Meeting review--Helicobacter pylori: basic mechanisms to clinical cure 2000.

The meeting Helicobacter pylori: Basic Mechanisms to Clinical Cure 2000, held in Bermuda from March 26 to 29, 2000, gathered physicians and scientists from all corners of the world. State-of-the-art reviews and the most recent developments in the field were presented. This article summarizes the highlights of this meeting, including important scientific and clinical developments.

Helicobacter pylori antibiotic resistance: trends over time.

Resistance to antibiotics can be a major problem in the treatment of bacterial infections. As the use of antibiotics increases, bacterial resistance to these agents is rising and in many cases is responsible for the failure of treatment regimens. Although the treatment of Helicobacter pylori infection requires the use of more than one antibiotic to obtain adequate eradication rates, the efficacy of the currently used antibiotic combinations has been shown to be decreased by resistance to one of the antibiotics. The use of antibiotics in regiments for the treatment of H pylori is increasing in many countries, including Canada. This increase is both in the use of these antibiotics alone for the treatment of nongastrointestinal infections and in their use in association with proton pump inhibitors for the treatment of H pylori infection. In several European and Asian countries, where resistance to antibiotics is being monitored, it has been demonstrated the H pylori resistance to metronidazole and to clarithromycin increased throughout the 1990s. Thus far, the data available in Canada do not show increased resistance to either of these antibiotics. As for other antibiotics used in the treatment of H pylori infection such as tetracycline and amoxicillin, the rate of resistance to these agents is still very low and does not constitute a significant problem. Because the efficacy of the regimens used in the treatment of H pylori infection is compromised by resistance to the antibiotics used, it is important that H pylori resistance rate in Canada and throughout the world continue to be monitored. Only with such reliable data can the most optimal regimens be recommended.

Methodological validation and clinical usefulness of carbon-14-urea breath test for documentation of presence and eradication of Helicobacter pylori infection.

UNLABELLED: A simple [14C]urea breath test (C-14-UBT) was validated with aims of determining accuracy in documenting both the presence and proof of eradication of Helicobacter pylori infection. METHODS: Fifty-six dyspeptic patients had endoscopy with biopsies and C-14-UBT. Eleven biopsy-proven H. pylori-negative patients allowed C-14-UBT normal value determination. Forty-three patients with recurrent peptic ulcer disease and biopsy-proven H. pylori infection were included in an antimicrobial eradication protocol. Endoscopy with biopsies and C-14-UBT were done again 8 wk after initiation of treatment in 35 patients. For C-14-UBT, 185 kBq (5 microCi) of [14C]urea was swallowed. Breath samples obtained up to 20 min were counted to calculate AS20, [(% 14CO2 dose excreted/mmol of CO2) x kg] at 20 min. Combined histologic and microbiologic analyses of antral biopsies were used as a gold standard. RESULTS: The positivity value was set as AS20 > 0.33% (mean + 3 s.d. of AS20 in H. pylori-negative patients). Diagnosis of H. pylori infection was correct with C-14-UBT in 55/56 patients (44 true-positive, 11 true-negative and 1 false-negative; sensitivity = 98%; specificity = 100%). As a proof of eradication, C-14-UBT correctly classified 33/35 patients (5 true-positive, 28 true-negative and 2 false-positive; sensitivity = 100%; specificity = 93%). The C-14-UBT global performance yielded sensitivity, specificity and accuracy of 98%, 95% and 97%, respectively. A significant correlation (r = 0.84) was found between AS20 and the number of H. pylori colonies on culture. CONCLUSION: This C-14-UBT is highly accurate both for diagnosis and proof of eradication of H. pylori infection and reflects the antral bacterial load. It is simple, fast and inexpensive, and it is therefore suitable for clinical practice.

Stimulation of duodenal muscle contraction by porcine or canine motilin in the dog in vivo.

