Preparation of a monoclonal antibody to rat alpha-CGRP for in vivo immunoneutralization of peptides.
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Animals↗
Biomedical subjects
J H Walsh
Publications and source records attributed to J H Walsh.
Helicobacter pylori: selection of patients for treatment.
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Gastritis↗
Species differences in the immunoreactive patterns of calcitonin gene-related peptide in the pancreas.
In the pancreas, calcitonin gene-related peptide (CGRP) immunoreactivity has been described in nerve fibers and in distinct types of islet cells. This unique, apparently species-specific cell-type expression prompted the present investigation to clarify further the pattern of CGRP immunoreactivity in different mammalian species (i.e., different strains of rats, mice, guinea pigs, rabbits, cats, dogs, pigs, and humans) commonly used for functional and anatomical studies of the pancreas by means of immunohistochemistry using three different CGRP antibodies. In each species, CGRP-immunoreactive neurites innervate the exocrine and endocrine compartments, the vasculature, and the intrapancreatic ganglia, where they form dense networks encircling unstained cell bodies. The only exception is the pig pancreas, where the islets appear to be devoid of immunoreactive fibers. The overall density of immunoreactive pancreatic axons in different species is as follows: rat, mouse, and rabbit greater than guinea pig greater than or equal to pig and cat much greater than dog and human. CGRP-immunoreactive endocrine cells appear to be restricted to the rat pancreas, where they form a subpopulation of somatostatin-containing D cells. In contrast, in mouse, guinea pig, cat, dog, and human pancreas, a homogeneous staining of the core of the islets, where insulin-producing B cells are located, was visualized in sections incubated with the rabbit CGRP antiserum at 4 degrees C, but not at 37 degrees C (an incubation temperature that does not affect the islet cell staining in the rat nor the fiber labeling in any species). Furthermore, the staining of islet B cells was not reproducible with all the CGRP antibodies used, all of which comparably stain nerve fibers in each species, and islet D cells in the rat. Immunoreactive islet cells were not visualized in pig and rabbit pancreas. These results are consistent with the hypothesis that the expression of CGRP in nerve fibers is a common feature of mammalian pancreas, whereas its expression in endocrine cells appears to be restricted to the D cells of the rat pancreas.
Animals↗
Autonomous proliferation of colon cancer cells that coexpress transforming growth factor alpha and its receptor. Variable effects of receptor-blocking antibody.
Four human colon adenocarcinoma cell lines, SNU-C1, SNU-C4, SNU-C5, and NCI-H716, that are capable of proliferating autonomously in serum-free medium containing no added peptide growth factors were identified. All four cell lines show epidermal growth factor (EGF) receptors (EGFRs), express transforming growth factor alpha (TGF-alpha) messenger RNA, and release anti-TGF-alpha-immunoreactive molecules. The blocking anti-EGFR monoclonal antibody (mAb) 225 blocks autonomous proliferation of SNU-C1 and SNU-C4 cells. In both of these cell lines, the inhibitory effect of mAb 225 is reversible by the addition of EGF, TGF-alpha, or conditioned medium from any of the four cell lines. In contrast, autonomous proliferation of SNU-C5 and NCI-H716 cells is not inhibited by mAb 225 and is not affected by exogenous EGF, TGF-alpha, or conditioned medium. Together, these data confirm the previous finding that anti-EGFR antibodies can inhibit the proliferation of some carcinoma cell lines that coexpress TGF-alpha and EGFR. However, here it is shown that the mechanisms of autonomous proliferation of colon carcinoma cell lines are heterogeneous and not always sensitive to antibody disruption of TGF-alpha/EGFR autocrine interactions.
Adenocarcinoma↗
Transforming growth factor alpha receptor binding sites in the canine gastrointestinal tract.
Transforming growth factor alpha (TGF-alpha) interacts with the same plasma membrane receptor as epidermal growth factor and is likely to play a role in proliferative and trophic processes of gastrointestinal tissues. The distribution of receptor binding sites for TGF-alpha was examined in the canine gastrointestinal tract (distal esophagus, stomach fundus, descending duodenum, jejunum, ileum, transverse colon) by tissue section autoradiography. 125I-TGF-alpha yielded a labeling pattern comparable to that of 125I-epidermal growth factor. Specific binding sites were particularly abundant in the mucosa in each region, with the highest concentration in the esophagus, colon, and stomach, as assessed by computer assisted densitometry. The density of binding sites was moderate in the stomach muscularis mucosae, low in the external muscle layer, and very low to undetectable in the submucosa throughout the gastrointestinal tract. In most cases, the greatest density within the individual regions was detected in the area characterized by the highest proliferative rate. Lymphoid aggregates were not labeled. In conclusion, TGF-alpha receptor binding sites are present throughout the gastrointestinal tract with differential patterns in the various regions; they are principally distributed to the mucosa and predominantly located to proliferative cell areas. These results are consistent with a role of this factor in regional regulation of proliferation and differentiation in the gut.
