Investigations to extend viability of a rainbow trout primary gill cell culture.

The primary culture of fish gill cells can provide functional, cell diverse, model in vitro platforms able to tolerate an aqueous exposure analogous to in vivo tissues. The utility of such models could be extended to a variety of longer term exposure scenarios if a method could be established to extend culture viability when exposed to water for longer periods. Here we report findings of a series of experiments to establish increased longevity, as monitored by culture transepithelial electrical resistance (TEER) and concurrent histological developments. Experimental cultures improved TEER during apical freshwater exposure for a mean of twelve days, compared to previous viabilities of up to 3 days. Cultures with larger surface areas and the use of trout serum rather than foetal bovine serum (FBS) contributed to the improvement, while perfusion of the intact gill prior to cell harvest resulted in a significantly faster preparation. Detailed scanning electron microscopy analysis of cultures revealed diverse surface structures that changed with culture age. Cultures grown on membranes with an increased porosity, collagen coating or 3D structure were of no benefit compared to standard membranes. Increased culture longevity, achieved in this study and reported for the first time, is a significant breakthrough and opens up a variety of future experimentation that has previously not been possible. The extended viability facilitates exploration of in vitro chronic or pulse-exposure test paradigms, longer term physiological and environmental monitoring studies and the potential for interactive co-culture with other organoid micro-tissues.

A toxic diet: transfer of contaminants to offspring through a parental care mechanism.

The transfer of maternal contaminants to offspring during oogenesis and gestation is documented in many animals, and in mammals, contaminants may pass from mother to offspring during lactation. Although other non-mammalian vertebrates provide parental care in the form of nutritive secretions for offspring to feed from, the potential for toxicant transfer during non-mammalian parental care is rarely considered. The discus fish, Symphysodon spp., employs an unusual parental care strategy where fry feed on parental epidermal mucus for several weeks after hatching. This strategy has the potential to act as a method of contaminant transfer. In discus adults, both waterborne and dietary toxicants are sequestered and secreted into their epidermal mucus, the food on which fry depend. To determine whether parents could channel these contaminants directly to offspring, we exposed parents to aqueous cadmium (Cd) and recorded the subsequent feeding behaviour and Cd content of fry. Fry continued to feed normally from contaminated mucus and accumulated significant tissue concentrations of Cd. In conclusion, this parental care mechanism of the discus fish can expose offspring to harmful contaminants during the sensitive early stages of life and highlights that parent to offspring contaminant transfer after birth may be more widespread than previously thought.

Changes in plasma fibronectin isoform levels predict distinct clinical outcomes in critically ill patients.

INTRODUCTION: Concentrations of the total pool of fibronectin in plasma (TFN), and the subset of this pool that contains the alternatively spliced EDA segment (A(+)FN), are both affected by disease processes, and the latter pool has gained a reputation as a biomarker for vascular injury. We therefore wished to determine if changes in either FN pool correlate with clinical outcomes in critically ill individuals. METHODS: We analyzed a database for 57 patients with major trauma (n = 33) or sepsis syndrome (n = 24) in which plasma levels of TFN and A(+)FN had been measured at intervals, along with clinical parameters. Logistic regression analysis was performed to detect associations between predictive variables and three clinical outcomes: 1) the acute respiratory distress syndrome (ARDS), 2) milder acute lung injury designated acute hypoxemic respiratory failure (AHRF), and 3) survival to hospital discharge. RESULTS: An increase in plasma TFN during the first 24 hours of intensive care unit (ICU) observation was negatively associated with progression to ARDS (odds ratio 0.98 per 1 microgram (µg)/ml increase, 95% CI (0.97, 1.00)) and AHRF (OR 0.97 per 1 µg/ml increase, (0.95, 0.99)), whereas an increase in A(+)FN over the first 24 hours was positively associated with progression to AHRF (OR 1.65 per 1 µg/ml increase, (1.04, 2.62)). Additionally, the ratio of the partial pressure of oxygen in arterial blood (PaO(2)) to the percentage of oxygen in inspired air (FIO(2)) after 24 hours was positively associated with survival (OR 1.01 per 1 unit increase in ratio, (1.00, 1.03)), along with change in A(+)FN (OR 1.30 per 1 µg/ml increase, (0.90, 1.88)). CONCLUSIONS: Different FN isoforms may constitute predictive covariate markers for distinct clinical outcomes in critically ill patients. The data also suggest that early TFN accumulation in the circulation may confer a clinical benefit to patients at risk for acute lung injury.

Accumulation of dietary and aqueous cadmium into the epidermal mucus of the discus fish Symphysodon sp.

The discus fish Symphysodon sp. is an Amazonian cichlid with a unusual form of parental care where fry obligately feed from parental mucus for the first few weeks of life. Here, we investigated the possible impact of environmental cadmium on this species, particularly with respect to mucus contamination. We exposed groups of fish to cadmium either through their food (400 mg kg(-1)) or through the water (3 µgl(-1)) for 4 weeks, and measured tissue concentrations and ATPase activities at weekly intervals. Cadmium significantly accumulated in all tissues (except for muscle) after 7 days, and tissue concentrations increased until the end of the experiment. Significant alterations in ATPase activities of intestine and kidney were observed at day 7 and 14, but no alterations in gill ATPase activities occurred. The epidermal mucus showed a high accumulation of cadmium from both exposures, but particularly from the diet, indicating that dietary cadmium can be transferred from gut to mucus. Combining this data with approximations of fry bite volumes and bite frequencies, we constructed daily estimates of the cadmium that could potentially be consumed by newly hatched fry feeding on this mucus. These calculations suggest that feeding fry might consume up to 11 µgg(-1)day(-1), and hence indicate that this species' dependency on parental mucus feeding of fry could make them particularly susceptible to cadmium contamination of their native habitat.

