Distribution of 131I-labeled Bothrops erythromelas venom in mice

Bothrops erythromelas is responsible for many snake bites in northeastern Brazil. In the present study we determined the in vivo distribution of the venom following its subcutaneous injection into mice. B. erythromelas venom and albumin were labeled individually with 131I by the chloramine T method, and separated in a Sephacryl® S-200 column. The efficiency of labeling was 68%. Male Swiss mice (40-45 g), which had been provided with drinking water containing 0.05% KI over a period of 10 days prior to the experiment, were inoculated dorsally (sc) with 0.3 ml (2.35 x 105 cpm/mouse) of 131I-venom (N = 42), 131I-albumin or 131I (controls, N = 28 each). Thirty minutes and 1, 3, 6, 12, 18 and 24 h after inoculation, the animals were perfused with 0.85% NaCl and skin and various organs were collected in order to determine radioactivity content. There was a high rate of venom absorption in the skin (51%) within the first 30 min compared to albumin (20.1%) and free iodine (8.2%). Up to the third hour after injection there was a tendency for venom and albumin to concentrate in the stomach (3rd h), small intestine (3rd h) and large intestine (6th h). Both control groups had more radioactivity in the digestive tract, especially in the stomach, but these levels decreased essentially to baseline by 12-18 h postinjection. In the kidneys, the distribution profiles of venom, albumin and iodine were similar. Counts at 30 min postinjection were low in all three groups (1.37, 1.86 and 0.77, respectively), and diminished to essentially 0% by 12-18 h. Albumin tended to concentrate in muscle until the 3rd h postinjection (1.98%). There was a low binding of labeled venom in the liver (<0.54%), thyroid (<0.11%) and lungs (<0.08%), and no iodinated venom was detected in brain, heart, diaphragm, spleen or bladder. The low venom binding observed in most internal organs, comparable to that of albumin, suggests that B. erythromelas venom does not specifically target most internal organs. That is, the systemic effects of envenomation are mainly due to an indirect action

Feeding manipulation elicits different proliferative responses in the gastrointestinal tract of suckling and weanling rats

Food deprivation has been found to stimulate cell proliferation in the gastric mucosa of suckling rats, whereas the weanling period has been reported to be unresponsive in terms of proliferative activity. In the present study we analyze regional differences in the effect of milk or food deprivation on cell proliferation of the epithelia of the esophagus and of five segments of small intestine in suckling, weanling and newly weaned Wistar rats of both sexes. DNA synthesis was determined using tritiated thymidine to obtain labeling indices (LI); crypt depth and villus height were also determined. Milk deprivation decreased LI by 50% in the esophagus (from 15 to 8.35%) and small intestine (from 40 to 20%) of 14-day-old rats. In 18-day-old rats, milk and food deprivation decreased LI in the esophagus (from 13 to 5%) and in the distal segments of the small intestine (from 36-40 to 24-32%). In contrast, the LI of the epithelia of the esophagus (5%) and of all small intestine segments (around 30%) of 22-day-old rats were not modified by food deprivation. Crypt depth did not change after treatment (80 to 120 µm in 14- and 22-day-old rats, respectively). Villus height decreased in some small intestine segments of unfed 14- (from 400 to 300 µm) and 18-day-old rats (from 480 to 360 µm). The results show that, contrary to the stomach response, milk deprivation inhibited cell proliferation in the esophagus and small intestine of suckling rats, demonstrating the regional variability of each segment of the gastrointestinal tract in suckling rats. In newly weaned rats, food deprivation did not alter the proliferation of these epithelia, similarly to the stomach, indicating that weanling is a period marked by the insensitivity of gastrointestinal epithelia to dietary alterations

Studies on the intrinsic nervous system of the wild rodent Calomys callosus digestive tract. II: The submucous plexus

