Biomedical subjects

A E Ellis

Publications and source records attributed to A E Ellis.

At least 55 records · Page 3Linked to original sources

PubMed2000

The gills are an important site of iNOS expression in rainbow trout Oncorhynchus mykiss after challenge with the gram-positive pathogen Renibacterium salmoninarum.

Following injection challenge of rainbow trout with the Gram-positive pathogen Renibacterium salmoninarum, serum nitrate levels increased indicative of NO production. The timing and amount of nitrate produced varied with the virulence of the bacterial strain used, with the highest levels seen in fish challenged with the most virulent (autoaggregating) strain. Immunization with a killed R. salmoninarum preparation in Freund's incomplete adjuvant significantly increased nitrate levels after challenge. Inducible nitric oxide synthase (iNOS) transcript expression was detectable in rainbow trout tissues after injection challenge with R. salmoninarum, and its induction in the gills was both quick (between 3 and 6 hr) and relatively prolonged (lasting several days). iNOS expression in the kidney was also seen at a later stage (24 hr) but appeared to switch off relatively rapidly. Bath challenge with R. salmoninarum also induced iNOS expression in gill, and a variable expression in the gut and kidney also occurred. These results highlight the importance of the gills, not only as a point of entry of pathogens but also as a tissue capable of mounting an immune response.

Animals↗

PubMed1999

Peroxide-inducible catalase in Aeromonas salmonicida subsp. salmonicida protects against exogenous hydrogen peroxide and killing by activated rainbow trout, Oncorhynchus mykiss L., macrophages.

Aeromonas salmonicida subsp. salmonicida expresses a single cytoplasmically located catalase which was found to be inducible by exposure to 20 microM hydrogen peroxide in mid-exponential phase resulting in a 4 fold increase in activity. Subsequent exposure to 2 mM peroxide in late-exponential/early-stationary phase resulted in further induction of catalase activity which increased to 20 fold higher levels than those found in uninduced cultures. Exponentially induced cultures were protected against subsequent exposure to 10 mM peroxide which was lethal to non-induced cultures. Bacteria subjected to induction in mid-exponential and early-stationary phase were resistant to 100 mM peroxide, although viability was greatly reduced. Growth of the bacterium under iron-restricted conditions had no effect on the peroxide induction of catalase. As current evidence indicates, the latter is an iron-co-factored heme catalase, this result suggests that catalase induction has a high priority in the metabolism of iron. Furthermore, exposure to peroxide also induces expression of periplasmic MnSOD. A. salmonicida MT423 was resistant to normal rainbow trout macrophages, but was susceptible to killing by activated macrophages. However, if catalase was induced by prior exposure to 20 microM peroxide during mid-exponential phase, A. salmonicida was resistant to killing by activated macrophages. The ability of A. salmonicida to upregulate periplasmic MnSOD and cytoplasmic catalase production under iron restricted conditions and low level peroxide (conditions expected to exist during the early stages of an infection) may be vital for its ability to withstand attack by phagocytic cells in vivo.

Aeromonas↗

PubMed1999

Binding of soluble immune complexes to fractionated Atlantic salmon (Salmo salar L.) leucocytes.

Binding of a fluorescent-labelled soluble immune complex to different types of Atlantic salmon leucocytes was investigated using flow cytometry. Peripheral blood leucocytes (PBL) were separated into sIg+ and sIg enriched populations by magnetic activated cell sorting, blood neutrophils were identified by electronic gating, and kidney macrophages were selected by plastic-adherence. About 60% of both sIg+ and sIg- enriched PBL, 44% of neutrophils and 34% of macrophages bound the soluble immune complexes.

Animals↗

PubMed1999

Vibrio anguillarum resistance to rainbow trout (Oncorhynchus mykiss) serum: role of O-antigen structure of lipopolysaccharide.

The sensitivity of Vibrio anguillarum to the bactericidal effect of rainbow trout serum was investigated with different strains of serogroups O1 and O2a, which are the most frequently found serogroups in clinical outbreaks of vibriosis. All of the V. anguillarum strains were able to activate complement in rainbow trout serum, but smooth strains of V. anguillarum serogroup O1 were resistant to complement-mediated killing in the absence of specific antibodies. In the case of V. anguillarum serogroup O2a strains, 80% of the analyzed strains were resistant to rainbow trout serum even when specific antibodies were present. Analysis of the lipopolysaccharide structures of the tested V. anguillarum strains showed a positive correlation between the O-antigen size of the lipopolysaccharide and resistance to serum killing. The classical complement pathway was responsible for the antibody-dependent serum killing of susceptible V. anguillarum strains. When serum-resistant V. anguillarum serogroup O2a strains were grown in glucose-enriched Lennox L broth, they produced lipopolysaccharide molecules with fewer high-molecular-weight O-antigen units than did strains grown in broth without the addition of glucose. Strains grown in glucose-enriched medium became sensitive to rainbow trout serum killing, indicating that the high-molecular-weight O-antigen side chains prevented the activated complement from damaging the bacterium.

