Ulcer Region Research Articles

Mouse model of Helicobacter pylori infection: studies of gastric function and ulcer healing.

Helicobacter pylori infection in humans is a major risk factor for peptic ulcer, but studies on the relation between H. pylori infection and gastric pathology are limited due to a deficiency of convenient animal models resembling this infection in humans. We studied the effects of inoculation of conventional BALB/c mice with CagA and VacA positive (type I) H. pylori or CagA and VacA negative H. pylori (type II) strains on gastric secretion and healing of chronic acetic acid-induced ulcers in mouse stomachs. The ulcer area, gastric blood flow, plasma interleukin (IL)-1beta and IL-12, as well as plasma gastrin and gastric luminal somatostatin were determined. Gastric mucosal biopsy samples were also taken for assessment of the presence of viable H. pylori using a rapid urease test, H. pylori-culture and the RT-PCR analysis of the signal for H. pylori CagA. Gastric acid and pepsin secretion was reduced by over 50% immediately after H. pylori inoculation and accompanied by a significant increment in plasma gastrin and fall in gastric luminal somatostatin content observed over all test days, particularly in mice infected with type I H. pylori. The area of ulcers in vehicle-treated controls decreased significantly starting from day 2 after ulcer induction and then continued to decline for a further 14 days to heal almost completely after 28 days. In contrast, the ulcers were present until day 28 in all mice infected with type I or type II H. pylori strains, being significantly larger, especially with type I H. pylori infection. The gastric blood flow at the ulcer margin and ulcer crater in vehicle-treated mice gradually increased with decreasing ulcer size, after 14 and 28 days reaching a value which was not significantly different from that in vehicle-administered mice. In contrast, the gastric blood flow in type I H. pylori and, to a lesser extent, in type II H. pylori infected mice was significantly lower than in vehicle controls, both at the margin and at the crater of ulcers at all tested days. Histological changes such as oedema or congestion of surface epithelium were found after 7 days whereas mucosal inflammatory infiltration appeared after 14 days with a further increase after 28 days, especially in type I H. pylori and to a lesser extent in type II H. pylori infected mice. Plasma IL-1beta and IL-12 were significantly elevated at all tested days of ulcer healing and their increments were significantly higher in type I than in type II H. pylori infection. Conventional mice with gastric ulcers can be successfully infected by both toxigenic and nontoxigenic H. pylori strains, and this infection causes an immediate suppression of gastric secretion and markedly delays the healing of ulcers due to the fall in mucosal microcirculation in the ulcer region, cytokine release and an impairment in the gastrin-somatostatin link that appears to be independent of gastritis and more pronounced with infection of toxigenic than nontoxigenic strains.

Expression of fibroblast growth factor-2 transcripts in the healing of acetic acid-induced gastric ulcers.

Basic fibroblast growth factor-2 (FGF-2) has an important role in angiogenesis, and has been demonstrated to promote ulcer healing. However, the actual part played by FGF-2 in the process of ulcer healing is not well understood. In this study, we investigated expression of FGF-2 transcripts at each stage of gastric ulcer healing, using an acetic acid model in rats. We made ulcers in the rat stomach by direct application of acetic acid, and 3 days and 1, 2, 3, 4, 8 weeks after treatment, we examined expression of FGF-2 transcripts by in situ hybridization. On day 3, FGF-2 transcripts were detected in mononuclear cells infiltrating the submucosal layer around the ulcer. After 1 and 2 weeks, expression of FGF-2 transcripts was prominent in spindle-shaped mesenchymal cells and endothelial cells, which proliferated in the ulcerative region. Some of the spindle-shaped cells which expressed FGF-2 transcripts also showed immunoreactivity for alpha-smooth muscle actin. After 3 and 4 weeks, FGF-2 expression was seen mainly in endothelial cells of vessels. These results suggest that different cells produce FGF-2 during the process of gastric ulcer healing, and some of the spindle-shaped cells expressing FGF-2 transcripts in the early phase are myofibroblasts.

Pythiosis of the digestive tract in dogs from Oklahoma.

