The antigenic and immunogenic properties of synthetic peptide immunocontraceptive vaccine candidates based on gamete antigens.

In this study we have investigated the immunogenicity and antigenicity of synthetic immunocontraceptive vaccine candidates containing T- and B-cell epitopes arranged in different geometries. Two epitopes were selected, a B-cell epitope from fox sperm lactate dehydrogenase C(4) and a B-cell epitope from murine zona pellucida protein 3. Both were immunogenic in BALB/c mice when coupled to defined T helper cell determinants, eliciting antibodies which bound to the corresponding B-cell epitope and also to the recombinant protein. Because each of these proteins represent important components in the fertilisation process the results encourage further investigation of synthetic immunocontraceptive vaccines.

Antigen-specific systemic and reproductive tract antibodies in foxes immunized with Salmonella typhimurium expressing bacterial and sperm proteins.

Attenuated Salmonella typhimurium strains are potential 'safe' delivery vectors of an oral immunocontraceptive vaccine for the European red fox (Vulpes vulpes). In the present study, model bacterial (Escherichia coli heat-labile enterotoxin B subunit, LTB) and fox sperm (fSP10) antigens were expressed in S. typhimurium SL3261 (delta aroA) under the control of the trc promoter. Adult female foxes were given three oral immunizations with SL3261 containing either LTB (SL3261/pLTB), fSP10 (SL3261/pFSP10) or a control plasmid (pKK233-2 or pTrc99A). All foxes raised serum (IgG) and vaginal (IgG and IgA) antibodies against S. typhimurium lipopolysaccharide (LPS). Each fox that received SL3261/pLTB raised high titre LTB-specific serum and vaginal IgG antibodies. However, only one of four foxes immunized with SL3261/pFSP10 raised an anti-fSP10 immune response, in the form of low titre serum and vaginal IgG antibodies. No vaginal IgA antibodies were raised against either LTB or fSP10 in these experiments. The immune responses against recombinant LTB and fSP10 resulted chiefly from the initial dose of antigen in the inocula and were minimally influenced by continued in vivo antigen expression. This study demonstrates for the first time in the female red fox that oral Salmonella can elicit specific systemic and reproductive tract antibodies against heterologous, recombinant proteins.

A bait-delivered immunocontraceptive vaccine for the European red fox (Vulpes vulpes) by the year 2002?

An orally-delivered immunocontraceptive vaccine is being developed for the control of fox populations. A number of genes (PH-20, LDH-C4, ZP3) encoding gamete proteins have been cloned, produced in recombinant expression systems and used in fertility trials to test the efficacy of these antigens. As the immunocontraceptive vaccine will be delivered in a bait, there is a requirement for a greater understanding of the immune responses of the reproductive mucosa in canids, and the assessment of the best vaccine delivery system that will evoke a mucosal antibody response. Several vaccine delivery systems including microencapsulated antigens, and both vaccinia virus and bacterial vectors are being investigated. Oral administration of Salmonella typhimurium recombinants expressing different fox sperm antigens stimulates both systemic IgG responses to the antigen and a mucosal immune response within the female reproductive tract in the fox, indicating that salmonella may have potential with respect to the oral delivery of antigen. The enhancement of mucosal immune responses to orally-delivered vaccines is also being examined, research focussing on the possible use of fox-specific cytokines or the beta-subunit of cholera toxin in forming part of the vaccine construct.

Changes in the concentration of fructose in the blood of piglets of different ages after doses of fructose, fructose plus glucose, and sucrose.

We investigated the hydrolysis of sucrose in the small intestine and the subsequent absorption and metabolism of fructose in sucking piglets by measuring temporal changes in the concentration of fructose in the plasma following the administration of physiological amounts of these carbohydrates. Calculations of the area under the curve for fructose in the plasma showed that there was no age limit to the piglets' ability to absorb fructose. However, there was a limit to the amount of fructose that the younger piglets could get from a dose of sucrose. Indeed, we demonstrated that there was a positive linear correlation between a piglet's capacity to hydrolyse sucrose and the age of the piglet up to 15 d of age (r 0.98). The half-life for fructose was 495, 103, 38, 49 and 28 min in 2-, 5-, 7-, 10- and 15-d-old piglets respectively and, thus, there was only limited utilization of fructose in the younger piglets. However, there were 13.0- and 1.4-fold increases in the elimination rate of fructose from the plasma of piglets from 2 to 7 d and from 7 to 15 d respectively, consistent with the reported increase in the deposition of fat in piglets of a similar age range. Hence, the effective metabolism of fructose may be partially dependent on the amount of adipose tissue present and the phosphorylation of this monosaccharide by hexokinase (EC 2.7.1.1) in this tissue.

Maintenance of villus height and crypt depth, and enhancement of disaccharide digestion and monosaccharide absorption, in piglets fed on cows' whole milk after weaning.

The aims of the present study were (a) to maintain the structure and function of the small intestine of the piglet after weaning, and (b) to compare the capacity in vivo of sucking and weaned piglets to digest oral boluses of lactose and sucrose and absorb their monosaccharide products. Piglets were fed on cows' whole milk ad libitum every 2 h for 5 d after weaning. Physiological doses of lactose plus fructose (treatment LAC+FRU) and sucrose plus galactose (treatment SUC+GAL) were administered on day 27 of lactation and on the fifth day after weaning, after which time piglets were killed. Villus height and crypt depth were maintained (P > 0.05) by feeding cows' milk after weaning. The areas under the curves (AUC) for galactose and glucose, adjusted for live weight and plasma volume, increased (P < 0.05) after weaning. Despite the enhancement of gut function after weaning, the galactose index (GalI:AUC for galactose ingested as lactose divided by the AUC for the same dose of galactose ingested as the monosaccharide) and fructose index (FruI: AUC for fructose ingested as sucrose divided by the AUC for the same dose of fructose ingested as the monosaccharide), which are indices of digestive and absorptive efficiency, both decreased after weaning. This apparent anomaly may be reconciled by increased growth, and hence surface area, of the small intestine between weaning and slaughter such that 'total' digestion and absorption most probably increased despite apparent decreases in GalI and FruI. Positive correlations (P < 0.05) between villus height and GalI are consistent with the maximum activity of lactase occurring more apically along the villus. Significant linear relationships (P < 0.05) were recorded between villus height at the proximal jejunum and adjusted AUC for galactose and glucose following treatment LAC+FRU, and between villus height at the proximal jejunum and adjusted glucose AUC following treatment SUC+GAL. These relationships suggest that maximum digestion and absorption occurs at increasing distances along the crypt:villus axis in the weaned pig.