Animals↗

PubMed1998

Neutrophil and macrophage responses to inflammation in the peritoneal cavity of rainbow trout Oncorhynchus mykiss. A light and electron microscopic cytochemical study.

The neutrophil and macrophage responses that accompany inflammation were studied in the peritoneal cavity of rainbow trout Oncorhynchus mykiss using light and electron microscopic cytochemistry. Neutrophils of inflammatory peritoneal exudates were alpha-naphthyl butyrate esterase-negative, peroxidase-positive and rich in cytoplasmic glycogen granules. Macrophages were poor in glycogen, esterase-positive and usually peroxidase-negative. Some peroxidase-positive macrophages were due to the transfer to macrophages of neutrophilic peroxidase. The ultrastructural double labelling for glycogen-peroxidase or esterase/peroxidase was most useful for precisely characterising neutrophils and macrophages in the inflamed peritoneal cavities and for correctly labelling peroxidase-positive macrophages. Intraperitoneal injection of casein, Incomplete Freund's Adjuvant (IFA) and live or formol-killed Yersinia ruckeri resulted in a rapid influx of neutrophils, peaking at 24 to 48 h post-injection and reaching values, in the case of live bacteria, 500 x those in the resting, unstimulated peritoneal cavity. Peritoneal macrophages also increased, but the response was slower (peak at 5 d) and with more modest increases in number (7.5 x). Neutrophil and mononuclear cells returned to normal values after 15 d in the case of casein and bacteria, but continued above base values 30 d after the injection of IFA. Conversely, after the injection of phosphate buffered saline, India ink or with sham-injections, very moderate neutrophil and and macrophage responses subsided in a few hours. Phagocytosis of bacteria was studied by light microscopy of preparations stained for peroxidase by a new method which allows for the simultaneous observation of intracellular bacteria and peroxidase staining. When Y. ruckeri was injected into resting peritoneal cavities, bacteria were ingested by the resident macrophages. When the bacteria were injected into cavities with high numbers of neutrophils (due to the previous injection of casein), more neutrophils than macrophages contained bacteria. Results show that the macrophages are the resident phagocytes of the peritoneal cavity of trout, while neutrophils are present in that body cavity in significant numbers only in situations of inflammation and only as long as the inflammation persists.

Animals↗

PubMed1998

Siderophore production by Aeromonas salmonicida subsp. salmonicida. Lack of strain specificity.

Siderophore production, presence of iron-regulated outer membrane proteins and siderophore specificity was determined among 17 isolates of Aeromonas salmonicida subsp. salmonicida obtained from Spain and Scotland. All grew in the presence of ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA) and siderophore production was detected using chrome azurol S (CAS) agar, confirming the presence of a high-affinity siderophore iron-uptake mechanism. The Arnow test confirmed that all isolates produced a catechol siderophore. Cross-feeding assays with indicator bacteria showed the absence of anguibactin, enterobactin, 2,3-dihydroxybenzoic acid (DHBA) and the hydroxamate siderophore, aerobactin, in the iron-restricted supernants of a representative isolate which cross fed 15/17 A. salmonicida isolates tested. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of the same 2 major iron-regulated outer membrane proteins (IROMPs) in all isolates when grown in iron-restricted conditions and siderophore strain specificity as assessed by cross-feeding experiments was not apparent. Thus, with respect to IROMP and siderophore production A. salmonicida appears to be a homogeneous species.

Aeromonas↗

PubMed1998

Isolation of rainbow trout neutrophils with an anti-granulocyte monoclonal antibody.

A magnetic cell sorting system has been optimised for the purification of rainbow trout neutrophils using a monoclonal antibody (E3D9) raised against Atlantic salmon neutrophils. The purified neutrophils have good viability (85%) and purity (approximately 92%), and were functional in respiratory burst and migration assays. The isolated neutrophils responded rapidly to PMA stimulation, producing levels of superoxide anion (4.85 nmols superoxide min-1/10(6) cells) approximately twice as high as macrophages from the same species. In the migration assay, there was a four-fold increase in migrating cells using the purified neutrophils compared with unfractionated blood leucocytes, and a relatively high neutrophil migratory activity was seen in the absence of serum.