Enteric pythiosis was diagnosed in nine dogs in Oklahoma. Eight dogs had anorexia and weight loss. Two of these dogs had diarrhea; two dogs exhibited vomiting and diarrhea; and one dog had vomiting. One dog presented with dysphagia. Seven dogs had either a palpable or radiographically visible abdominal mass. These seven dogs had localized regions of mucosal ulceration and thickened gastric or intestinal walls with some involvement of the adjacent mesentery or omentum. Two dogs had enlarged regional mesenteric lymph nodes. One dog that presented with dysphagia had an oropharyngeal mass involving the larynx and cranial esophagus. Microscopically, there was transmural chronic sclerosing and granulomatous to pyogranulomatous inflammation with arteritis. Pythium spp. were identified in all specimens by immunohistochemistry.

Anti-ulcer effects of antioxidants: effect of probucol

We investigated the effect of probucol, a lipid-lowering agent with antioxidant properties, on HCl plus ethanol-induced gastric mucosal injury and on the healing of acetic acid-induced gastric ulcers in rats. When the free radical-scavenging activity of probucol was measured by an electron spin resonance technique, the agent (10 −5–10 −3 M) scavenged both superoxide anions and hydroxyl radicals. Oral administration of probucol (250–1000 mg/kg) dose dependently prevented the HCl plus ethanol-induced gastric mucosal injury and the increase in thiobarbituric acid-reactive substances, an index of lipid peroxidation, in the injured mucosa. Repeated oral administration of probucol (250–1000 mg/kg twice daily) dose dependently accelerated the healing of acetic acid-induced gastric ulcers. In addition, probucol already inhibited the increase in the content of thiobarbituric acid-reactive substances in the ulcerated region before the ulcer-healing effect of this agent was recognized. These results suggest that probucol may partly protect gastric mucosa from acute gastric mucosal injury and promote the healing of chronic gastric ulcers by its antioxidant activity.

Roles of hepatocyte growth factor and its receptor Met during gastric ulcer healing in rats

BACKGROUND & AIMS: It is unclear which growth factors are primarily responsible for stimulating gastric ulcer healing. The roles of hepatocyte growth factor (HGF) and Met/HGF receptor during gastric ulcer healing were studied in rats. METHODS: HGF and Met/HGF receptor were located and quantified by in situ hybridization and immunohistochemistry during experimental gastric ulcer healing. The in vivo effects of exogenous recombinant human HGF on cell proliferation and ulcer healing were assessed and compared with those of placebo and omeprazole treatment. RESULTS: Compared with intact oxyntic mucosa, messenger RNA (mRNA) of HGF and met was substantially greater in the ulcerated region on days 3 and 15. HGF mRNA was located in stromal cells between the regenerative glands and in the arterial vessels of submucosal tissue, whereas met mRNA was located in the epithelial cells of the regenerative glands. After cryoinjury, immunoreactivity for the Met/HGF receptor was absent on day 3, reappeared on day 8, and was overexpressed on day 15. Exogenous recombinant human HGF had no effect on the ulcer healing parameters over days 3-8, but it did increase epithelial cell proliferation in the ulcer margin over days 8-15. CONCLUSIONS: These data suggest that HGF mediates specific tissue interactions between mesenchyme and epithelia during gastric ulcer healing. (Gastroenterology 1997 Dec;113(6):1858-72)

Juxtalumenal location of plaque necrosis and neoformation in symptomatic carotid stenosis.