The submucous plexus of the normal small and large intestine of Calomys callosus was studied by NADH and AChE histochemical techniques and by transmission and scanning electron microscopy. The plexus contains (mean ± SD) 7,488 ± 293 neurons/cm2 in the duodenum, 5,611 ± 836 in the jejunum, 2,741 ± 360 in the ileum, 3,067 ± 179 in the cecum, and 3,817 ± 256 in the proximal colon. No ganglia or nerve cell bodies were seen in the esophagus, stomach, distal colon or rectum. The neurons are pear-shaped with a round or oval nucleus and the neuronal cell profile areas were larger in the large intestine than in the small intestine. Most of the neurons display intense AChE activity in the cytoplasm. AChE-positive nerve fibers are present in a primary meshwork of large nerve bundles and in a secondary meshwork of finer nerve bundles. At the ultrastructural level, the ganglia are irregular in shape and covered with fibroblast-like cells. The nucleoplasm of the neurons is finely granular with a few condensations of chromatin attached to the nuclear envelope. In the neuropil numerous varicosities filled with vesicles of different size and electron densities are seen. The pre- and post-synaptic membrane thickenings are asymmetric. Characteristic glial cells with oval nuclei and few organelles are numerous. These data provide a detailed description of this submucosal meshwork.

Variations in gastric emptying of liquid elicited by acute blood volume changes in awake rats

We have observed that acute blood volume expansion increases the gastroduodenal resistance to the flow of liquid in anesthetized dogs, while retraction decreases it (Santos et al. (1991) Acta Physiologica Scandinavica, 143: 261-269). This study evaluates the effect of blood volume expansion and retraction on the gastric emptying of liquid in awake rats using a modification of the technique of Scarpignato (1980) (Archives Internationales de Pharmacodynamie et de Therapie, 246: 286-294). Male Wistar rats (180-200 g) were fasted for 16 h with water ad libitum and 1.5 ml of the test meal (0.5 mg/ml phenol red solution in 5% glucose) was delivered to the stomach immediately after random submission to one of the following protocols: 1) normovolemic control (N = 22), 2) expansion (N = 72) by intravenous infusion (1 ml/min) of Ringer-bicarbonate solution, volumes of 1, 2, 3 or 5% body weight, or 3) retraction (N = 22) by controlled bleeding (1.5 ml/100 g). Gastric emptying of liquid was inhibited by 19-51.2% (P<0.05) after blood volume expansion (volumes of 1, 2, 3 or 5% body weight). Blood volume expansion produced a sustained increase in central venous pressure while mean arterial pressure was transiently increased during expansion (P<0.05). Blood volume retraction increased gastric emptying by 28.5-49.9% (P<0.05) and decreased central venous pressure and mean arterial pressure (P<0.05). Infusion of the shed blood 10 min after bleeding reversed the effect of retraction on gastric emptying. These findings suggest that gastric emptying of liquid is subject to modulation by the blood volume.

Functional characterization and localization of AQP3 in the human colon

Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98% identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells.

Inhibition of gastric emptying and intestinal transit in anesthetized rats by a Tityus serrulatus scorpion toxin

The effects of a fraction (T1) of Tityus serrulatus scorpion venom prepared by gel filtration on gastric emptying and small intestinal transit were investigated in male Wistar rats. Fasted animals were anesthetized with urethane, submitted to tracheal intubation and right jugular vein cannulation. Scorpion toxin (250 µg/kg) or saline was injected iv and 1 h later a bolus of saline (1.0 ml/100 g) labeled with 99m technetium-phytate (10 MBq) was administered by gavage. After 15 min, animals were sacrificed and the radioactivity remaining in the stomach was determined. Intestinal transit was evaluated by instillation of a technetium-labeled saline bolus (1.0 ml) through a cannula previously implanted in the duodenum. After 60 min, the progression of the marker throughout 7 consecutive gut segments was estimated by the geometric center method. Gastric retention of the liquid test meal in rats injected with scorpion toxin (median: 88%; range: 52-95%) was significantly higher (P<0.02) than in controls (54%; 21-76%), an effect which was not modified by gastric secretion blockade with ranitidine. The progression of the isotope marker throughout the small intestine was significantly slower (P<0.05) in rats treated with toxin (1.2; 1.0-2.5) than in control animals (2.3; 1.0-3.2). Inhibition of both gastric emptying and intestinal transit in rats injected with scorpion toxin suggests an increased resistance to aboral flow, which might be caused by abnormal neurotransmitter release or by the local effects of venom on smooth muscle cells.