Animals↗

PubMed1998

Defined deletion mutants demonstrate that the major secreted toxins are not essential for the virulence of Aeromonas salmonicida.

The importance of the two major extracellular enzymes of Aeromonas salmonicida, glycerophospholipid: cholesterol acyltransferase (GCAT) and a serine protease (AspA), to the pathology and mortality of salmonid fish with furunculosis had been indicated in toxicity studies. In this study, the genes encoding GCAT (satA) and AspA (aspA) have been cloned and mutagenized by marker replacement of internal deletions, and the constructs have been used for the creation of isogenic satA and aspA mutants of A. salmonicida. A pSUP202 derivative (pSUP202sac) carrying the sacRB genes was constructed to facilitate the selection of mutants. The requirement of serine protease for processing of pro-GCAT was demonstrated. Processing involved the removal of a short internal fragment. Surprisingly, pathogenicity trials revealed no major decrease in virulence of the A. salmonicida delta satA::kan or A. salmonicida delta aspA::kan mutants compared to the wild-type parent strains when Atlantic salmon (Salmo salar L.) were challenged by intraperitoneal injection. Moreover, using a cohabitation model, which more closely mimics the natural disease, there was also no significant decrease in the relative cumulative mortality following infection with either of the deletion mutants compared to the parent strain. Thus, although these two toxins may confer some competitive advantage to A. salmonicida, neither toxin is essential for the very high virulence of A. salmonicida in Atlantic salmon. This first report of defined deletion mutations within any proposed extracellular virulence factor of A. salmonicida raises crucial questions about the pathogenesis of this important fish pathogen.

Acyltransferases↗

PubMed1997

Detection of specific and 'constitutive' antibody secreting cells in the gills, head kidney and peripheral blood leucocytes of dab (Limanda limanda).

The relative immunological importance of the gills of fish was investigated in terms of antibody production by enumerating antibody secreting cells (ASC) in the gills, head kidney and blood of dab (Limanda limanda) using the ELISPOT assay. The contribution of 'constitutive' ASC in the gill appeared more substantial than that of elicited specific ASC. The gills were found to contain a mean (+/- SD) of 4227 +/- 1029 'constitutive' ASC/10(6) cells which was fewer than the head kidney which contained a mean (+/- SD) of 15617 +/- 3723 'constitutive' ASC/10(6) cells but more than peripheral blood leucocytes which contained a mean (+/- SD) of 2650 +/- 212 'constitutive' ASC/10(6) cells. The number of specific anti-human gamma globulin (HGG) ASC following parenteral or oral administration of HGG was also determined. Anti-HGG ASC were detected in all three tissues following parenteral immunization, peaking simultaneously, 4 weeks post-immunization. The strongest response was found in the head kidney. After oral immunization, responses were much weaker: again the head kidney was the most active but the gill response was barely detectable. These data were complemented by measurement of specific antibody in the serum by ELISA. Serum antibody titres following immunization were found to correlate closely with the number of specific ASC in the head kidney following parenteral immunization whereas serum antibody titres after oral administration of antigen most closely followed the number of specific ASC in the blood. In the light of these data it is suggested that the primary immunological role of the leucocytes in the gill may be in the earliest stages of defence against infection.

Animals↗

PubMed1997

In vivo production of A-protein, lipopolysaccharide, iron-regulated outer membrane proteins and 70-kDa serine protease by Aeromonas salmonicida subsp. salmonicida.

Using specific immunostaining of Western blots, the in vivo expression of several putative virulence factors of Aeromonas salmonicida subsp. salmonicida was demonstrated in infected muscle tissue of Atlantic salmon and rainbow trout. Three virulent isolates of A. salmonicida were used. One isolate was chosen because in vitro it was apparently a non-producer of the 70-kDa serine protease. Infected furuncle tissue was centrifuged and samples of the pellet and supernatant probed for evidence that the components of interest were bacterial cell-associated or secreted. The A-protein was detected in pelleted furuncle material but not in the supernatant. Lipopolysaccharide, both high and low molecular mass, was present in the pellet but only high molecular mass lipopolysaccharide was detected in the furuncle supernatant. Iron-regulated outer membrane proteins were detected in the furuncle pellet. The 70-kDa serine protease was detected in the furuncle supernatant of both protease-producing strains. However, whilst the protease-deficient isolate was demonstrated to produce low levels of the 70-kDa protease when grown in vitro under iron restricted conditions, none could be detected in vivo.

Aeromonas↗

PubMed1997

N-acetylcysteine improves indocyanine green extraction and oxygen transport during hepatic dysfunction.

