Chronic treatment of female tammar wallabies with deslorelin implants during pouch life: effects on reproductive maturation.

The present study reports on attempts to delay puberty in a model marsupial species using the gonadotrophin-releasing hormone (GnRH) agonist deslorelin. Female tammar wallaby pouch young received deslorelin (5 mg) or placebo implants (n=8/group) when they were 193±2 days old. Sexual maturity was significantly delayed in deslorelin-treated animals, with the first successful production of offspring in treated and control animals occurring at 813±62 and 430±42 days of age, respectively. This delay was associated with a period of retarded pouch and teat development. Progesterone concentrations remained at basal levels throughout the first breeding season, indicating the absence of luteal cycles in treated females. Recovery and maturation of the hypothalamic-pituitary axis was a gradual process. Treated animals failed to respond to GnRH challenge at 12 months of age and had a reduced LH response at 18 months of age, before attaining full responsiveness by 24 months of age. Despite this apparent pituitary recovery by 24 months of age, as evidenced by complete teat eversion and LH responsiveness to GnRH, the time to first parturition was significantly delayed beyond this time in three females. This suggests that there may be longer-lasting effects at the level of the ovary and/or on FSH secretion. The significant delay in the onset of sexual maturation in response to chronic GnRH agonist treatment in this model marsupial species may be of practical significance to the management of fertility in captive and semi-free range marsupial populations.

Applications of GnRH in the control and management of fertility in female animals.

Gonadotropin releasing hormone (GnRH) has long been recognized as a potential target for the control and management of fertility in female animals. Attempts to apply GnRH-based technology to manage fertility have focussed on the development of GnRH agonists, antagonists and vaccines. All of these methods have potential, but the widespread application of these technologies has been limited to date. The greatest advance in the use of GnRH-based technology for long-term fertility control in recent years has been the development and commercialization of depot formulations that release GnRH agonists for periods of up to 1 year. These products have a broad range of potential applications in production and domestic animal management. The further development and commercialization of GnRH vaccines has been hampered by the variability of response between individual animals. The need to use adjuvant and multiple boosters also make this a less attractive option than the current GnRH agonist technology. However, GnRH vaccines have the advantage that they do not induce the initial stimulatory response that follows GnRH agonist administration. GnRH antagonists and GnRH-toxin conjugates show promise but are in an earlier phase of development. To date, no depot or long-acting formulations of antagonists have been developed. GnRH-toxin conjugates have yet to achieve permanent sterilization, but further dose-response trials may advance this approach.

Augmentation by eosinophils of gelatinase activity in the airway mucosa: comparative effects as a putative mediator of epithelial injury.

1. We have studied the release of gelatin-degrading enzymes from isolated sheets of bronchial mucosa in the presence and absence of eosinophils. 2. Isolated sheets of bovine bronchial mucosa released gelatin-degrading activity in similar amounts from both the apical and basolateral aspects of the tissue. Gelatinolytic activity could not be increased by treatment of the mucosal sheets with calcium ionophore, A23187. 3. The activity of the released gelatinases could be inhibited by chelation of divalent cations or by the matrix metalloproteinase inhibitors, BB-94 and BB-250. However, inhibitors of serine proteinases, or of cysteine proteinases were without effect. In zymography, major bands of gelatin-degrading activity consistent with gelatinases A and B were identified. 4. Addition of guinea-pig eosinophils to the basolateral aspect of bronchial mucosa for 60 min resulted in an increase in the gelatinolytic activity of the conditioned medium, irrespective of whether the eosinophils were stimulated with ionophore A23187 or not. However, only ionophore-stimulated eosinophils reacted to produce sufficient tissue damage to increase the transepithelial flux of serum albumin. 5. Purified eosinophils were a poor source of gelatinolytic activity, indicating that when interacting with the bronchial mucosa their effect is to increase the apparent release and/or activation of gelatinases derived from the airway mucosa. 6. After organomercurial activation, recombinant human progelatinase A increased the permeability of the bronchial mucosa to mannitol. However, the activity of enzyme and duration of exposure required to do this were greater than the amounts of gelatinase activity detected during eosinophil-mediated injury. Sheets of airway mucosa were also resistant to injury evoked by high concentrations of hydrogen peroxide or plasmin. 7. Collectively, these results suggest that if gelatinases are involved in eosinophil-mediated injury and repair of the bronchial mucosa, they require other mediators to act in concert to bring about outright epithelial cell detachment. This does not preclude the possibility that gelatinases are crucial in rendering the airway mucosa hyperfragile.

