Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones.

Members of the ATP-binding cassette (ABC) transporter family (P-glycoproteins, Half-transporters and Multidrug Resistant Proteins) potentially play a role in the development of anthelmintic resistance. The aim of this study was to investigate the possible involvement of ABC transporters in anthelmintic resistance in the bovine parasite, Cooperia oncophora. Partial sequences of 15 members of the ABC transporter protein family were identified, by mining a transcriptome dataset combined with a degenerate PCR approach. Reverse transcriptase PCR showed that most of the ABC transporters identified were constitutively transcribed throughout the life cycle of C. oncophora. Constitutive differences in gene transcript levels between a susceptible and resistant isolate were only observed for Con-haf-9 and Con-mrp-1 in eggs of the resistant isolate, while no differences were observed in L3 or the adult life stage. Analysis of resistant adult worms, collected from calves 14 days after treatment with either ivermectin or moxidectin, showed a significant 3- to 5-fold increase in the transcript levels of Con-pgp-11 compared to non-exposed worms. Interestingly, a 4-fold transcriptional up-regulation of Con-pgp-11 was also observed in L3 of the resistant isolate, after in vitro exposure to different concentrations of ivermectin, whereas this effect was not observed in exposed L3 of the susceptible isolate. The results suggest that the worms of this particular resistant isolate have acquired the ability to up-regulate Con-pgp-11 upon exposure to macrocyclic lactones. Further work is needed to understand the genetic basis underpinning this process and the functional role of PGP-11.

Presence of helodermin-like peptides of the VIP-secretin family in mammalian salivary glands and saliva.

Helodermin is a biologically active peptide isolated from the venom of the Gila monster lizard (Heloderma suspectum) whose structure is related to that of vasoactive intestinal peptide and secretin. Using a specific radioimmunoassay based on antisera prepared by immunizing rabbits with natural helodermin, we demonstrated the presence of helodermin-like material in mammalian salivary glands, including parotid, submaxillary and sublingual glands from rat and dog, and parotid and submaxillary glands from man. All helodermin-like materials had an apparent molecular mass of 4-12 kDa. Dog saliva, collected after pilocarpine stimulation, revealed similar immunoreactivity with a major component around 6 kDa.

Failure to pentagastrin administration to restore postprandial acid secretion from Heidenhain pouches after antrectomy in dogs.

In four dogs with Heidenhain pouche, acid outputs from the pouch were measured in response to feeding boiled liver with or without simultaneous administration of pentagastrin. Antrectomy was performed and the experiments were repeated. Antrectomy decreased the acid response to feeding by 90 percent. The preantrectomy response was restored by infusing pentagastrin but near maximal doses were needed and the maximal response to feeding plus pentagastrin could not be restored. These results indicate that the decrease of the acid secretory response to feeding from Heidenhain pouches after antrectomy cannot be explained fully through suppression of endogenous gastrin release. Antrectomy probably interferes with the intestinal phase of gastric secretion.

Effect of cimetidine, ranitidine and omeprazole on postprandial gastrin and somatostatin release in conscious dogs.

During a first series of experiments, the gastrin responses to a meal were measured and compared to the responses seen after administration of cimetidine (2.5 mg/kg/h) or omeprazole (2 mg/kg). During a second series of experiments the effects of cimetidine (2.5 mg/kg/h), ranitidine (0.5 mg/kg/h) and omeprazole (2 mg/kg) on post-prandial gastrin and somatostatin release were determined in experiments during which the intragastric pH was maintained close to 6.4. During a third series of experiments, the effects of cimetidine (2.5 mg/kg/h) and omeprazole (2 mg/kg) on basal gastrin and somatostatin release were estimated. Postprandial gastrin release was increased by cimetidine and by omeprazole. When acidification of the gastric content was prevented by intragastric titration, postprandial gastrin release was increased by about 100%. No further increase was observed when the animals were concomitantly treated with cimetidine, ranitidine or omeprazole. Intragastric titration did not alter postprandial somatostatin release. Concomitant administration of H2 blockers decreased the somatostatin response to the meal, while concomitant administration of omeprazole did not alter this release. No significant changes were observed in basal gastrin or somatostatin levels after administration of cimetidine or omeprazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of vagotomy and atropine on plasma somatostatin response to a meal in conscious dogs.