OBJECTIVES: To investigate whether the beneficial systemic hemodynamic effects of N-acetylcysteine, an agent that increases cyclic guanosine monophosphate (cGMP) concentration in fulminant hepatic failure, are present in a range of liver disorders and what concurrent effect this agent has on the hepatic-splanchnic circulation. SETTING: Liver Failure Unit, King's College Hospital, London, UK. PATIENTS: Fifteen patients with hepatic dysfunction who were mechanically ventilated, either after liver transplantation or during an acute or decompensated chronic liver disorder. INTERVENTIONS: Prostacyclin was administered at a continuous infusion rate of 5 ng/kg/min for 60 mins. After a washout period, the hemodynamic effects of this infusion were compared with the effects present during infusion of N-acetylcysteine at 150 mg/kg in 250 mL of 5% dextrose in water over 15 mins and then 50 mg/kg in 250 mL of 5% dextrose for 45 mins at an infusion rate of 62.5 mL/hr. MEASUREMENTS AND MAIN RESULTS: Following N-acetylcysteine infusion, the baseline oxygen delivery (DO2) increased from 667 +/- 154 to 751 +/- 166 (SD) mL/min/m2, and oxygen consumption (VO2) improved in 13 of 15 patients (150 +/- 30 to 169 +/- 25 mL/min/m2) (p< .01). Indocyanine green clearance, as determined by a fiberoptic physiologic monitoring system, also improved in 13 of 15 patients (7.3 +/- 4.2% to 11.8 +/- 4.0% [mean change 100%, 95% confidence interval 9 to 256]) (p = .002). Patients who were defined as responders in relation to systemic hemodynamics (VO2 of >10% from baseline [n = 6; 40%]) had a significantly lower baseline consumption compared with that of nonresponders (133 vs. 162 mL/min/m2, p = .04). No clear relationship between the increments in VO2 and indocyanine green clearance was observed (r2 = .21; p = .08). Prostacyclin resulted in moderate improvements in systemic DO2 (but not VO2) and a nonsignificant increase in indocyanine green clearance. CONCLUSION: N-acetylcysteine increases systemic VO2 in a proportion of patients with a wide variety of hepatic disorders. In addition, N-acetylcysteine elicits an improvement in indocyanine green clearance. These properties may be clinically useful in a range of critical illnesses where systemic or hepatic-splanchnic circulations are compromised.

Acetylcysteine↗

PubMed1997

Immunization with bacterial antigens: furunculosis.

Although the nature of the antigens and the immune responses they elicit to achieve immunity to furunculosis are still not well defined, the currently available vaccines comprising A. salmonicida bacterins emulsified in oil adjuvants and delivered by intraperitoneal injection provide remarkably high levels of long-lasting protection. Despite some concern over side-effects, these vaccines have been adopted by most Atlantic salmon farmers over the last four years, transforming a situation where furunculosis outbreaks were becoming catastrophic to one where losses from the disease are negligible. Present evidence indicates that antibody responses to the polysaccharide capsule and iron regulated outer membrane proteins are associated with protection. Furthermore, cell-mediated immune responses involving antigen-induced release of cytokines from lymphocytes and the resultant activation of macrophages with the ability to kill the pathogen are also considered important protective mechanisms. Vaccines comprising whole A. salmonicida cultures grown under iron-restricted conditions and delivered by injection in an oil adjuvant are expected to induce prolonged stimulation of all the above responses. While these vaccines are suitable and effective for administration to salmon smolts there is still a need for mass vaccination by immersion or oral routes for salmonid fry. Effective means of achieving this are still required.

Aeromonas↗

PubMed1996

c-Fos antisense blocks acquisition and extinction of conditioned taste aversion in mice.

Conditioned taste aversion is an unusual form of associative learning in which long delays between conditional stimulus taste (CS) and unconditioned stimulus illness (US) suggest stimulus encoding by novel mechanisms. Recent data suggest that stimulus inputs are encoded by inducible bZIP proteins, the kinetics of which match the temporal features of the CS and US in taste aversion learning. Blockade of US-induced c-Fos translation in the brain stem by antisense oligonucleotides specifically blocks both acquisition and extinction of a learned taste aversion, but does not impair sensory processing of either CS or US, suggesting that c-Fos antisense blocks associative events within NTS necessary to support taste aversion learning.

Animals↗

PubMed1996

Development of preterm and full-term infant ability on AB, recall memory, transparent barrier detour, and means-end tasks.