Stimulated eosinophils and proteinases augment the transepithelial flux of albumin in bovine bronchial mucosa.

1. The apical to basolateral transmucosal flux of albumin has been measured in isolated sheets of bovine bronchial and tracheal mucosa. Under resting conditions the net unidirectional flux in the bronchial mucosa was not significantly different from that measured previously for the basolateral to apical vector. In contrast, the apical to basolateral flux in the tracheal mucosa was significantly lower than that measured in the opposite direction. 2. Addition of guinea-pig peritoneal eosinophils to the apical side of the tissues had no significant effect on the transmucosal flux of albumin in either the bronchial or tracheal mucosa. 3. When eosinophils were stimulated with the ionophore A23187 or by opsonic adherence to tissues treated with a guinea-pig anti-bovine airway epithelium antibody, the bronchial mucosal sheets that had been exposed showed a significant increase in the transmucosal flux of albumin. However, tissues from the tracheal mucosa were resistant to the effects of stimulated eosinophils. 4. Histologically, sheets of mucosa from bovine main bronchi that had been exposed to stimulated eosinophils were characterized by epithelial injury consisting of loss of columnar epithelium from the underlying basal cell layer and biomatrix. Much less evidence of cellular injury was observed in tracheal tissues. 5. Bacterial collagenases applied to the apical side of the sheets were shown to increase the permeability of the bronchial mucosa to albumin and to produce histological changes that had similarities with the pattern of damage produced by stimulated eosinophils. 6. These observations demonstrate that the ability of eosinophils to injure the bronchial mucosa is independent of the side of the tissue on which they are present. Furthermore, key aspects of the injury process may be reproduced, at least in part, by metalloproteinases.

In vitro modulation of the eosinophil-dependent enhancement of the permeability of the bronchial mucosa.

1. Basolateral to apical albumin flux has been measured in sheets of bovine bronchial and tracheal mucosa mounted in vitro. 2. Addition of guinea-pig peritoneal eosinophils or neutrophils to the basolateral side of such tissues had no significant influence on the transmucosal flux of albumin in either the bronchial or tracheal mucosa. 3. Stimulation of eosinophils or neutrophils by the calcium ionophore A23187, or by their presentation to an opsonized airways mucosa, resulted in a significant increase in the transbronchial flux of albumin. This effect was seen after only 60 min incubation of the leucocytes with the bronchial mucosa, and was no greater when the contact time was extended to 180 min. Incubation of bronchial mucosal tissues with 1 mg ml-1 polyarginine for 3 h produced a significant increase in albumin flux, but was ineffective at 0.5 mg ml-1. 4. In contrast to the bronchial mucosa, the tracheal mucosa appeared resistant to the effects of stimulated eosinophils and neutrophils. 5. The lipoxygenase inhibitor AA-861 failed to influence the ability of eosinophils to augment the transmembrane flux of albumin. However, insertion of a Millipore filter mask between the eosinophils and the bronchial mucosa significantly inhibited the eosinophil-dependent enhancement of mucosal permeability. 6. The broad spectrum antiproteinase alpha 2-macroglobulin achieved almost total ablation of the action of stimulated eosinophils in the bronchial mucosa. These results suggest that proteinases may make a significant contribution to the genesis of epithelial injury, whereas leukotrienes do not.

The metabolism of prostaglandin D2. Evidence for the sequential conversion by NADPH and NAD+ dependent pathways.

Cell-free 100,000 g supernatants from human lung converted PGD2 into a product which on HPLC was indistinguishable from 9 alpha, 11 beta-PGF2. The rate of reaction was relatively slow (0.30 +/- 0.03 pmol/min/mg protein). In the presence of NAD+, 9 alpha, 11 beta-PGF2 was itself metabolized at a rate of 1.46 +/- 0.3 pmol/min/mg protein. The product of this reaction was less polar than the substrate and eluted with an HPLC retention time similar to that seen in our previous study where it was identified by GC/MS as being 15-keto-9 alpha, 11 beta-PGF2. There was no evidence for the formation of 13,14-dihydro-15-keto-9 alpha, 11 beta-PGF2. The sequence of metabolism of PGD2 was further established in cell-free supernatants prepared from guinea-pig liver and kidney which have previously been shown to be rich in both 11-ketoreductase and 15-hydroxy-prostaglandin dehydrogenase (PGDH) activities. Reactions contained both NAD+ and a NADPH-regenerating system and demonstrated the sequential metabolism of PGD2 to 9 alpha, 11 beta-PGF2 and ultimately 13,14-dihydro-15-keto-9 alpha, 11 beta-PGF2. Under these conditions the rate of the C-15 oxidation reaction was slower than that of 11-ketoreduction. These observations provide further support for our previous suggestions that the 11-ketoreduction of PGD2 is followed by a PGDH-type reaction, and that these reactions are likely to occur sequentially in vivo provided that the appropriate cofactors are present.