Motilin is a 22 amino acid polypeptide stimulating intestinal muscle contraction. Structure analysis of motilin purified from hog and from dog intestinal mucosa reveals different amino acids in positions 7, 8, 12, 13, and 14. Previous in vitro experiments suggested this species-related structural heterogeneity could generate different bioactive characteristics for these two peptides. This study was designed to compare the stimulatory mechanism of canine and porcine motilins on dog duodenal motility in vivo, testing the hypothesis that canine motilin stimulates a receptor located on the intestinal muscle, while porcine motilin is acting on intrinsic nerves regulating intestinal muscle contraction. Synthetic porcine and canine motilins were administered through a catheter inserted in the caudal pancreatic duodenal artery to stimulate the contraction of a close irrigated duodenal segment. In acute experiments performed on anaesthetized animals, both peptides induced a similar motor stimulation that was inhibited by tetrodotoxin and atropine. In experiments in conscious animals, canine and porcine motilins were equally potent in inducing premature phase III of the migrating motor complex, and the action of both peptides was abolished by atropine or hexamethonium. This study reveals that the structural heterogeneity between porcine and canine motilin does not influence the bioactivity of both peptides in the dog in vivo and suggests that circulating endogenous motilin stimulates intestinal muscle contraction through intrinsic excitatory nerves.

Regulation of plasma motilin by opioids in the dog.

In the first part of this study, we compared the effects of morphine and trimebutine, two opioid receptor agonists, on small intestinal motility and plasma motilin in dogs. Morphine (100 micrograms/kg iv for 10 min) induced first a typical vomiting myoelectric profile followed subsequently by a migrating electrical activity mimicking phase III of the migrating myoelectric complex; trimebutine (5 mg/kg iv for 10 min) initiated only a migrating phase III-like activity. Despite their different initial contractile effects, both agents induced a significant and similar rise in plasma motilin that preceded the beginning of the premature phase III. In the second portion of the study, naloxone, an opioid receptor antagonist, was infused to verify the influence of endogenous opiates on plasma motilin and on the migrating motor complex. Naloxone (2 mg/kg, then 0.5 mg.kg-1.h-1 iv) delayed significantly the cyclic recurrence of plasma motilin peak increases and of the phase IIIs. In some animals, where naloxone abolished the phase IIIs, the amplitude of the motilin peak increases was significantly diminished. These results suggest 1) that opioid administration increases plasma levels of motilin by a mechanism that is independent of the intestinal contractile activity, and 2) that endogenous opioids could be physiological inducers of plasma motilin increases in the conscious dog.

Molecular heterogeneity of human motilinlike immunoreactivity explained by the processing of prepromotilin.

Results of studies on the molecular forms of canine motilin suggest that this hormone might be synthesized as a higher molecular weight precursor, as is the case for most other biologically active peptides. Chromatographic analysis of human duodenal mucosa extracts and of human serum, using motilin-specific antibodies, also shows the presence of multiple forms of motilinlike immunoreactivity. Cell free translation of human duodenal messenger ribonucleic acid (mRNA) and immunoprecipitation of nascent peptides with motilin antibodies confirm that motilin is synthesized as a 14-15-kilodalton polypeptide precursor. Sequence of the human intestinal motilin complementary deoxyribonucleic acid (cDNA) demonstrates that this precursor bears a 25-amino acid signal peptide, a single dibasic cleavage site immediately following the 22 amino acids of human motilin, and a 65-amino acid polypeptide (motilin-related peptide) following this dibasic processing site. Southern analysis of human genomic kilobases DNA demonstrates that only one motilin gene is contained within 3.5-4 of human genomic DNA. Northern analysis of human intestinal RNA reveals one species of motilin mRNA of an estimated 700 nucleotides in length. These results suggest that (a) only one gene encodes the synthesis of human motilin in the different tissues where this polypeptide is synthesized; (b) human intestinal motilin is translated from one mRNA species and the resulting precursor is processed within the duodenal mucosa by sequential proteolytic cleavage first at a site within the motilin-related peptide, which results in the liberation of a 6-kilodalton form of motilinlike immunoreactivity, and then at the dibasic amino acid cleavage site, thus freeing motilin 22 from its precursor.

Indium-111 leukocyte scanning of the abdomen. Analysis of its value for diagnosis and management of inflammatory bowel disease.