In 4 conscious dogs with gastric fistulas the somatostatin responses to a meal were measured and compared to the responses seen after i.v. infusion of atropine sulfate (20 and 50 micrograms.kg-1.h-1) or cimetidine (8 mg.kg-1.h-1). The experiments were repeated after truncal vagotomy. The somatostatin responses to bombesin (0.5 micrograms.kg-1.h-1) were also measured before and after vagotomy. Vagotomy decreased basal and postprandial somatostatin levels and reduced the somatostatin responses to feeding during the first 30-min period following the ingestion of the meal but not during subsequent periods. Bombesin-induced somatostatin release was increased after vagotomy. Atropine decreased the somatostatin responses to the meal before and after vagotomy. Cimetidine had no significant effect. These studies suggest that, in conscious dogs, somatostatin released into the circulation is partly under vagal control and that, as for gastrin release, vagal pathways for stimulation and inhibition are present. Our studies also suggest that cholinergic mechanisms are involved in the control of postprandial somatostatin release.

Treatment of high-output gastric fistulas with omeprazole.

Two patients with high-output gastrocutaneous fistulas were treated with total parenteral nutrition and gastric antisecretory drugs. IV administration of omeprazole decreased acid output in one patient in a rapid and significant way, resulting in the spontaneous healing of the fistula after eight days of treatment. In the second patient, omeprazole caused a marked reduction in the acidity of the fluid in the fistula, which also closed without surgical operation. The long-standing decrease in acid output induced by intravenous omeprazole may be very useful for promoting spontaneous closure of high-output gastrocutaneous fistula.

Effect of met-enkephalin on acid secretion from gastric fistulas and Heidenhain pouches in dogs stimulated by pentagastrin, pentagastrin plus bethanechol, or a meal.

In dogs with denervated Heidenhain pouches and gastric cannulas the authors studied the action of met-enkephalin on acid secretion stimulated by a liquid meal, pentagastrin or pentagastrin + bethanechol. Serum gastrin levels were determined during the feeding experiments. Meal-stimulated and pentagastrin-stimulated acid secretion from the gastric fistula were inhibited by met-enkephalin. A rise in pentagastrin-induced acid secretion was observed in the denervated stomach. No significant changes were observed in the postprandial acid response from the pouch to the meal, nor from the main stomach or the pouch during stimulation with pentagastrin + bethanechol. Gastrin release was decreased by met-enkephalin during the first hour following feeding and increased during the second hour. Our data indicate that met-enkephalin can either inhibit or stimulate acid secretion in dogs. The inhibition occurs only in the innervated fundic mucosa, and might be explained by a decrease in gastrin release and by a decrease in vagal tone.

Increase in gastrin and somatostatin cell numbers in the antrum of dogs after small bowel resection.

A proximal 50 p. 100 resection of the small bowel was performed in six dogs while six control animals underwent a simple intestinal transsection and reanastomosis. The pre- and postoperative serum gastrin levels were measured in both groups before and after a standard test meal. The stomach was resected in both groups 6 to 8 weeks after the operation. A morphometric method using specific antigastrin and antisomatostatin antibodies was used to estimate the density and the total number of antral gastrin and somatostatin cells. No variation in basal serum gastrin was found after surgery. A 50 p. 100, though not significant, increase in the postprandial gastrin response was observed after intestinal resection whereas a significant (P less than 0.01) 50 p. 100 increase in the gastrin cell density was found in the antral mucosa of these dogs. At the same time, a remarkable hyperplasia of the antral somatostatin cell population was observed in the animals submitted to small bowel resection.