Genetics, biotechnology and population management of over-abundant mammalian wildlife in Australasia.

Wildlife management involves regulation of population numbers of wild vertebrate species. In some cases there are too many animals and in others there are too few. Genetic issues arise in both instances. The historical and genetic evidence for the number of mammals that were in the founder populations of successful colonizing species in Australia and New Zealand is reviewed here. Small numbers have often given rise to large populations, despite the concomitant loss of genetic variability. Restriction of the number of over-abundant and pest species by either physical or chemical methods frequently constitutes very strong artificial selection, which leads to rapid genetic change; an example of major importance in the two countries is sodium monofluoroacetate (compound 1080). Pathogenic agents, surgical sterilization, hormonal contraceptives and translocation have all been used with varying degrees of success. The strengths and weaknesses of these techniques are assessed. A method that has received much attention is immunocontraception. We argue that this attempt to use the animals' own immune system to modulate reproduction is incompatible with the basic biological function of protection against infectious disease. Immune function genes are highly variable in vertebrates, and so often genetic change in the population subjected to immunocontraception is likely to be even more rapid than is the case with lethal agents. Selection for failure to respond to the immunocontraceptive will occur, and will change immune function in general. Poor scientific description of ecosystem complexity makes it difficult to predict the consequences of immunocontraception on wildlife populations.

Chronic treatment of male tammar wallabies with deslorelin implants during pouch life: effects on development, puberty, and reproduction in adulthood.

The present study evaluated the effects of chronic GnRH agonist (deslorelin) treatment on sexual maturation in the male tammar wallaby. Slow-release deslorelin or placebo implants were administered to male pouch young (n = 10/group) when they were between 180 and 200 days old, to determine if disruption of the pituitary-testicular axis during development altered the timing of sexual maturation or had long-term effects on adult reproductive function. Deslorelin treatment caused retardation of testicular growth and reduced the serum FSH and testosterone concentrations between 12 and 24 mo of age. Maturation of the hypothalamic-pituitary-testicular axis was also delayed in treated animals at 13 and 19 mo of age. Despite these alterations in the pattern and timing of neuroendocrine development, sexual maturation was not permanently blocked in these animals and deslorelin-treated animals reached sexual maturity at the same age as treated animals, as evidenced by a fully functional pituitary-testicular axis and proven fertility at 25 mo of age. The ability of the treated animals to reach puberty at the same time as control animals, despite delayed maturation of the hypothalamic-pituitary-testicular axis, suggests that puberty in the male tammar wallaby is additionally regulated by other, gonadotropin-independent factors.

Long-term effects of deslorelin implants on reproduction in the female tammar wallaby (Macropus eugenii).

The contraceptive and endocrine effects of long-term treatment with implants containing the GnRH agonist deslorelin were investigated in female tammar wallabies (Macropus eugenii). Fertility was successfully inhibited for 515 +/- 87 days after treatment with a 5 mg deslorelin implant (n = 7), while control animals gave birth to their first young 159 +/- 47 days after placebo implant administration (n = 8). The duration of contraception was highly variable, ranging from 344 to 761 days. The strict reproductive seasonality in the tammar wallaby was maintained once the implant had expired. This inhibition of reproduction was associated with a significant reduction in basal LH concentrations and a cessation of oestrous cycles, as evidenced by low progesterone concentrations. There was evidence to suggest that some aspect of either blastocyst survival, luteal reactivation, pregnancy or birth may be affected by deslorelin treatment in some animals. These results show that long-term inhibition of fertility in the female tammar wallaby is possible using slow-release deslorelin implants. The effects of deslorelin treatment were fully reversible and there was no evidence of negative side effects. Slow-release GnRH agonist implants may represent a practicable method for reproductive management of captive and semi-wild populations of marsupials.

Long-term effects of unresolved sexual trauma.