Indium-111 leukocyte scanning of the abdomen (IAS) was performed in 10 patients with ulcerative colitis and in 39 patients with Crohn's disease involving the small intestine (in 25 occasions) and/or the colon (17 cases). Radionuclide uptake by the gut was seen in 84% of the patients with active inflammation. We compared the extent of the disease displayed by IAS with the findings obtained by either radiological or endoscopic studies or at surgery. In two-thirds of the patients, the IAS gave an accurate evaluation of the extent of the disease (sensitivity 68%). False-positive IASs were not seen in small bowel disease (specificity 100%), but were observed on 4 occasions on the colon (specificity 86%). The intensity of the radionuclide uptake could not be correlated with the clinical activity of the disease as evaluated by the Crohn's disease activity index. These results suggest that IAS is not superior to the standard procedures used to detect and localize inflammatory bowel disease and that IAS cannot replace these techniques. However, the simplicity of IAS and the complete lack of complications associated with its use render it useful in the evaluation of the extent and distribution of inflammation in some patients, mainly those with severe disease in whom standard diagnostic procedures would be contraindicated.

Motilin-like-immunoreactivity in intestine and brain of dog.

Motilin-like-immunoreactivity was detected in various regions of canine intestinal tract and brain. Its content in the brain was much smaller than in the gut. Its regional distribution was not uniform in both organs. On gel chromatography (G-50 SF), intestinal extracts revealed a main molecular form of motilin-like-immunoreactivity corresponding to motilin 1-22, while, in the brain, it eluted predominantly with the void volume. Further characterization of this later substance does not suggest it is strongly related to motilin. Putative motilin precursors of 14 kd and 6 kd are detectable in small concentration in intestinal mucosa.

Comparative stimulation of motilin duodenal receptor by porcine or canine motilin.

Motilins purified from porcine and canine intestine differ in their amino acid composition in positions 7-8-12-13-14. We studied in vitro the contractile response of longitudinal duodenal muscles from various animals (guinea pig, rabbit, dog) to porcine and canine synthetic motilins. Both substances failed to elicit contraction of the guinea pig duodenum but were active and equally potent on rabbit muscle. In dogs, porcine motilin was inactive at the concentrations tested (up to 10(-4) M) whereas canine motilin induced duodenal contractions in a dose-response fashion (mean dose required to induce half-maximal response: 4.82 +/- 0.25 X 10(-5) M). The contraction generated by synthetic canine motilin (10(-5) M) was not influenced by atropine, hexamethonium, tetrodotoxin, naloxone, or sodium nitroprusside (all used at 10(-4) M) but was blocked by verapamil (10(-4)). Our study shows that species-related structural alterations in motilin molecules generate different bioactive capacities in some animal species, suggests that the middle portion of the molecule is important for its bioactive expression, suggests the presence of motilin receptors on canine duodenal muscle, and suggests that an influx of extracellular calcium is involved in the canine duodenal muscle contraction elicited by canine motilin.

Effect of trimebutine on intestinal motility and plasma motilin in the dog.

In a previous report, trimebutine was shown to induce premature periods of phase III activity in fasting dogs, and its action was blocked by naloxone. In this study, we observed that trimebutine (5 mg X kg-1 iv) could induce premature phase IIIs in canine small intestine during interdigestive and digestive periods; trimebutine-induced phase IIIs were migrating along the small intestine faster than spontaneous activity fronts and; trimebutine-induced phase IIIs were accompanied by sharp rises in concentrations of plasma motilin. To further elucidate the trimebutine-stimulatory mechanism, we verified its effects on the release of various circulating peptides that influence intestinal motility: short-interval blood sampling during trimebutine infusion revealed that plasma motilin increases induced by trimebutine preceded the beginning of phase III in proximal duodenum; and gastrin and insulin postprandial releases were abolished by trimebutine. Therefore, trimebutine, by its simultaneous but opposite effects on various peptides that individually carry positive (e.g., motilin) or negative (e.g., gastrin and insulin) influences on the generation of activity fronts, could alter the equilibrium between stimulatory and inhibitory forces in such a way that, in some circumstances (e.g., postprandial period), stimulatory mechanisms become predominant.

Nonparallel secretion of enzymes by the rabbit pancreas.