Animals↗
Intestinal fat does not inhibit gastric function through a hormonal somatostatin mechanism in dogs.
In awake dogs with chronic gastric, duodenal, and jejunal fistulas, F(ab)1 fragments of somatostatin monoclonal antibody (mAb S607) were administered intravenously (IV) to test the hypothesis that intraintestinal lipid inhibits peptone-stimulated gastric acid secretion and emptying by a hormonal somatostatin mechanism. Plasma somatostatin was increased significantly by duodenal and jejunal perfusion with 20% lipid. Somatostatin administered IV caused dose-dependent inhibition of meal-stimulated gastric acid secretion and gastric emptying similar to that seen after intestinal perfusion with lipid. Administration of mAb S607 F(ab)1 fragments significantly reversed somatostatin (400 pmol.kg-1.h-1, IV)-induced inhibition of peptone-stimulated acid output and gastric emptying. Acid output inhibited by intraduodenal lipid was reversed partially after F(ab)1 administration, but the inhibitory effect of intrajejunal lipid was not altered. Inhibition of acid secretion by IV somatostatin and by intraintestinal fat was not caused by a decrease in circulating gastrin concentrations. Gastric emptying delayed by intraintestinal lipid was unaffected by antibody administration. Somatostatin does not appear to be a major hormonal mediator of intestinal fat-induced inhibition of gastric acid secretion or delayed gastric emptying in dogs.
Animals↗
Cholecystokinin receptor antagonist MK-329 blocks intestinal fat-induced inhibition of meal-stimulated gastric acid secretion.
MK-329, a selective type A cholecystokinin (CCK) receptor antagonist, was given to dogs to test the hypothesis that CCK is one of the principal physiological enterogastrones mediating fat-induced decreases in gastric acid secretion. Gastric acid secretion in response to 300 mL 8% peptone meals was measured by intragastric titration to pH 5.5 in six awake dogs with chronic gastric, duodenal, and jejunal fistulas. Gastric emptying was measured by a dye-dilution technique. During the last hour of peptone stimulation, the intestine was perfused with either control solution or 20% lipid (Intralipid; Kabi Vitrum, Alamedo, CA) intraduodenally or intrajejunally. Compared with control perfusions, mean gastric acid outputs were decreased significantly after lipid perfusion of the duodenum (47% of control) and jejunum (24% of control). Similarly, mean gastric emptying rates were significantly less after lipid perfusion of the duodenum (56%) and jejunum (26%). Oral pretreatment with MK-329 (1 mg/kg) significantly reversed the inhibition of gastric acid output caused by lipid perfusion of the duodenum and jejunum, but fat-induced inhibition of gastric emptying was not significantly affected. These studies provide evidence for an important inhibitory role for CCK as an enterogastrone but do not implicate CCK as being important in fat-induced delayed gastric emptying of a liquid meal in dogs.
Animals↗
Helicobacter pylori secretes a chemotactic factor for monocytes and neutrophils.
Helicobacter pylori is associated with an inflammatory reaction in the stomach and duodenum, yet the mechanism of this inflammatory infiltrate is unknown. The ability of Helicobacter pylori to secrete a factor that attracts leucocytes is investigated. Helicobacter pylori conditioned supernatant attracted neutrophils and monocytes with 50-100% of the activity of control chemotactic factor, 10(-8) M formyl-methionine-leucine-phenylalanine. Strains derived from individuals with ulcer or non-ulcer associated H pylori infections displayed similar chemotactic activity. Preliminary characterisation shows that the factor has a molecular weight of less than 3000, is heat stable, is acid resistant, and can be diluted at least 10-fold. Checkerboard analysis confirmed that the activity was chemotactic rather than chemokinetic. This chemotactic activity could play a role in the pathogenesis of Helicobacter pylori gastritis.
Chemotactic Factors↗
Intestinal acid inhibits gastric acid secretion by neural and hormonal mechanisms in rats.
