A comparison of bovine serum albumin and chicken ovalbumin as supplements for the serum-free growth of chinook salmon embryo cells,CHSE-214.

Chicken ovalbumin and bovine serum albumin (BSA) were compared as supplements to the basal medium, L-15, for the serum-free cultivation of the Chinook Salmon Embryo cell line, CHSE-214. Unlike L-15 alone, ovalbumin and some commercial BSA preparations allowed cell proliferation and development of confluent monolayer cultures. However, only a fatty acid-free BSA (2%) supported continuous proliferation for two years through approximately 15 subcultivations. For this, subcultivation was achieved with non enzymatic cell dissociation solutions. Also, new serum-free subcultivation techniques were developed that utilized avian egg white trypsin inhibitors to terminate the action of either bovine or cod trypsin. Finally, CHSE-214 were successfully cryopreserved in 2% BSA, allowing all cell cultivation steps to be performed in the absence of FBS.     

Analysis and characterisation of IL-1beta processing in rainbow trout, Oncorhynchus mykiss

Mammalian IL-1beta is produced as a biologically inactive 31 kDa precursor, which is converted to the active 18 kDa form by proteolytic processing. Synthesis and processing of native piscine IL-1beta is poorly understood. In the present study, the native IL-1beta precursor or mature peptides were detected at sizes of approx. 29 kDa and 24 kDa in cell lysates of a rainbow trout macrophage cell line RTS-11, with or without LPS stimulation, by Western blot analysis using a polyclonal antibody against the putative trout mature IL-1beta (rmIL-1beta) produced in Escherichia coli. Processing of the 29 kDa precursor into a 24 kDa mature peptide was confirmed by analysis of such proteins using a monoclonal conjugate (Ni-NTA-HRP) against 6 histidines in lysates of the RTS-11 cells transfected with an expression plasmid containing the IL-1beta precursor molecule tagged with 6 histidines at its C terminus. Only the recombinant mature 24 kDa) IL-1beta/HIS protein was purified from the culture supernatants of the transfected cells, indicating the molecule is cleaved to be secreted. These findings strongly suggest that the trout IL-1beta molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1beta despite the lack of a clear ICE cut site.     

Effect of corticosteroids on viability and proliferation of the rainbow trout monocyte/macrophage cell line,RTS11

Cortisol at 1,000 and 100 ng/ml, and less consistently at 10 ng/ml, inhibited increases in cell number and 3H-thymidine incorporation by cultures of the rainbow trout (Oncorhynchus mykiss) monocyte/macrophage cell line, RTS11. Cell viability was not altered by cortisol, although a small decline in the capacity of cultures to reduce the redox dye, Alamar Blue was observed. In cortisol-treated cultures, more round and fewer spread cells were evident. Similar results were observed with dexamethasone but not cortisone. The glucocorticoid receptor antagonist, RU-486, prevented the effects of cortisol on RTS11 proliferation, and shape. In co-culture with the spleen stroma cell line (RTS34st) or in medium conditioned by RTS34st, the proliferation of RTS11 was enhanced. Treating RTS11/RTS34st co-cultures or RTS11 cultures in RTS34st conditioned medium with cortisol did not inhibit RTS11 proliferation. Overall these experiments suggest that proliferation of rainbow trout macrophages is regulated by cortisol, but the effect is modulated by the cellular micro-environment, possibly through the release of cytokines.     