Since nonparallel secretion of enzymes by the exocrine pancreas has been demonstrated with several experimental models, we were interested in verifying a recent claim that enzyme secretion remained strictly proportional (parallel) upon stimulation of the in vivo rabbit pancreas. Pancreatic juice was collected by extraduodenal cannulation of the pancreatic duct, in two different protocols. In the first protocol the administration of pentobarbital induces a mild anesthesia. Under this condition, amylase and chymotrypsin secretion remained parallel after cholecystokinin stimulation. In a second protocol, a deeper and constant anesthesia was attained with Fluothane resulting in a lower basal protein output than in the first protocol. Pancreatic secretion was collected under intravenous secretin perfusion (4.5 clinical units X kg-1 X h-1). After stabilization and basal collection periods, pancreatic secretion was stimulated with an i.v. bolus injection of either cholecystokinin (2 Ivy dog units/kg), caerulein (0.1 micrograms/kg), or carbachol (6 micrograms/kg). Upon stimulation of the pancreas, protein output increased an average of 30-fold and there was a concomitant 20-25% decrease in the ratio of the specific activities of amylase to chymotrypsin which resulted from a greater increase in the specific activity of chymotrypsin in pancreatic juice after stimulation of secretion. Thus, under appropriate conditions, nonparallel secretion of enzymes by the exocrine pancreas can be demonstrated in yet another experimental model. Furthermore, the proportion of amylase and chymotrypsin activities in pancreatic juice are once more shown to be dependent, up to a threshold, upon the rate of protein output by this exocrine gland.

Translational control of protein synthesis in isolated pancreatic acini: role of CCK8, carbachol, and insulin.

We wished to determine whether the stimulation of protein synthesis by CCK8, carbachol, and insulin in isolated rat pancreatic acini resulted from translational or transcriptional induction of protein synthesis, and whether these hormones had similar or different effects on the rates of synthesis of individual enzymes. Isolated pancreatic acini were prepared from streptozocin-treated rats by collagenase digestion, mechanical dissociation, and centrifugation through a bovine serum albumin (BSA) cushion. Sixty-minute incubations, with maximally effective doses of CCK8, carbachol, and insulin, produced a 50, 90, and 100% increase, respectively, in the rate of protein synthesis. After inhibition of transcription with actinomycin D, the hormones still produced a 23, 50, and 61% increase, respectively, in the rate of protein synthesis. The study of the effect of the three hormones and the combination of CCK8 and insulin on the rate of synthesis of trypsinogen, chymotrypsinogen, lipase, and amylase, purified by isoelectric focusing, demonstrated that the hormones induced similar effects on the pattern of enzyme synthesis, and that they all induced the rate of synthesis of chymotrypsinogen slightly more than that of the other enzymes studied. We conclude that the hormones studied exert similar posttranscriptional influences in the regulation of protein synthesis in the pancreatic acinar cell.

Structure of the two related elastase genes expressed in the rat pancreas.

We have isolated and characterized rat genomic DNA fragments bearing the two secretory elastase genes that are expressed in the exocrine pancreas. The complete exonic sequences for each of the genes as well as considerable intronic and flanking sequences are reported. Each elastase gene is interrupted by seven intervening sequences which are located at corresponding positions within the two genes, with one exception: the third intron of the elastase II gene has shifted one codon in the 5' direction. The placement of introns within the amino acid coding domains in part may reflect the formation of the progenitor serine protease gene by the duplication of an exon encoding a characteristic polypeptide structure comprising three beta sheets. The activation peptides of the zymogens and the signal peptides, which form discrete functional domains in the protein precursors, are encoded by separate exons. In addition to the TATAA box, the two genes share considerable sequence similarity in the 5'-proximal flanking regions (up to approximately 450 base pairs upstream); however, a number of gaps must be introduced to optimize the sequence alignment. The similarities are largely confined to six oligonucleotide regions with greater than 70% sequence conservation. The elastase I gene has a perfect repeating copolymer (GT)24 located 427-379 nucleotides upstream from the start of transcription. The elastase II gene has a similar GT-rich region (52/55 G or T) located 384-330 nucleotides upstream. Comparison of the 5'-flanking regions of the two elastase genes with those of pancreatic chymotrypsin and trypsin I and II reveals that one of the six conserved oligonucleotide regions is generally conserved for these genes as well. This conserved region contains putative enhancer core sequences.