[Basal concentrations and postprandial integrated flows of gastrin in patients with atrophic gastritis or duodenal ulcer. Limits of diagnostic usefulness]. / Concentrations basales et débits intégrés post-prandiaux de gastrine chez des malades atteints de gastrite atrophique ou d'ulcère duodénal. Limites à l'utilité diagnostique.

Maximal acid outputs were determined during intravenous pentagastrin tests (6 micrograms/kg/h) in 119 male subjects: 17 controls, 74 patients with duodenal ulcer and 28 with atrophic gastritis. Basal and postprandial serum gastrin levels were also determined in order to estimate the integrated gastrin response to the meal. In patients with atrophic gastritis the maximal acid output was decreased (p less than 0.01) and the integrated gastric response was increased (p less than 0.01) but the basal gastrin levels in these patients did not differ from that of controls. An integrated gastrin response greater than 2.5 ng/ml/100 min was observed in 89 p. 100 of patients with atrophic gastritis. An integrated gastrin response smaller than 2.5 ng/ml/100 min was observed in 76 p. 100 of controls. The maximal acid output was smaller than 20 mmol/l in all patients with atrophic gastritis but was greater than this value in all controls. In duodenal ulcer patients, the measured parameters were not significantly different from control values. The measure of the integrated gastrin response which reflects the presence of an antral endocrine hyperactivity may be useful to detect patients with atrophic gastritis, but this test is less sensitive and less specific than the determination of the maximal acid output.

[Interactions between secretin and somatostatin on acid secretion and gastric emptying in dogs]. / Interactions entre la sécrétine et la somatostatine sur la sécrétion acide et la vidange gastrique chez le chien.

Secretin and somatostatin are two peptides released by H+ ions. The fact that blood levels of these peptides increase during the postprandial period makes them the most probable candidates for gastric acid secretion retrocontrol . The aim of our study was to determine whether a potentiation of inhibitory effects exists when the two peptides are administered simultaneously. Seven dogs were provided with a gastric fistula. Gastric acid secretion was stimulated by intragastric infusion of a bactopeptone solution or by pentagastrin (1 microgram X kg-1 X h-1). Acid outputs and gastric emptying rates were measured at regular intervals. The tests were repeated during intravenous infusion of secretin (0.125 micrograms X kg-1 X h-1), somatostatin (0.2 micrograms X kg-1 X h-1) or both peptides together at the same doses. Both secretin and somatostatin inhibited acid secretion and gastric emptying. The inhibition was not greater when both peptides were administered together. Under the conditions used, no potentiation or additive effects between the two peptides were observed on gastric acid secretion or gastric emptying.

[Physiological control and pharmacological inhibition of digestive secretions]. / Contrôle physiologique et inhibition pharmacologique des sécrétions digestives.

The pharmacological treatment of alimentary tract fistulas was for a long time limited to the use of atropine and related substances. More recently H2 blockers, pirenzepine, omeprazole and loperamide have been used. It is likely that in the coming years specific and long acting derivatives of somatostatin and enkephalins inhibiting digestive secretions will be synthetized.

[Treatment of resectable cancer of the stomach]. / Traitement du cancer gastrique réséquable.

The results of 631 cancers of the stomach treated by gastrectomy between 1970 and 1983 were closely reviewed in six departments of surgery. This review showed that the prognostic factors determined by resectability, extension of the tumor and lymph node involvement are considered to be reliable following the various pre- and postoperative examinations. In addition, it was shown that most of the gastrectomies were performed following conventional procedures and that the widespread lymph node dissections are not applied in a systematic manner. The postoperative mortality is very low (4.4%) for all the gastrectomies and even lower for the radical gastrectomies (3%). The 5 year survival rate for all the gastrectomies is 15%; this rate amounts to 36% for the gastrectomies performed with a curative aim. In the discussion, the adjuvant radiotherapy and chemotherapy are discussed and their timeliness is shown.