The functional characterisation of CK-1, a putative CC chemokine from rainbow trout (Oncorhynchus mykiss)

Recently a number of cytokine homologs have been cloned in teleost fish, including several that resemble chemokines, but to date few have been confirmed using functional assays. Chemokines are a family of cytokines that are able to induce chemotaxis in leucocytes. In this study CK-1, a rainbow trout chemokine, was functionally characterised. Recombinant CK-1 is able to attract rainbow trout peripheral blood leucocytes (PBL) in a micro-chemotaxis chamber. A greater number of PBLs migrated in response to CK-1 than to negative controls, either media alone or equivalent concentrations of beta2M, while comparable numbers migrated to the positive control, recombinant human C5a. The tissue distribution of CK-1 mRNA was also assessed by Northern blotting of RT-PCR and showed that expression is constitutive in the liver and gut, and is inducible by intraperitoneal injection of phytohemagglutinin in PBL and the head-kidney. Continuous cell lines generated from the gut and pituitary gland of the rainbow trout also express CK-1 message, whilst Southern analysis shows that CK-1 is a single copy gene. Finally, CK-1 shows the greatest amino acid similarity CCL20/LARC/Mip-3alpha as well as similar gene structure and expression pattern.     

Redox-active metals in commercial preparations of lipopolysaccharide: implications for studies of cellular responses to bacterial products

The mechanisms by which lipopolysaccharide (LPS) activates cells have been the subject of intense investigation for many years. Whereas much information on this process has been collected for mammalian species, little is known about the signalling path-ways operative in other animals. One general mode of cellular activation that has been recently pro-posed for pathways independent of the primary mammalian LPS receptor, CD14, involves reactive oxygen species (ROS) as intermediates in LPS-induced signalling pathways. Therefore, we used 2',7'-dichlorodihydrofluorescein, a fluorogenic probe of redox activity, to examine LPS-induced oxidative responses of a macrophage-like cell line from the rainbow trout, RTS11. Lipopolysaccharide dose-dependently increased oxidation of this probe by RTS11 cells, and a variety of other cell lines. This process was inhibited by catalase, superoxide dismutase and NG-methylarginine citrate, an inhibitor of nitric oxide synthases, suggesting the involvement of a diverse assortment of cellular ROS. More careful dissection of this phenomenon led us to conclude that the increase in oxidation was, in fact, due almost entirely to metals, particularly copper, in some LPS preparations, which is something to consider when experimenting with LPS.     

Developmental competence of juvenile calf oocytes in vitro and in vivo: influence of donor animal variation and repeated gonadotropin stimulation

Juvenile calf oocytes represent an untapped source of germ plasm for reproduction. Reports on the developmental competence of calf oocytes have been controversial. In this research, oocytes were recovered after gonadotropin stimulation from Holstein calves (N = 10) at 2-3 mo of age (2-mo cycle) and again at 4-5 mo of age (4-mo cycle). The in vitro developmental competence was measured, and prestimulation follicle numbers (for 2-mo cycle) and poststimulation follicle numbers (both cycles) were obtained. The number of antral follicles doubled after stimulation (23.4 +/- 6.1 vs. 55.1 +/- 16.1) for the 2-mo cycle and for the 4-mo cycle (47.4 +/- 12.4). The number of follicles observed prior to stimulation in the 2-mo cycle was found to be highly correlated with the poststimulation oocyte recovery for both collection cycles (r = 0.95, 2-mo cycle; r = 0.81, 4-mo cycle). The majority (90-96%) of recovered oocytes were found to be usable for in vitro maturation and fertilization; of these, 41-42% cleaved and 10-11% developed to morulae or blastocysts. Eighty-four in vitro-produced embryos were transferred to synchronized recipients and resulted in 11 pregnancies, leading to 7 live (4 males, 3 females) and 2 dead (one male, one female) calves at full term. No significant differences were observed between the 2-mo and 4-mo collection cycles; however, 73% of the total pregnancies resulted from the 2-mo cycle. All pregnancies resulted from embryos of high-responding donors. The high correlation between the number of follicles prior to stimulation and the poststimulation response suggests the possibility of screening calves prior to stimulation for routine embryo production.     