Variations in gastric compliance induced by acute blood volume changes in anesthetized rats

The impact of acute volume imbalances on gastric volume (GV) was studied in anesthetized rats (250-300 g). After cervical and femoral vessel cannulation, a balloon catheter was positioned in the proximal stomach. The opposite end of the catheter was connected to a barostat with an electronic sensor coupled to a plethysmometer. A standard ionic solution was used to fill the balloon (about 3.0 ml) and the communicating vessel system, and to raise the reservoir liquid level 4 cm above the animals' xiphoid appendix. Due to constant barostat pressure, GV values were considered to represent the gastric compliance index. All animals were monitored for 90 min. After a basal interval, they were randomly assigned to normovolemic, hypervolemic, hypovolemic or restored protocols. Data were compared by ANOVA followed by Bonferroni's test. Mean arterial pressure (MAP), central venous pressure (CVP) and GV values did not change in normovolemic animals (N = 5). Hypervolemic animals (N = 12) were transfused at 0.5 ml/min with a suspension of red blood cells in Ringer-lactate solution with albumin (12.5 ml/kg), which reduced GV values by 11.3% (P<0.05). Hypovolemic rats (N = 12) were bled up to 10 ml/kg, a procedure that increased GV values by 15.8% (P<0.05). In the restored group (N = 12), shed blood replacement brought GV values back to basal levels in bled animals (P>0.05). MAP and CVP values increased (P<0.05) after hypervolemia but decreased (P<0.05) with hypovolemia. In conclusion, blood volume level modulates gastric compliance, turning the stomach into an adjustable reservoir, which could be part of the homeostatic process to balance blood volume.

Functional dyspepsia: relationship between clinical subgroups and Helicobacter pylori status in Western Turkey

The etiology of functional dyspepsia is not known. The objective of the present study was to determine the characteristics of functional dyspepsia in Western Turkey. We divided 900 patients with functional dyspepsia into three subgroups according to symptoms: ulcer-like (UL), 321 (35.6%), motility disorder-like (ML), 281 (31.2%), and the combination (C) of these symptoms, 298 (33.1%). All patients were submitted to endoscopic evaluation, with two biopsies taken from the cardia and corpus, and four from the antrum of the stomach. All biopsy samples were studied for Helicobacter pylori (Hp) density, chronic inflammation, activity, intestinal metaplasia, atrophy, and the presence of lymphoid aggregates by histological examination. One antral biopsy was used for the rapid urease test. Tissue cagA status was determined by PCR from an antral biopsy specimen by a random sampling method. We also determined the serum levels of tumor necrosis factor-alpha (TNF-alpha) and gastrin by the same method. Data were analyzed statistically by the Kolmogorov-Smirnov test and by analysis of variance. Hp and cagA positivity was significantly higher in the UL subgroup than in the others. The patients in the ML subgroup had the lowest Hp and cagA positivity and Hp density. The ML subgroup also showed the lowest level of Hp-induced inflammation among all subgroups. The serum levels of TNF-alpha and gastrin did not reveal any difference between groups. Our findings show a poor association of Hp with the ML subgroup of functional dyspepsia, but a stronger association with the UL and C subgroups.

Decreased gastric tone and delayed gastric emptying precede neutrophil infiltration and mucosal lesion formation in indomethacin-induced gastric damage in rats

Gastric antral dysmotility has been implicated in the pathogenesis of indomethacin-induced gastric damage, but the relationship between gastric motor abnormalities and mucosal lesions has not been extensively studied. We investigated whether changes in gastric tone and gastric retention correlate with mucosal lesions and neutrophil migration in indomethacin-induced gastric damage in rats. Indomethacin, either 5 or 20 mg/kg (INDO-5 and INDO-20), was instilled into the stomach, and then gastric damage, neutrophil migration, gastric tone and gastric retention were assessed 1 or 3 h later. Gastric damage was calculated as the sum of the lengths of all mucosal lesions, and neutrophil migration was measured by assaying myeloperoxidase activity. Gastric tone was determined by a plethysmometric method, and gastric retention of either saline or Sustacal® was evaluated by a scintigraphic method. Gastric damage was detectable 3 h after either INDO-5 or INDO-20, but not after 1 h. Neutrophil migration was significantly higher 3 h after INDO-20 as compared with INDO-5 or control group, but not after 1 h. Values of gastric tone 1 and 3 h after either INDO-5 (1 h = 1.73 ± 0.07 ml; 3 h = 1.87 ± 0.03 ml) or INDO-20 (1 h = 1.70 ± 0.02 ml; 3 h = 1.79 ± 0.03 ml) were significantly lower than in controls (1 h = 1.48 ± 0.05 ml; 3 h = 1.60 ± 0.06 ml). Gastric retention of saline was higher 1 h after INDO-5 (58.9 ± 3.3%) or INDO-20 (56.1 ± 3.1%) compared to control (45.5 ± 1.7%), but not after 3 h. There were no differences concerning gastric retention of Sustacal® between the various groups. Indomethacin induced decreased gastric tone and delayed gastric emptying, which precede mucosal lesion and neutrophil infiltration. These results indicate that there is no relationship between these gastric motor abnormalities and mucosal lesion in indomethacin-induced gastropathy.