Biparental mucus feeding: a unique example of parental care in an Amazonian cichlid.

Vertebrates display a wide variety of parental care behaviours, including the guarding of offspring pre and post nutritional independence as well as the direct provision of nutrients during the early development period. The Amazonian cichlid Symphysodon spp. (discus fish) is unusual among fish species, in that both parents provide offspring with mucus secretions to feed from after hatching. This extensive provision of care, which can last up to a month, imposes a physiological demand on both parents and gives rise to conflict between the parent and offspring. Here, we investigated the relationship between parents and offspring during a breeding cycle, determining both mucus composition (total protein, cortisol, immunoglobulin, and Na(+), K(+) and Ca(2+) concentrations) and the behavioural dynamics of the parent-offspring relationship. Over the course of a breeding cycle, a significant increase in offspring bite rate was recorded, with a concomitant increase in the frequency of turns the male and female parent took at caring for their young. A peak in mucus antibody provision was seen as offspring reached the free-swimming stage, suggesting a role analogous to colostrum provision in mammals. Mucus protein content was lowest during the second and third weeks of free swimming, and a weaning period, similar to that seen in mammalian parental care, occurred when the offspring had been free swimming for ∼3 weeks. In many ways, the parental behaviour of discus fish is more similar to mammalian and avian parental care than other fish species, and represents an exciting aquatic model for studying the parent-offspring conflict.

Impaired reproduction in three-spined sticklebacks exposed to ethinyl estradiol as juveniles.

To investigate the population-level effects of exposure to environmental endocrine disrupters, a mesocosm-scale study was carried out in which the reproductive performance of groups of free-spawning three-spined sticklebacks, Gasterosteus aculeatus, exposed as juveniles to a model estrogen, was assessed. Juvenile sticklebacks were exposed to ethinyl estradiol (EE(2)) at measured concentrations of (mean +/- SEM) 1.75 +/- 0.37 ng L(-1) and 27.7 +/- 1.08 ng L(-1) for 4 wk posthatch and then reared thereafter in pristine lake water until they reached adulthood. Exposure to the higher EE(2) concentration resulted in the occurrence of ovotestis among males, whereas no gonadal abnormalities were evident among males exposed to the lower concentration of EE(2). In addition, when spawning was allowed in the mesocosm environment, fewer nests were built per male, and fewer eggs were deposited per nest, in the group exposed to 27.7 ng L(-1). Males from this group also exhibited a less intense nuptial coloration than control males. In the group exposed to 1.75 ng L(-1) EE(2) posthatch, significantly fewer nests were built than in the control group. These results demonstrate that the timing of exposure to estrogenic contaminants, in developmental terms, is critically important. Short-term exposure to estrogens as juveniles can clearly influence reproductive performance as adults, despite all growth and development subsequent to the exposure period taking place in an estrogen-free environment. In addition, these results suggest that reproductive dysfunction can occur even in fish with no gross abnormalities in gonadal structure. This suggests that the absence of gonadal intersex is not a reliable indicator of the reproductive potential, or estrogen-exposure history, of fish populations or the only important factor involved in compromising the reproduction of estrogen-exposed fish.

Building a rapid response team.

The use of rapid response teams is a relatively new approach for decreasing or eliminating codes in acute care hospitals. Based on the principles of a code team for cardiac and/or respiratory arrest in non-critical care units, the rapid response teams have specially trained nursing, respiratory, and medical personnel to respond to calls from general care units to assess and manage decompensating or rapidly changing patients before their conditions escalate to a full code situation. This article describes the processes used to develop a rapid response team, clinical indicators for triggering a rapid response team call, topics addressed in an educational program for the rapid response team members, and methods for evaluating effectiveness of the rapid response team.

Plasma levels of fibronectin bearing the alternatively spliced EIIIB segment are increased after major trauma.

Using Western-blot analysis and enzyme-linked immunosorbent assay (ELISA) of N-deglycosylated samples, we have observed that plasma levels of fibronectin (FN) bearing the alternatively spliced EIIIB segment (EIIIB(+) FN) increase in patients after admission to the intensive-care unit (ICU) for acute major trauma. Although not increased at the first sampling ("0 hour"), taken within 24 hours of ICU admission, levels measured 24, 48, and 72 hours later were significantly increased compared with levels obtained in healthy controls. Furthermore, average concentrations at these latter time points were significantly increased relative to the 0-hour sampling. EIIIB(+) FN levels then decreased in plasma samples taken 1 month after hospital discharge, such that no significant difference was found between ELISA-measured values at this time and 0 hour or control values. On the basis of comparisons with previous measurements in these samples, it is apparent that after acute major trauma, circulating levels of soluble EIIIB(+) FN exhibit temporal changes that are qualitatively similar to those encountered for FN isoforms bearing the alternatively spliced EIIIA segment (EIIIA(+) FN), yet different from those observed for the total pool of circulating FN. This is the first report of measurement of FN in clinical plasma samples with antibodies specific for the highly conserved EIIIB segment. Like EIIIA(+) forms of FN, EIIIB(+) FNs are recognized as soluble bloodborne markers for vascular tissue injury.