The responses of blood galactose to oral doses of lactose, galactose plus glucose and milk to piglets.

The capacity of intestinal lactase (EC 3.2.1.23) of piglets to hydrolyse lactose in vivo was investigated by measuring the response of blood galactose to doses of lactose, galactose plus glucose and both whole and skimmed milk. Following the administration of oral doses of lactose dissolved in water to piglets from 2 to 18 d of age the adjusted galactose area under the curve (AUC) was between 1.12 and 1.36 arbitrary units, while following a dose of galactose plus glucose dissolved in water it was between 1.56 and 1.98 arbitrary units. Whereas these results suggest that the rate of digestion of lactose appeared to limit the amount of galactose reaching the peripheral blood after a dose of lactose dissolved in water, there was no significant correlation between the capacity of piglets to hydrolyse physiological amounts of lactose and the age of the piglets (2- to 18-d-old piglets; r 0.11). Following oral doses of sow's milk containing either lactose, or galactose plus glucose, the adjusted galactose AUC values were 0.94 and 1.00 arbitrary units respectively, in 10-d-old piglets. Thus, the limitation to the digestion of lactose observed when it was present in water was not evident for lactose in sow's milk. Since there was no significant difference between the adjusted galactose AUC following a dose of whole milk (0.95 arbitrary units) and that following a dose of skimmed milk (1.03 arbitrary units), the presence of fat in sow's milk did not appear to affect the utilization of lactose by the sucking piglets.

Progressive changes in milk protein gene expression and prolactin binding during lactation in the tammar wallaby (Macropus eugenii).

Changes in milk protein gene expression and specific prolactin binding were quantified in mammary tissue from the tammar wallaby (Macropus eugenii) at different stages of lactation. The transition from early (phase 2) lactation to late (phase 3) lactation was characterized by the induction of the gene for late lactation protein, a novel whey protein. During the same period, the levels of beta-lactoglobulin and beta-casein gene expression increased, whereas there was no change in the levels of expression of alpha-lactalbumin and alpha-casein genes. Prolactin binding in the mammary gland doubled during the latter half of phase 2 of lactation but declined significantly during the transition to phase 3 of lactation. These changes in prolactin binding resulted from changes in the number of receptors and not from a change in the affinity of the receptor for prolactin. Treatment of membranes with concanavalin A increased the number of prolactin-binding sites by 40% in membranes from phase 2 mammary tissue but decreased binding by 40% in membranes from phase 3 tissue, indicating that significant changes had occurred in the membranes of cells during this period. The tammar wallaby can secrete phase 2 and phase 3 milk from adjacent mammary glands (asynchronous concurrent lactation) and the developmental changes in milk protein gene expression and prolactin binding observed during lactation were reflected in these individual glands. Taken collectively, these findings suggest that mammary development and milk secretion in the tammar wallaby are regulated by both endocrine and local (intramammary) mechanisms.

The response in the blood of piglets to oral doses of galactose and glucose and intravenous administration of galactose.

The kinetics of the response in the blood of piglets to physiological oral intakes of galactose and glucose, and intravenous administration of galactose are described. Following the intravenous administration of galactose to 2- and 10-d-old piglets (n 7), the half-life was 7.98 (SD 0.75) and 7.99 (SD 1.89) min respectively, and efficient elimination rate was 9.09 (SD 2.15) and 8.75 (SD 0.79)% per min respectively. The turnover of galactose in the piglets was 100.3 micrograms/min per kg body weight. These observations demonstrate that galactose was rapidly removed from the blood of the piglets. While the dosing and sampling procedures stimulated hyperglycaemia, they had no effect on the concentration of galactose in the peripheral plasma. The galactose area under the curve (adjusted to the plasma volume of the animal) following a dose of either galactose or galactose plus glucose was 1.75 (SD 0.15) and 1.95 (SD 0.14) arbitrary units respectively in 2-d-old piglets and 1.96 (SD 0.26) and 1.98 (SD 0.10) arbitrary units respectively in 10-d-old piglets. Since the presence of glucose did not lower the adjusted area under the curve for galactose in the peripheral blood, the effect of glucose on the metabolism of galactose in piglets was more like that reported for rats than that for man, guinea-pigs or mice. It is suggested that the galactose moiety of lactose may make an important contribution to the replenishment of liver glycogen in the neonatal piglet.

Etodolac (1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-acetic acid): a potent antiinflammatory drug. Conformation and absolute configuration of its active enantiomer.

The active (+) enantiomer of the antiinflammatory agent etodolac (1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]-indole-1-acetic acid) has been assigned an S absolute configuration on the basis of a crystallographic analysis of the (S)-(-)-borneol ester of (-)-etodolac, and the conformation of etodolac has been determined by a crystallographic analysis of (+/-)-etodolac. Analyses of the solid-state conformation, as well as energy-minimized conformations obtained by molecular mechanics calculations, have failed to provide a basis for identifying a probable receptor-site conformation.