Assessing resistance against macrocyclic lactones in gastro-intestinal nematodes in cattle using the faecal egg count reduction test and the controlled efficacy test.

The main objective of the present study was to evaluate the accuracy of the faecal egg count reduction test (FECRT) to assess the resistance status of ivermectin (IVM)-resistant isolates of the cattle nematodes Ostertagia ostertagi and Cooperia oncophora, using the controlled efficacy test (worm counts) as a reference. The second objective was to investigate whether both IVM-resistant isolates showed side-resistance against moxidectin (MOX) under controlled conditions. Thirty male Holstein calves were experimentally infected with 25,000 L3 of an IVM-resistant O. ostertagi isolate and 25,000 L3 of an IVM-resistant C. oncophora isolate. Twenty-eight days later the calves were randomly divided into 2 treatment groups and 1 untreated control group. Animals in groups 1 and 2 received MOX (Cydectin(®) 1%, Pfizer) and IVM (Ivomec(®) 1%, Merial) respectively, by subcutaneous injection at a dose rate of 0.2mg/kg bodyweight. Faecal samples were collected 7 and 14 days after treatment and animals were necropsied 14/15 days post-treatment. Both the FECRT and the controlled efficacy test demonstrated that the O. ostertagi and C. oncophora isolates were resistant against IVM, with efficacies below 90%. The IVM-resistant O. ostertagia isolate was still susceptible to MOX treatment, as shown by over 99% reduction in egg counts and worm burden. The FECRT suggested borderline resistance against MOX in the IVM-resistant C. oncophora isolate, with egg count reductions between 97% (95% CI: 76; 100) at day 7 and 86% (95% CI: 49; 96) at day 14. However, the controlled efficacy test clearly showed MOX-resistance, with a decrease of only 31% (95% CI: -12; 57) in C. oncophora worm numbers. After MOX treatment, a significantly lower number of eggs per female C. oncophora worms was counted compared to the control group (43% reduction). Due to this reduced fecundity, the FECRT may fail to detect MOX-resistance.

Altered avr-14B gene transcription patterns in ivermectin-resistant isolates of the cattle parasites, Cooperia oncophora and Ostertagia ostertagi.

Ivermectin (IVM) resistance is an emerging problem for the control of gastrointestinal nematodes of cattle such as Cooperia oncophora and Ostertagia ostertagi. Although there is still a poor understanding of the molecular basis of macrocyclic lactone (ML)-resistance, it is clear that IVM exerts its activity by binding to glutamate-gated chloride (GluCl) channels within the parasite's neuromuscular system. One of the GluCl genes (avr-14) encodes, via alternative splicing, two subunits, AVR-14A and AVR-14B; the latter is suggested to be the main target for IVM. The genomic DNA (gDNA) sequence of avr-14 in C. oncophora contains 21 exons separated by 20 introns and spans approximately 10 kb of gDNA. Intron 13 contains a sequence with high homology to a mammalian mariner transposase. The L256F polymorphism in the avr-14 gene, which was shown to be associated with IVM resistance in a UK isolate of C. oncophora, was not found in the IVM-resistant C. oncophora and O. ostertagi isolates investigated in this study. However, genetic analyses on C. oncophora indicated a loss in allelic diversity of the avr-14 gene in the resistant isolates compared with the susceptible isolate. This suggests that the avr-14 gene, or another genetically linked locus, is under selection in these Belgian C. oncophora isolates. Comparison of the full-length avr-14B coding sequence in the susceptible and resistant C. oncophora isolates did not show any polymorphisms specifically linked to IVM resistance, although a decrease in the number of avr-14B isoforms was observed in the resistant isolates compared with the susceptible one. Measuring the transcription levels of avr-14B in adult male and female C. oncophora and O. ostertagi worms showed significantly lower levels in resistant worms compared with susceptible ones. Whether the down-regulation of this IVM target actually contributes to the resistance mechanism in these worms remains unclear.