To determine the relative contributions of neural reflexes and intestinal hormones to the inhibition of gastric acid secretion by intestinal acidification, rats with an extrinsically denervated, transplanted segment of jejunum, and those with an innervated segment of jejunum, were studied. Postoperatively, meal-stimulated gastric acid secretion was measured. When the acid secretory response to intragastric liver extract reached a plateau, graded concentrations of hydrochloric acid or saline were instilled into the jejunal segments. Gastric acid secretion was inhibited by intrajejunal acid (pH 2.5) by 79% in the innervated rats and by 64% in the transplanted group. Thus at a pH of 2.5 there was a 15% greater maximum inhibition of plateau acid response in the innervated rats than in the transplanted rats, presumably because of the extrinsic neural contribution. To examine the hormonal mediators, the effects of a somatostatin monoclonal antibody and a CCK-A receptor antagonist (L 364718) on acid-induced inhibition of gastric acid secretion were studied in transplanted rats. Treatment with a somatostatin monoclonal antibody or with L 364718 reduced the acid-induced (pH 2.5) inhibition of gastric acid secretion by 93 and 27%, respectively. Jejunal acidification inhibits gastric acid secretion in the rat by both neural and hormonal mechanisms. The hormonal mechanism is mediated by somatostatin and CCK.
Acids↗
Role of gastrin, histamine, and acetylcholine in the gastric phase of acid secretion in anesthetized rats.
To determine the relative contributions of gastrin, histamine, and cholinergic stimulation to the gastric phase of acid secretion, peptone-stimulated acid output was measured in urethan-anesthetized pylorus-ligated rats after intravenous administration of gastrin monoclonal antibody, cimetidine, and atropine. Intragastric peptone stimulated acid secretion four-fold over basal, which was associated with a significant increase in plasma gastrin levels. Gastrin immunoneutralization and simultaneous H2- and muscarinic-receptor blockade demonstrated that approximately 40% of peptone-stimulated acid output as attributed to endogenous gastrin through a histamine-dependent pathway, whereas 20% of acid output was accounted for by a cholinergic component. Another 10% of titratable acid was omeprazole-insensitive and presumably due to intragastric digestion of peptone. Therefore, approximately 30% residual acid output in response to peptone could not be accounted for by known acid stimulatory mechanisms. In rats given somatostatin monoclonal antibody to block the tonic inhibitory effect of endogenous somatostatin, residual acid output was a similar fraction of meal-stimulated acid output. In contrast, gastric distension induced by intragastric instillation of saline stimulated acid secretion to 1.5-fold over basal. Although 60% of distension-induced acid secretion could be inhibited by either H2 blockade or gastrin immunoneutralization, acid output returned to basal levels after simultaneous muscarinic blockade. These results indicate that gastrin, through a histaminergic pathway, is the principal mediator of meal-stimulated acid secretion in anesthetized rats. Approximately 30% of acid output was due to other unidentified mechanisms, such as chemical secretagogues, a direct effect of amino acids, or novel peptides.
Acetylcholine↗
Cholecystokinin inhibits gastric acid secretion through type "A" cholecystokinin receptors and somatostatin in rats.
The purpose of this study was to determine whether selective antagonism of type "A" cholecystokinin (CCK) receptors blocks inhibition of gastric acid secretion produced by CCK and whether this inhibition is mediated through either a somatostatin-dependent pathway or a vago-vagal reflex. Intravenous infusion of CCK (0.04-10 nmol.kg-1.h-1) dose dependently inhibited pentagastrin-stimulated gastric acid secretion in urethan-anesthetized rats, with a 50% inhibitory dose of 0.9 nmol.kg-1.h-1 and a maximum inhibition of approximately 50%. Blockade of type A CCK receptors using the selective type A receptor antagonist MK-329 completely reversed the inhibitory effect produced by a maximal dose (4 nmol.kg-1.h-1) of CCK. Immunoneutralization of endogenous somatostatin by administration of somatostatin monoclonal antibody abolished the inhibition produced by CCK. Concentrations of somatostatin in portal venous plasma were significantly increased after CCK administration; the increase in somatostatin was blocked by pretreatment with MK-329. In contrast, CCK-induced inhibition of gastric acid secretion was unaltered after perivagal capsaicin treatment. These results indicate that CCK inhibits gastric acid secretion in rats by activation of type A CCK receptors and through release of endogenous somatostatin.
Animals↗
Regulation of gastric acid secretion by gastrin in duodenal ulcer patients and healthy subjects.