The role of IFN-gamma and IL-4 in gastric mucosa inflammation associated with Helicobacter heilmannii type 1 infection

Although Helicobacter heilmannii infection is less common than H. pylori infection in humans, it is considered to be of medical importance because of its association with gastritis, gastric ulcer, carcinoma, and mucosa-associated lymphoid tissue lymphoma of the stomach. However, there have been no studies evaluating the role of the Th cell response in H. heilmannii gastric infection. We evaluated the participation of pro-inflammatory and anti-inflammatory cytokines, IFN-gamma and IL-4, in H. heilmannii gastric infection in genetically IFN-gamma- or IL-4-deficient mice. The serum IFN-gamma and IL-4 concentrations were determined by ELISA. The gastric polymorphonuclear infiltrate was higher (P = 0.007) in H. heilmannii-positive than in H. heilmannii-negative wild-type (WT) C57BL/6 mice, whereas no significant inflammation was demonstrable in the stomach of H. heilmannii-positive IFN-gamma-/- C57BL/6 mice. The degree of gastric inflammatory cells, especially in oxyntic mucosa, was also higher (P = 0.007) in infected IL-4-/- than in WT BALB/c mice. Serum IFN-gamma levels were significantly higher in IL-4-/- than in WT BALB/c mice, independently of H. heilmannii-positive or -negative status. Although no difference in serum IFN-gamma levels was seen between H. heilmannii-positive (11.3 ± 3.07 pg/mL, mean ± SD) and -negative (11.07 ± 3.5 pg/mL) WT BALB/c mice, in the group of IL-4-/- animals, the serum concentration of IFN-g was significantly higher in the infected ones (38.16 ± 10.5 pg/mL, P = 0.04). In contrast, serum IL-4 levels were significantly decreased in H. heilmannii-positive (N = 10) WT BALB/c animals compared to the negative (N = 10) animals. In conclusion, H. heilmannii infection induces a predominantly Th1 immune response, with IFN-gamma playing a central role in gastric inflammation.

Chemotherapy versus support cancer treatment in advanced gastric cancer: a meta-analysis

The aim of the present study was to compare the efficacy of chemotherapy and support treatment in patients with advanced non-resectable gastric cancer in a systematic review and meta-analysis of randomized clinical trials that included a comparison of chemotherapy and support care treatment in patients diagnosed with gastric adenocarcinoma, regardless of their age, gender or place of treatment. The search strategy was based on the criteria of the Cochrane Base, using the following key words: 1) randomized clinical trials and antineoplastic combined therapy or gastrointestinal neoplasm, 2) stomach neoplasm and drug therapy, 3) clinical trial and multi-modality therapy, 4) stomach neoplasm and drug therapy or quality of life, 5) double-blind method or clinical trial. The search was carried out using the Cochrane, Medline and Lilacs databases. Five studies fulfilled the inclusion criteria, for a total of 390 participants, 208 (53%) receiving chemotherapy, 182 (47%) receiving support care treatment and 6 losses (1.6%). The 1-year survival rate was 8% for support care and 20% for chemotherapy (RR = 2.14, 95% CI = 1.00-4.57, P = 0.05); 30% of the patients in the chemotherapy group and 12% in the support care group attained a 6-month symptom-free period (RR = 2.33, 95% CI = 1.41-3.87, P < 0.01). Quality of life evaluated after 4 months was significantly better for the chemotherapy patients (34%; RR = 2.07, 95% CI = 1.31-3.28, P < 0.01) with tumor mass reduction (RR = 3.32, 95% CI = 0.77-14.24, P = 0.1). Chemotherapy increased the 1-year survival rate of the patients and provided a longer symptom-free period of 6 months and an improvement in quality of life.

Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

Novel mechanisms of growth hormone regulation: growth hormone-releasing peptides and ghrelin

Growth hormone secretion is classically modulated by two hypothalamic hormones, growth hormone-releasing hormone and somatostatin. A third pathway was proposed in the last decade, which involves the growth hormone secretagogues. Ghrelin is a novel acylated peptide which is produced mainly by the stomach. It is also synthesized in the hypothalamus and is present in several other tissues. This endogenous growth hormone secretagogue was discovered by reverse pharmacology when a group of synthetic growth hormone-releasing compounds was initially produced, leading to the isolation of an orphan receptor and, finally, to its endogenous ligand. Ghrelin binds to an active receptor to increase growth hormone release and food intake. It is still not known how hypothalamic and circulating ghrelin is involved in the control of growth hormone release. Endogenous ghrelin might act to amplify the basic pattern of growth hormone secretion, optimizing somatotroph responsiveness to growth hormone-releasing hormone. It may activate multiple interdependent intracellular pathways at the somatotroph, involving protein kinase C, protein kinase A and extracellular calcium systems. However, since ghrelin has a greater ability to release growth hormone in vivo, its main site of action is the hypothalamus. In the current review we summarize the available data on the: a) discovery of this peptide, b) mechanisms of action of growth hormone secretagogues and ghrelin and possible physiological role on growth hormone modulation, and c) regulation of growth hormone release in man after intravenous administration of these peptides.

Expression of TERT in precancerous gastric lesions compared to gastric cancer

The objective of this study was to determine the levels of TERT mRNA and TERT protein expression in stomach precancerous lesions such as intestinal metaplasia (IM) and gastric ulcer (GU) and compare them to gastric cancer (GC). Real-time PCR was performed to detect TERT mRNA expression levels in 35 biopsies of IM, 30 of GU, and 22 of GC and their respective normal mucosas. TERT protein was detected by immunohistochemistry in 68 samples, 34 of IM, 23 of GU, and 11 of GC. Increased TERT mRNA expression levels were observed in a significant number of cases, i.e., 46% of IM, 50% of GU, and 79% of GC. The relative mean level of TERT mRNA after normalization with the β-actin reference gene and comparison with the respective adjacent normal mucosa was slightly increased in the IM and GU groups, 2.008 ± 2.605 and 2.730 ± 4.120, respectively, but high TERT mRNA expression was observed in the GC group (17.271 ± 33.852). However, there were no statistically significant differences between the three groups. TERT protein-positive immunostaining was observed in 38% of IM, 39% of GU, and 55% of GC. No association of TERT mRNA and protein expression with Helicobacter pylori infection or other clinicopathological variables was demonstrable, except for the incomplete type vs the complete type of IM. This study confirms previous data of the high expression of both TERT mRNA and protein in gastric cancer and also demonstrates this type of changed expression in IM and GU, thus suggesting that TERT expression may be deregulated in precursor lesions that participate in the early stages of gastric carcinogenesis.

Polymorphism of the aryl-hydrocarbon receptor gene in intron 10 of human cancers

Polychlorinated dibenzo-p-dioxins (PCDDs) and related halogenated aromatic hydrocarbons (e.g., PCDFs), often called "dioxins", are ubiquitously present environmental contaminants. Some of them, notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are among the most toxic synthetic compounds known. The biological effects of dioxins are mediated via the aryl hydrocarbon receptor (AhR). Mutations in the AhR transactivation domain are linked to sensitivity to the acute lethality of TCDD. We present here a study of AhR gene polymorphism in normal and cancer human tissues affecting pre-mRNA splicing in the AhR gene-coding transactivation domain region (exon 10, intron 10, exon 11 region), previously shown to be associated with AhR dysfunction. We tested 126 pairs of normal and cancer tissue samples from liver, lung, stomach, kidney, mucous, breast, and pancreas of 49 males and 77 females (45-70 years of age). We used in vitro splicing assay, RT-PCR and sequencing methods. Our results showed that in an in vitro system it is possible to reconstitute cellular pre-mRNA splicing events. Tested cancer tissues did not contain mutations in the AhR transactivation domain region when the DNA sequences were compared with those from normal tissues. There were also no differences in AhR mRNA splice variants between normal and malignant breast tissues and no polymorphisms in the studied regions or cDNA.