Opercular epithelial cells: a simple approach for in vitro studies of cellular responses in fish.

This study evaluated the efficacy of fish opercular external (skin) and inner (opercular membrane) epithelium as an in vitro model for toxic and other substances studies. The rainbow trout (Oncorhynchus mykiss) operculum was cultured in 12-well dishes containing sterile Leibovitz 15 (L-15) supplemented with glutamine medium during 24h at 9 degrees C, and the effect of copper, a toxic agent, and/or cortisol, an endogenous agent, on the epithelial cells was analyzed using light microscopy techniques. The opercula were submitted to four treatments: (i) control (Cont), L-15 medium only, (ii) 0.28 microM cortisol (Cort), (iii) 100 microM CuSO(4) (Cu), and (iv) 0.28 microM cortisol+100 microM CuSO(4) (Cort-Cu). The tissue condition after 24h incubation was analyzed by staining the mucous cells for neutral and acid mucosubstances. Cellular necrosis was evaluated by measuring the lactate dehydrogenase (LDH) leakage at 12 and 24h incubation. Cellular proliferation, apoptosis, metallothionein (MT) and glucocorticoid receptor (GR) expression were evaluated by immunohistochemistry. The LDH leakage was higher and the proliferating cell nuclear antigen (PCNA) positive-stained cells were lower in Cu treatment in both, epidermis and opercular membrane. Apoptotic cells in the opercular membrane were higher in the Cort and Cort-Cu treatments while, in the epidermis, they were higher in Cu and Cort-Cu treatments. GR-positive stained cells decreased significantly in all treatments in both epithelia and the MT-positive cells increased in the Cu and Cort-Cu treated groups. Copper showed to be a potent toxic stressor killing the cells via necrosis, decreasing the number of PCNA-positive cells and inducing MT synthesis while cortisol did not affect the MT synthesis, although it might stimulate apoptosis. The results are evidence that the opercular epithelia serve as a suitable model for studying in vitro effects of toxic agents, as well as endogenous factors on the cellular responses without interference of the physiological state of fish being useful to predict in vivo toxicity.

Proteome analysis of highly immunoreactive proteins of Helicobacter pylori.

BACKGROUND: Identification of the immunoreactive proteins of Helicobacter pylori is important for the development of both diagnostic tests and vaccines relating to the organism. Our aim was to determine whether there are significant differences between human IgG and IgA reactivities to individual H. pylori proteins, and whether patterns of immunoreactivity are sustained across different strains of H. pylori. METHOD: The total complement of protein from seven strains of H. pylori was resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins were transferred electrophoretically onto polyvinylene difluoride (PVDF) membranes, which were probed with sera pooled either from H. pylori-infected patients, or noninfected (control) patients. Highly immunoreactive proteins were detected using chromogenic enzyme-antibody conjugates recognising either serum IgG or IgA. These proteins were then characterised by tryptic peptide-mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Highly immunoreactive proteins were detected which were common to all seven strains, and recognised by both immunoglobulin subclasses. The proteins appear to be localised in five groups. Protein analysis established that these groups encompass multiple isoforms of chaperonin HspB (two subgroups); urease beta-subunit UreB; elongation factor EF-Tu; and flagellin FlaA. The pattern of highly immunoreactive proteins was strongly conserved across the seven strains. CONCLUSION: These results suggest that within a tightly defined region on the H. pylori proteome map there are five groups of proteins that are highly reactive to both IgG and IgA. Our analysis suggests it is unlikely that the highly immunoreactive clusters harbour any significant proteins other than isoforms of HspB, UreB, EF-Tu and FlaA, and that, with the partial exception of FlaA, these clusters are strongly conserved across all seven strains.

Stress response to waterborne Cu during early life stages of carp, Cyprinus carpio.

Following in vitro fertilization, eggs/embryos and larvae of the common carp (Cyprinus carpio) were exposed to 0 (control), 0.3 or 0.8 micromol.l(-1) Cu in artificially prepared fresh water for 168 h. The total amounts of Cu, Na, Ca, adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH) and cortisol were measured in homogenates of eggs (up to 60 h post fertilization, hpf), isolated embryos (between 60 hpf and hatching) and free-swimming larvae. Only in embryos of eggs exposed to 0.8 micromol.l(-1) Cu a significant accumulation of Cu was observed as well as a concurrent increase in the incidence of spinal cord deformation and larval mortality. Further, when exposed to 0.8 micromol.l(-1) Cu, the whole-body Ca and Na contents were lower at 48 and 72 hpf compared to the controls and those exposed to 0.3 micromol.l(-1) Cu. Conversely, in larvae (>72 hpf) exposed to 0.3 micromol.l(-1) Cu, the Ca content was elevated from 96 hpf onwards. At 48 hpf and onwards, the whole-body ACTH and cortisol contents of the embryos exposed to 0.8 micromol.l(-1) Cu were higher than those in either controls or those exposed to 0.3 micromol.l(-1) Cu. By 96 hpf, ACTH and cortisol contents of the group exposed to 0.3 micromol.l(-1) Cu also surpassed those in controls. The alpha-MSH content in both Cu exposed groups was lower than in controls from 48 hpf onwards. It thus appears that ACTH cells and MSH cells in early life stages of carp exposed to waterborn Cu respond differently; we conclude that in prehatch carp pituitary corticotropes and interrenal cortisol producing cells respond to the chemical stressor Cu and that the resulting hormonal changes provide a sensitive diagnosis for stress as well as toxicity tests.