To examine the role of gastrin as a major mediator of meal-stimulated acid secretion at low and high intragastric pH, gastric acid secretory responses after exogenous and endogenous stimulation were studied in relation to circulating plasma gastrin levels in 19 healthy control subjects and in 18 patients with inactive duodenal ulcer disease. Gastrin was given intravenously in stepwise fourfold-increasing doses from 3.1 to 800 pmol.kg-1.h-1 over consecutive 30-minute periods. Circulating plasma gastrin and acid secretion rates, measured by intragastric titration, were compared with the values obtained during endogenous stimulation by intragastric meals of 0.5, 1, 2, 4, and 8 g% peptone at either pH 5.5 or pH 2.5. The studies showed that circulating gastrin is a major regulator of acid secretion in the presence of peptone in both healthy controls and subjects with duodenal ulcers. Patients with duodenal ulcers had higher acid secretion rates in response to endogenous and exogenous stimulation. In duodenal ulcer subjects and healthy controls, acid secretion in response to higher doses (2-8 g%) of peptone was inhibited at low intragastric pH. This pH inhibition could be fully explained by diminished gastrin release. Patients in the DU group differed from the controls by diminished inhibition of acid secretion at intragastric pH 2.5 when low doses (1 g%) of peptone meals were used. In summary, gastrin is a major regulator of endogenously stimulated acid secretion at high and low intragastric pH in healthy subjects. DU patients differ from healthy controls by higher total acid secretion rates and diminished inhibition of acid secretion when low concentrations of peptone are present in the stomach.
Adult↗
Ulcer recurrence following duodenal ulcer healing with omeprazole, ranitidine, or placebo: a double-blind, multicenter, 6-month study. The Omeprazole Duodenal Ulcer Study Group.
The present study determined whether the rate of relapse of duodenal ulcer was reduced after ulcer healing with omeprazole compared with ranitidine or placebo. It was made up of a double-blind, randomized, controlled multiple-center trial set within the United States. Patients were candidates if their duodenal or pyloric channel ulcer successfully healed in one of two large multicenter U.S. trials; one compared omeprazole, 20 mg once daily, before breakfast with ranitidine, 150 mg twice daily, and the other compared the same dose of omeprazole with placebo. Two hundred forty (73.8%) of the 325 patients with complete ulcer healing within 4 weeks of starting therapy who were eligible to enter the follow-up study were enrolled. There was no intervention. Endoscopic assessment of ulcer status was performed at 2, 4, and 6 months and whenever patients had symptoms thought to represent return of an ulcer. The lifetable relapse rates for duodenal ulcer according to initial ulcer therapy with omeprazole, ranitidine, or placebo were 76.7% [95% confidence interval (CI), 64%-89.3%], 59.8% (95% CI, 47.8-71.7%), and 50.4% (95% CI, 15.7%-85.2%), respectively. These rates were not statistically significantly different. Seventeen percent of recurrent ulcers occurred at a site different from that of the original ulcer. It is concluded that despite the more rapid rate of duodenal ulcer healing with omeprazole therapy, the rate of ulcer relapse appears similar and independent of whether ulcer healing was accelerated with omeprazole or ranitidine.
Adult↗
Physiology and pathophysiology of gastrin.
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Animals↗
Childhood leukaemia in Ireland.
In response to professional and public concern about health consequences, in particular cancer risk, from previous and current levels of ionising radiation in the Irish Sea, a study of incidence and mortality from acute lymphoid leukaemia (ALL) and other lymphoid malignancies in children was undertaken. Overall rates were similar to those found in other western populations and distribution of high rates was quite random over the country as a whole. There was a small but significant excess in incidence of ALL for the years 1974-76 in a narrow three mile wide strip along the east coast. It is not possible in the context of this study to postulate aetiological factors which might explain this finding.
Acute Disease↗
Regulation of rat gastric H+/K(+)-ATPase alpha-subunit mRNA by omeprazole.