Rape and child sexual abuse are common in our society, and the incidence is increasing. A variety of presenting complaints in adults may be signals of unresolved, remote sexual trauma. These include memory problems, confusion, impulsive or self-injurious behavior, unexplained somatic complaints and many others. Family physicians attentive to such signals can assist in relieving the toxic secret.

Effect of deslorelin implants on follicular development, parturition and post-partum oestrus in the tammar wallaby (Macropus eugenii).

The effect of treatment with slow release implants containing the GnRH agonist, deslorelin, was investigated in female tammar wallabies. Pouch young were removed from 16 wallabies presumed to be carrying quiescent blastocysts. Eight received a 5 mg deslorelin implant and eight received a placebo implant. Animals were caught daily from day 25 to day 30 and their pouches inspected for newborn young and their urogenital sinus checked for a copulatory plug. Treatment with deslorelin did not affect reactivation of a dormant blastocyst and subsequent birth in 4/8 animals, but post-partum mating was inhibited in these animals. Five control and five treated animals were killed within 0-48 h post partum and their reproductive tracts analysed. At autopsy, all five control animals had large preovulatory follicles but only one deslorelin-treated animal showed signs of follicular development. These differences were also reflected in the weights of the lateral vaginae, with treated animals showing no evidence of oestrogenic stimulation. The remaining three control and three treated animals were monitored for approximately 2 years. The long-term contraceptive effects of a single 5 mg deslorelin implant lasted for just under one year. These results indicate that slow release deslorelin implants inhibit follicular development in the female tammar wallaby for extended periods of time and may have potential application in reproductive management of captive marsupials in the kangaroo family.

Bacteriocin from Actinomyces odontolyticus with temperature-dependent killing properties.

A strain of Actinomyces odontolyticus, originally isolated from human dental plaque, produced a non-dialyzable, trypsin-sensitive substance that was bactericidal for certain strains of bifidobacteria at 42 degrees C but not at 37 degrees C. Detectable quantities of the bacteriocin were not produced in liquid media. Experimentally useful yields were obtained by extraction from pour plate cultures of producer cells. At 42 degrees C, exponential killing did not occur until indicator cells had doubled at least once. At 37 degrees C, the bacteriocin effected a transient bacteriostasis. Partially purified concentrates were obtained by diethylaminoethyl-cellulose chromatography, and such material was not inactivated by ribonuclease, deoxyribonuclease, or lipase. Pronase, trypsin, and exposure to 100 degrees C for 20 min completely abolished activity. Inhibitory activity was considerably reduced by exposure to a pH of either 3 or 11. Treatment of producer cells with curing agents did not induce a high frequency of non-bacteriocinogenic cells. The odontolyticin was adsorbed by susceptible, as well as resistant, bacteria.

The use of plasma as a replacement fluid in plasma exchange. Canadian Apheresis Group.

BACKGROUND: The Canadian Apheresis Group has maintained a national registry of apheresis activities for the past 16 years. Since 1991, the use of plasma as a replacement fluid in plasma exchange has been recorded. STUDY DESIGN AND METHODS: Six years of data from the registry on the use of plasma as a replacement fluid were analyzed. RESULTS: Plasma was used in more than 25 percent of all plasma exchange procedures. Of 41,519 plasma exchange procedures reported, 11,970 used plasma alone or in combination with albumin. In 1991, 1026 (78%) of these procedures used plasma appropriately for either thrombotic thrombocytopenic purpura (TTP) or adult hemolytic uremic syndrome (HUS); between 1992 and 1996, these numbers were 1043 (81%), 1570 (86%), 1171 (87%), 2192 (92%), and 2741 (90%), respectively. In the remaining procedures, frozen or cryosupernatant plasma was administered to 326 patients for a total of 40 diseases other than TTP or HUS. CONCLUSIONS: In those diseases for which plasma was administered as the sole replacement fluid, no disease appears to justify such treatment without the existence of an associated condition requiring specific replenishment of some plasma component. Further evaluation of the specific indications for the use of plasma as a replacement fluid in plasma exchange is required for diseases other than TTP or HUS.

Effects of a gonadotropin-releasing hormone agonist implant on reproduction in a male marsupial, Macropus eugenii.

