormality in the Skin

Imaging mass spectrometry (IMS) is a useful cutting edge technology used to investigate the distribution of biomolecules such as drugs and metabolites, as well as to identify molecular species in tissues and cells without labeling. To protect against excess water loss that is essential for survival in a terrestrial environment, mammalian skin possesses a competent permeability barrier in the stratum corneum (SC), the outermost layer of the epidermis. The key lipids constituting this barrier in the SC are the ceramides (Cers) comprising of a heterogeneous molecular species. Alterations in Cer composition have been reported in several skin diseases that display abnormalities in the epidermal permeability barrier function. Not only the amounts of different Cers, but also their localizations are critical for the barrier function. We have employed our new imaging system, capable of high-lateral-resolution IMS with an atmospheric-pressure ionization source, to directly visualize the distribution of Cers. Moreover, we show an ichthyotic disease pathogenesis due to abnormal Cer metabolism in Dorfman–Chanarin syndrome, a neutral lipid storage disorder with ichthyosis in human skin, demonstrating that IMS is a novel diagnostic approach for assessing lipid abnormalities in clinical setting, as well as for investigating physiological roles of lipids in cells/tissues.     

The effect of stress on the stomach of the European eel, Anguilla anguilla L.

The gastrointestinal tract is a system that is very sensitive to stress-like conditions in mammals. Fish display physiological reactions to stress that are fundamentally similar to those of the'General Adaptation Syndrome' in higher vertebrates. An investigation was therefore undertaken to determine if morphological changes equivalent to those affecting the stomachs of mammals also occur in fish under stress.
Conflict for social dominance served as the stressor. The unavoidable confrontation with a dominant fish in a tank proved to produce great stress for the subordinate eel. This was demonstrated by a number of physiological and haematological parameters.
Stressed eels have shrunken stomachs, the consistency of which is translucent and soft. The mucous membrane folds flatten or disappear. The mucous epithelium atrophies and during this process numerous residual bodies appear. The cell-to-cell contact loosens. The gastric glands degenerate developing large intracellular cavities, vacuolated ER and swollen mitochondria. The connective tissue proliferates replacing the necrotic glands. The submucosal vessels contract, thereby disturbing the circulation.
The consequences of the gastric atrophy (increased self-digestion, reduction of the immunological barrier, interference with protein digestion, endocrine function, etc.) are discussed.     

Development and homeostasis of the skin epidermis

The skin epidermis is a stratified epithelium that forms a barrier that protects animals from dehydration, mechanical stress, and infections. The epidermis encompasses different appendages, such as the hair follicle (HF), the sebaceous gland (SG), the sweat gland, and the touch dome, that are essential for thermoregulation, sensing the environment, and influencing social behavior. The epidermis undergoes a constant turnover and distinct stem cells (SCs) are responsible for the homeostasis of the different epidermal compartments. Deregulation of the signaling pathways controlling the balance between renewal and differentiation often leads to cancer formation.     

Piscinoodinium, a fish-ectoparasitic dinoflagellate, is a member of the class dinophyceae, subclass gymnodiniphycidae: convergent evolution with Amyloodinium

All dinoflagellates that infest the skin and gills of fish have traditionally been placed within the class Blastodiniphyceae. Their relatedness was primarily based upon a similar mode of attachment to the host, i.e., attachment disc with holdfasts. Results of recent molecular genetic analyses have transferred these parasites, including Amyloodinium, to the class Dinophyceae, subclass Peridiniphycidae. In our study, a small subunit rDNA gene from a parasitic dinoflagellate that has features diagnostic for species in the genus Piscinoodinium, i.e., typical trophont with attachment disc having rhizocysts, infesting the skin of freshwater tropical fish, places this organism within the dinophycean subclass Gymnodiniphycidae. This suggests a close relationship of Piscinoodinium spp. to dinoflagellates that include symbionts, e.g., species of Symbiodinium, and free-living algae, e.g., Gymnodinium spp. These molecular and morphological data suggest that evolution of this mode of fish ectoparasitism occurred independently in 2 distantly related groups of dinoflagellates, and they further suggest that the taxonomic status of parasites grouped as members of Piscinoodinium requires major revision.     

Effect of the daily ingestion of a purified anthocyanin extract from grape skin on rat serum antioxidant capacity

The aim of this work was to study the effect of the daily ingestion of a purified anthocyanin extract from red grape skin on rat serum antioxidant capacity (ORAC) and its safety for the intestinal epithelium. The study was carried out in rats orally administered with the extract for 10 days in either normal physiological conditions or exposed to a pro-oxidant chemical (CCl(4)). The oral administration of the extract significantly (P<0.05) enhanced the ORAC value of the deproteinised serum of about 50 % after 10 days of ingestion. Anthocyanin administration was also able to reverse completely the decrease in the serum ORAC activity induced by the CCl(4) treatment. Experiments with Ussing chamber mounted intestine allowed to exclude any toxicity of the extract for the intestinal epithelium. In conclusion, our results demonstrate that the purified anthocyanin extract from red grape skin enhances the total antioxidant capacity of the serum in either normal physiological condition or during oxidative stress induction, revealing a protective role against the decrease in the serum antioxidant capacity induced by a pro-oxidant compound.     

Spatial and temporal distribution of the adherens-junction-associated adhesion molecule A-CAM during avian embryogenesis

A-CAM (adherens-junction-specific cell adhesion molecule) is a calcium-dependent adhesion molecule which is associated with intercellular adherens junctions in various tissues (Volk & Geiger, 1986, J. Cell Biol. 103, 1441-1450 and 1451-1464). In the present report, we have investigated the distribution of A-CAM during avian morphogenesis by immunofluorescence microscopy and immunoblotting. A-CAM appeared at the onset of gastrulation on developing mesodermal and endodermal cells and was then expressed on tissues derived from the three primary germ layers. During embryonic life, A-CAM was constitutively expressed in a number of tissues including the central and peripheral nervous system, myocardium, muscles, notochord, skin and lens whereas it was found transiently in many tissues ranging from the nephritic tubules and the endoderm of visceral arches to ectodermal placodes. In the adult, in addition to the nervous system, A-CAM was restricted to the skin, lens, heart and testis, and exhibited an apparent molecular weight higher than the one found in the embryo. The prevalence and cell-surface modulation of A-CAM could frequently be correlated with morphogenetic events such as mesenchyme condensation into epithelia or cell clusters (e.g. formation of the somitic epithelium, kidney tubules and peripheral ganglia), dissociation of epithelia (e.g. dissociation of the somitic epithelium and segregation of neural crest from the neural tube), separation of cell populations (e.g. fibroblasts and myotubes in the heart) and reorganizations of epithelia (e.g. neurulation). In addition, using electron microscopy, the expression of A-CAM on the surface of aggregating and separating cells could be correlated with the formation and disappearance of adherens junctions. This precisely scheduled control of A-CAM correlated with early morphogenetic events during embryogenesis suggests that this CAM could play a crucial role in these processes.