Some but not All Tetrahymena Species Destroy Monolayer Cultures of Cells from a Wide Range of Tissues and Species

The activities of Tetrahymena corlissi, Tetrahymena thermophila, and Tetrahymena canadensis were studied in coculture with cell lines of insects, fish, amphibians, and mammals. These ciliates remained viable regardless of the animal cell line partner. All three species could engulf animal cells in suspension. However, if the animal cells were monolayer cultures, the monolayers were obliterated by T. corlissi and T. thermophila. Both fibroblast and epithelial monolayers were destroyed but the destruction of human cell monolayers was done more effectively by T. thermophila. By contrast, T. canadensis was unable to destroy any monolayer. At 4 °C T. thermophila and T. corlissi did not carryout phagocytosis and did not destroy monolayers, whereas T. canadensis was able to carryout phagocytosis but still could not destroy monolayers. Therefore, monolayer destruction appeared to require phagocytosis, but by itself this was insufficient. In addition, the ciliates expressed a unique swimming behavior. Tetrahymena corlissi and T. thermophila swam vigorously and repeatedly into the monolayer, which seemed to loosen or dislodge cells, whereas T. canadensis swam above the monolayer. Therefore, differences in swimming behavior might explain why T. corlissi has been reported to be a pathogen but T. canadensis has not.     

Development and practical applications of a method for repeated transvaginal, ultrasound-guided biopsy collection of the bovine ovary

In response to the increasing research into primordial and preantral follicular dynamics, a device for transvaginal, ultrasound-guided biopsy collection of the bovine ovary was developed and tested. The new device is based upon a commercially available Ovum Pick-up instrument and consists of a modified needle guidance system, which has been equipped with a trocar needle and caries a 60 cm long true-cut biopsy needle. Biopsies are captured in a 20mm long and 2mm wide specimen notch. In the present experiment, 10 cows were subjected to a twice weekly biopsy regime over a four-week period. A total of 208 attempts at biopsy collection were made, and 141 tissue samples collected (success rate of 68%). Through histological and immunological analyses, these tissue samples have been shown to contain primordial and preantral follicles. At the end of the trial period, several of the donor cows were slaughtered at timed intervals, and the ovaries were harvested for assessment of the damage inflicted by the repeated biopsy procedure. Post mortem ovaries were inspected macroscopically and examined by conventional histological staining. In ovaries retrieved 2 days after the last biopsy session, blood clots were macroscopically apparent throughout the ovaries. Histological examination showed increased infiltration of red blood cells in the ovarian stroma. Analysis from ovaries collected at subsequent slaughter points revealed reduced infiltration of blood, and clear indications of resumed antral follicle development were apparent towards the end of the first month after the trial period. We conclude that the biopsy sampling technique is a repeatable procedure which could serve as a renewable source of primordial and preantral follicles for culture, and as an in vitro model for the study of preantral follicular dynamics.     

Gliotoxin-induced cytotoxicity in three salmonid cell lines: cell death by apoptosis and necrosis

Epithelial (CHSE-214), fibroblast (RTG-2) and macrophage (RTS11) cell lines from Chinook salmon and rainbow trout were tested for their sensitivity to gliotoxin, a fungal metabolite. Gliotoxin treatment for 6 or 24 h caused cell viability to decrease in a dose-dependent manner, with effective concentrations (EC50s) being similar for the three cell lines but varying with exposure time. Under some exposure conditions, hallmarks of apoptosis were detected. Apoptosis was evaluated by the appearance of fragmented nuclei upon H33258 staining and of genomic DNA laddering into 180 bp oligomers. Gliotoxin induced cell detachment in RTG-2 and CHSE-214 cultures, under some conditions. These were the only cultures of these two cell lines in which apoptosis was detected, and apoptotic cells appeared more frequent in the detached population. At the highest concentration, 15 microM, the cells died by an alternative mode, likely necrosis. By contrast, in RTS11 cultures cell detachment was not observed, and apoptosis occurred over a wider concentration range, even 15 microM, reaching levels of over 90%. The preferential death by necrosis for epithelial cells (CHSE-214) and by apoptosis for macrophages (RTS11) could be a beneficial host response to gliotoxin-producing fungi, leading respectively to the development and then resolution of inflammation.