Modifications in pancreatic enzyme proportions following secretory stimulations.

The short-term influence of repeated secretory stimulations of the pancreas on pancreatic enzyme content was studied in the rat. The animals received 10 successive injections of either cholecystokinin-pancreozymin (CCK-PZ), secretin, CCK-PZ plus secretin, caerulein or pilocarpine. The pancreatic enzyme content was determined the next morning. CCK-PZ, with or without secretin, caerulein and pilocarpine had a similar influence increasing the chymotrypsinogen concentration in the pancreas twofold, while lipase and amylase concentrations increased by only 50 and 25%, respectively. Fasted and fed animals responded similarly. Secretin, a mostly ductal secretagogue, was without influence on the pancreatic enzyme composition. Thus, the mere stimulation of pancreatic protein secretion seems to result in a rapid change in enzyme composition in the pancreas.

Dietary regulation of pancreatic protein synthesis. I. Rapid and specific modulation of enzyme synthesis by changes in dietary composition.

Dietary adaptation of pancreatic protein synthesis and of pancreatic enzyme concentration, was studied over the first 24 h of exposure to a new diet. Rats were adapted to a carbohydrate-rich (G) or to a protein-rich diet (P) and were switched to the opposite regime after a 15 h fast. The evolution of the relative rate of synthesis of amylase, chymotrypsinogen and trypsinogen and of the pancreatic concentration of amylase and chymotrypsinogen were followed. Fasting caused important modifications in the relative rate of synthesis of the three enzymes in rats adapted to a P diet. Adaptative changes in the relative rate of synthesis of amylase, chymotrypsinogen and trypsinogen were seen within 2 h after the beginning of refeeding. These changes were followed by corresponding adaptative modifications in pancreatic contents 4 h after the beginning of refeeding. After 24 h of refeeding, significant adaptative changes had occurred in both the relative rates of synthesis and in enzyme concentrations. Thus exocrine pancreatic protein synthesis can be modulated as early as 2 h after refeeding and this modulation is followed by adaptative changes in pancreatic enzyme content.

Dietary regulation of pancreatic protein synthesis. II. Kinetics of adaptation of protein synthesis and its effect on enzyme content.

The kinetics of the adaptative changes in the relative rates of synthesis and pancreatic concentrations of amylase, chymotrypsinogen and trypsinogen were studied over 10 days of adaptation to a carbohydrate-rich (G), or a protein-rich (P) diet. During adaptation to the P diet, 60% of the adaptative decrease of the amylase to chymotrypsinogen ratio of incorporation was complete within 24 h of feeding and plateau values were obtained after five days. Adaptation to the G diet was only 20% complete after 24 h and plateau values were obtained later than with the P diet. The evolution of the ratio of concentrations of amylase and chymotrypsinogen followed those of incorporation in the adaptation to both diets. These results support the determinant role of adaptative changes in the rates of synthesis of individual enzymes on the dietary adaptation of enzyme proportions in the pancreas. The differences in the kinetics of adaptation to the two diets suggest that different mechanisms are involved in the adaptative regulation of protein synthesis to a carbohydrate-rich diet or a protein-rich diet.

Pancreatic enzyme secretion: nonspecific stimulation by duodenal mucosal extracts.

Felber et al. (Lancet 2: 185-188, 1974) reported that duodenal extracts obtained from rats fed a given meal induced the specific secretion of related pancreatic enzymes. Such an observation challenges the generally accepted theory of parallel secretion of pancreatic enzymes. Although these experiments were faithfully reproduced, no induction of specific enzyme secretion could be obtained. Moreover, comparison of the secretagogue potency of different preparations of duodenal extract, both in vivo (anesthetized rat) and in vitro (pancreatic lobules), demonstrated that in the original extraction procedure most of the secretagogue activity was lost. Finally, even the fully active extract failed to induce specific enzyme secretion. It is therefore unlikely that duodenal extracts from rats fed a specific meal can induce selective secretion of the related enzyme.