The H+/K(+)-ATPase is the dimeric enzyme responsible for H+ secretion by the gastric parietal cells. The present study examined the response of rat fundic mRNA levels of H+/K(+)-ATPase alpha-subunit and somatostatin to the inhibition of H+/K(+)-ATPase enzyme activity and gastric pH elevation by oral omeprazole administration. Omeprazole inhibits the alpha-subunit of H+/K(+)-ATPase covalently and stabilizes stimulated morphology of the parietal cell. After a single administration of omeprazole (100 mg/kg), H+/K(+)-ATPase alpha-subunit mRNA levels increased significantly by 57% at 3 h and remained elevated for 6 h, returning to the basal level by 24 h. After multiple administrations of omeprazole (100 mg/kg per day, every 24 h for 3 days), H+/K(+)-ATPase alpha-subunit mRNA levels were already elevated at the time of the last dose, reached maximum at 6 h (95% increase above control), and returned to the pre-treatment level after 36 h. Nuclear run-on assay indicated H+/K(+)-ATPase gene transcription was significantly increased by omeprazole pretreatment in vivo. In contrast, a significant decrease in fundic somatostatin mRNA occurred at 12 h after a single dose, and the inhibition was more pronounced and lasted longer after multiple doses of omeprazole. These data indicate that omeprazole, while effectively inhibiting H+/K(+)-ATPase activity, induces H+/K(+)-ATPase gene expression in the parietal cells. An inverse relationship exists between the regulation of somatostatin gene expression in fundic D-cells and H+/K(+)-ATPase gene expression. The increase in H+/K(+)-ATPase alpha-subunit mRNA could be due to alterations in extracellular gastrin/somatostatin ratios or could be induced by intracellular effects of omeprazole.
Actins↗
Preclinical evaluation of an anti-autocrine growth factor monoclonal antibody for treatment of patients with small-cell lung cancer.
We have evaluated an anti-autocrine growth factor monoclonal antibody for potential use in the treatment of patients with small-cell lung cancer. The monoclonal antibody, designated 2A11, binds to the C-terminal region of the autocrine growth factor gastrin-releasing peptide and neutralizes its growth-promoting effects in vitro and in vivo. Equilibrium-binding analysis demonstrated that the peptide binds to the antibody (dissociation constant = 1.5 x 10(-10) at least as avidly as it binds to the tumor peptide receptor. Pharmacokinetic studies in normal BALB/c mice demonstrated an initial clearance half-life (alpha t1/2) of 24.3 +/- 4 hours and a secondary clearance half-life (beta t1/2) of 1039.6 +/- 309 hours, and biodistribution studies revealed a distribution pattern which generally reflected blood flow. Single intravenous infusions of 2A11 (20 mg/20-25-kg dogs) into normal mongrel dogs with surgically created gastric fistulas antagonized the stimulatory effects of exogenously infused gastrin-releasing peptide or bombesin on plasma gastrin release and gastric acid secretion. Toxicology studies in normal dogs (with gastric fistulas) infused with 50 mg 2A11 intravenously three times a week for 4 weeks failed to reveal any adverse behavioral, clinical, or pathological effects. Four of six dogs developed an immune response to 2A11. Anti-idiotypic antibodies elicited in two cases did not mimic the functional effects of the peptide. We conclude that the concept of immunoblockade of an autocrine growth factor appears feasible in vivo.
Animals↗
Immunohistochemical study of cholecystokinin peptide in rat spinal motoneurons.
With the aid of indirect immunofluorescence histochemistry and sequence specific antibodies a possible localization of cholecystokinin (CCK) peptide in spinal motoneurons has been analyzed. To increase peptide levels, the sciatic nerve was ligated, and the area around the ligation was studied 24 hours later. For comparison, antisera raised against calcitonin gene-related peptide (CGRP) and substance P were employed. With CCK specific antisera (directed to the N-terminal portion of CCK-8 or the midportion of CCK-33) accumulation of peptide-like immunoreactivity (LI) was observed in large, dilated axonal swellings proximal to, but at some distance from, the ligature. Such accumulations were also observed with C-terminally directed CCK antiserum, but in addition numerous axons of smaller diameter extending up to the ligation contained this type of immunoreactivity. The latter antiserum is thought to cross-react with CGRP. In fact, this staining pattern was indistinguishable from the one seen after incubation with CGRP antiserum. In contrast substance P-LI could not be seen in the larger dilated axons but only in large numbers of thinner fibers close to the ligation. Double staining experiments revealed that the large dilations contained both CGRP- and CCK-specific LI. Distal to the ligation CGRP- and substance P- but no specific CCK-LI could be observed. The present findings support the view that CCK mRNA in spinal motoneurons is translated into CCK peptide, at least after axotomy, and that the peptide is transported into the motoneuron axon. However, compared to CGRP the CCK levels are presumably low, and the functional role of CCK peptide in motoneurons remains to be established.
Animals↗