This study evaluated the potential of slow-release GnRH agonist (deslorelin) implants to inhibit reproductive function in the male tammar wallaby. The specific aim was to measure the effects of graded dosages of deslorelin on testes size and plasma LH and testosterone concentrations. Adult male tammar wallabies were assigned to four groups (n = 6 per group) and received the following treatment: control, placebo implant; low dose, 5 mg deslorelin; medium dose, 10 mg; high dose, 20 mg. All dosages of deslorelin induced acute increases (P < 0.001) in plasma LH and testosterone concentrations within 2 h, with concentrations remaining elevated during the first 24 h but returning to pretreatment levels by Day 7. Thereafter, there was no evidence of a treatment-induced decline in plasma testosterone concentrations. There was no detectable difference in basal LH concentrations between treated and control animals, nor was there a significant change in testes width or length (P > 0.05). These results suggest that the male tammar wallaby is resistant to the contraceptive effects of chronic GnRH agonist treatment. Despite the maintenance of testosterone secretion, the majority of male tammars (10 of 17) failed to respond to a GnRH challenge with a release of LH between Days 186 and 197 of treatment. The failure of animals to respond to exogenous GnRH suggests a direct effect of deslorelin on the pituitary, resulting in a level of desensitization that was sufficient to inhibit a LH surge but insufficient to inhibit basal LH secretion. The variation between animals is believed to result from earlier recovery of some individuals, in particular those that received a lower dose, or individual resistance to the desensitization process.

Augmentation of permeability in the bronchial epithelium by the house dust mite allergen Der p1.

Sequence analyses have revealed the existence of homology between certain aeroallergens and proteolytic enzymes. This homology can be expressed functionally, but its significance to airway pathophysiology is unknown. Studies with Madin-Darby canine kidney cells and canine tracheal epithelial cells grown on plastic substrata or matrix proteins suggest that Der p1, a major allergen from the house dust mite Dermatophagoides pteronyssinus and a cysteine proteinase, or the unfractionated growth medium extract (SGME) from which it was purified, are both capable of causing cell detachment. The ability of both agents to produce functional changes in the barrier function of the epithelium was further demonstrated using isolated bovine airway preparations. Over a 3-h duration, both Der p1 and SGME elicited significant increases in the permeability of isolated sheets of bronchial mucosa to serum albumin. Exposure of isolated bronchial segments to luminally applied solutions of Der p1 resulted in histologic evidence of epithelial injury. Neither Der p1 nor SGME was active in these experimental systems unless chemically reduced, suggesting that the effect was initiated by cysteine proteinase activity. Similar augmentation of mucosal permeability and tissue injury was produced by bovine trypsin and collagenase from Clostridium histolyticum. In both the isolated mucosal sheet model and in cultured cells, the actions of Der p1 or SGME were associated with relatively little cytolysis, suggesting a specific action of the reagents on cell attachment. These results demonstrate a new functional activity of Der p1 that may be germane to the processes of allergen presentation, inflammatory cell activation, and chronic tissue injury.

Genomewide search in familial Paget disease of bone shows evidence of genetic heterogeneity with candidate loci on chromosomes 2q36, 10p13, and 5q35.

Paget disease of bone (PDB) is a common disorder characterized by focal abnormalities of increased and disorganized bone turnover. Genetic factors are important in the pathogenesis of PDB, and previous studies have shown that the PDB-like bone dysplasia familial expansile osteolysis is caused by activating mutations in the TNFRSF11A gene that encodes receptor activator of nuclear factor kappa B (RANK); however, linkage studies, coupled with mutation screening, have excluded involvement of RANK in the vast majority of patients with PDB. To identify other candidate loci for PDB, we conducted a genomewide search in 319 individuals, from 62 kindreds with familial PDB, who were predominantly of British descent. The pattern of inheritance in the study group as a whole was consistent with autosomal dominant transmission of the disease. Parametric multipoint linkage analysis, under a model of heterogeneity, identified three chromosomal regions with LOD scores above the threshold for suggestive linkage. These were on chromosomes 2q36 (LOD score 2.7 at 218.24 cM), 5q35 (LOD score 3.0 at 189.63 cM), and 10p13 (LOD score 2.6 at 41.43 cM). For each of these loci, formal heterogeneity testing with HOMOG supported a model of linkage with heterogeneity, as opposed to no linkage or linkage with homogeneity. Two-point linkage analysis with a series of markers from the 5q35 region in another large kindred with autosomal dominant familial PDB also supported linkage to the candidate region with a maximum LOD score of 3.47 at D5S2034 (187.8 cM). These data indicate the presence of several susceptibility loci for PDB and identify a strong candidate locus for the disease, on chromosome 5q35.