10 preterm and 10 full-term infants were tested longitudinally from 28 to 60 weeks of age on a modified version of the AB task, a nonreaching AB task, a Barrier Detour task, a Means-End task, and Perseveration in the Means-End task. Results show that age-corrected (age since conception) premature infants tolerated longer delays than full-term infants on the modified and nonreaching AB tasks. However, when compared by chronological age (age since birth), there were no group differences on either the reaching or nonreaching AB task. No group differences were found on Barrier Detour, Means-End, or Perseveration in either the age-corrected or chronological age comparisons. The results suggest that the function that mediates modified AB performance is one of memory and not of perseveration or means-end ability. Further, these findings suggest that current proposals about brain development based on single samples of infants may be tenuous. Finally, the results of this study suggest that development of the brain structure(s) that mediate modified AB performance is strongly influenced by experience in the postnatal environment.

Age Factors↗

PubMed1993

Effects of the acetylcholinesterase toxin of Aeromonas hydrophila on the central nervous system of fish.

The purified acetylcholinesterase (AcChE) toxin, crude extracellular products (ECP) or viable virulent Aeromonas hydrophila were injected intraperitoneally into rainbow trout in different sublethal and lethal doses. When fish showed signs of morbidity, brain tissue was excised and assayed for acetylcholinesterase activity. In all cases there was a large increase in AcChE activity (about 40-fold for purified AcChE-toxin). This was shown to be due to an accumulation of the fish's own AcChE and not the bacterial toxin. Nevertheless, the latter was detected in brain homogenates from fish in all treatment groups using a rabbit antiserum to the purified toxin to probe Western blots of brain homogenates, demonstrating that the toxin does gain access to brain tissue and is produced during in vivo infection. The results strongly suggest that this toxin plays a central role in the pathogenesis of A. hydrophila infection.

Acetylcholinesterase↗

PubMed1992

Purification and characterisation of an extracellular metalloprotease, serine protease and haemolysin of Aeromonas hydrophila strain B32: all are lethal for fish.

Three different lethal (for rainbow trout, Salmo gairdneri) extracellular toxins were purified by HPLC from the culture supernatants of Aeromonas hydrophila strain B32 which had been isolated from rainbow trout. A metalloprotease, MW 38 kDa, was stable at 56 degrees C for 10 min, had no cytotoxic activity and and LD50 of 150 ng/g fish. In narrow range isoelectric-focusing (IEF) the enzyme had 11 isomers with (pls) between 4.12 and 4.8. A serine protease (22 kDa) was stable at 56 degrees C for 10 min, possessed cytotoxic activity and had an LD50 of 150 ng/g fish. In IEF, multiple isomers possessed pls between 4.5-5.2. The haemolysin had alpha-haemolytic activity (68 kDa) multiple isomers in IEF with pl range 4.5-5.1 and an LD50 of 2 micrograms/g fish. It was stable after heating to 56 degrees C for 20 min, 60 degrees C for 10 min and possessed esterase activity on beta-naphthyl acetate. These latter properties suggest it may be a novel haemolysin distinct from alpha- and beta-haemolysin.

Acetylcholinesterase↗

PubMed1991

A novel method for specific visualisation of serum albumin in polyacrylamide gels by iodine staining.

Bovine serum, bovine serum albumins (delipidated or globulin free or Fraction V), rabbit serum, rabbit serum albumin, Atlantic salmon serum, purified Atlantic salmon serum albumin, human plasma alpha 2-macroglobulin, hemocyanin, trypsin inhibitor, bovine transferrin and bovine lactoferrin were examined by a novel method for specific visualisation of albumins. In native polyacrylamide gel electrophoresis the albumins were visualised by iodine staining as a transparent spot against a brown background whilst the other proteins could not be visualised. It is suggested that the brown background was due to penetration of the gel by iodine while the chemical binding of iodine by albumin produced a decolourisation reaction. This novel method provides a fast and simple approach to identifying serum albumin in polyacrylamide gels.

Animals↗

PubMed1991

Cultural conditions for Aeromonas hydrophila affect the production of haemolysins with differing host specificities.

Five strains of Aeromonas hydrophila were studied for production of haemolysin specific for erythrocytes of various animal species using three cultural methods. All the strains produced haemolysin for all the erythrocyte species when the organisms were cultured on blood agar. Using cellophane overlay method, all the strains produced haemolysin for fish erythrocytes and variable activity to mammalian erythrocytes. Only one strain produced haemolytic activity for various though not all of the erythrocyte species when grown in brain heart infusion broth. Data suggest that A. hydrophila produces multiple haemolysins with specificities for erythrocytes of different animals. This was confirmed for trout and horse erythrocyte targeted haemolysins, by using iso-electric focussing separation and by measuring the effect of addition of ammonium sulphate to the growth medium.

Aeromonas hydrophila↗