The structural features that underlie carotid plaque disruption and symptoms are largely unknown. We have previously shown that the chemical composition and structural complexity of critical carotid stenoses are related to plaque size regardless of symptoms. To further determine whether the spatial distribution of individual plaque components in relation to the lumen corresponds to symptomatic outcome, we evaluated 99 carotid endarterectomy plaques. Indications for operation were symptomatic disease in 59 instances (including hemispheric transient ischemic attack in 29, stroke in 19, and amaurosis fugax in 11) and angiographic asymptomatic stenosis > 75% in 40. Plaques removed after remote symptoms beyond 6 months were excluded. Histologic sections from the most stenotic region of the plaque were examined using computer-assisted morphometric analysis. The percent area of plaque cross-section occupied by necrotic lipid core with or without associated plaque hematoma, by calcification, as well as the distance from the lumen or fibrous cap of each of these features, were determined. The presence of foam cells, macrophages, and inflammatory cell collections within, on, or just beneath the fibrous cap was taken as an additional indication of plaque neoformation. The mean percent angiographic stenosis was 82% +/- 11% and 79% +/- 13% for the asymptomatic and symptomatic groups, respectively (p > 0.05). The necrotic core was twice as close to the lumen in symptomatic plaques when compared with asymptomatic plaques (0.27 +/- 0.3 mm vs 0.5 +/- 0.5 mm; p < 0.01). The percent area of necrotic core or calcification was similar for both groups (22% vs 26% and 7% vs 6%, respectively). There was no significant relationship to symptom production of either the distance of calcification from the lumen or of the percent area occupied by the lipid necrotic core or calcification. The number of macrophages infiltrating the region of the fibrous cap was three times greater in the symptomatic plaques compared with the asymptomatic plaques (1114 +/- 1104 vs 385 +/- 622, respectively, p < 0.009). Regions of fibrous cap disruption or ulceration were more commonly observed in the symptomatic plaques than in the asymptomatic plaques (32% vs 20%). None of the demographic or clinical atherosclerosis risk factors distinguished between symptomatic and asymptomatic plaques. These findings indicate that proximity of plaque necrotic core to the lumen and cellular indicators of plaque neoformation or inflammatory reaction about the fibrous cap are associated with clinical ischemic events. The morphologic complexity of carotid stenoses does not appear to determine symptomatic outcome but rather the topography of individual plaque components in relation to the fibrous cap and the lumen. Imaging techniques that precisely resolve the position of the necrotic core and evidence of inflammatory reactions within carotid plaques should help identify high-risk stenoses before disruption and symptomatic carotid disease.

A CASE OF PHYLLODES TUMOR OF THE RIGHT BREAST TREATED WITH AN EMERGENCY OPERATION

A 66-year-old woman was admitted to the hospital because of a rapidly growing right breast tumor since previous one year. The tumor was about 14cm in size and occupied the entire right breast. A bleeding ulcer was noticed in the D region of the right breast. The tumor and ulcer increased markedly within 4 days. The tumor with oozing blood protruded from the ulcer region and marked anemia was noted. Emergency operation was performed under the diagnosis of rapidly growing phyllodes tumor. Post-operative course was uneventful and anemia was improved until 17th postoperative day. The histological diagnosis was intermediate type phyllodes tumor. She has been well without any sign of recurrence.

PEPTIC ULCER: PATHOGENESIS, HEALING AND ANTI‐ULCER DRUGS

To investigate pathophysiological diversities in the repairing process of gastric ulcer, distribution density of basic fibroblast growth factor (bFGF)-positive fibroblasts and myofibroblasts and vascular endothelial cells, mucosal haemoglobin content, PAS-positive mucus amount and glandular index were compared in the peripheral region of an open ulcer (the unhealed group; n = 17), the central region of a red scar (the red scar group; n = 32) and the central region of the white scar (white scar group; n = 32). Density of bFGF-positive fibroblasts and myofibroblasts and vascular endothelial cells was highest in the unhealed group, followed by the red scar group, while the white scar group showed a low value close to control. Mucosal haemoglobin content was high in the red scar and unhealed groups. PAS-positive mucus amount in the unhealed and red scar groups showed lower values compared with that in the white scar group. Glandular index in the unhealed group was the lowest, followed by the red scar group, while the white scar group neared control values. Statistically significant correlations were observed between the density of bFGF-positive "fibroblasts and myofibroblasts' and density of bFGF-positive vascular endothelial cells, between the density of bFGF-positive vascular endothelial cells and mucosal haemoglobin content and between the PAS-positive mucus amount and glandular index. Discriminant analysis demonstrated that the unhealed group could be distinguished from the red and white scar groups, based on glandular index, density of bFGF-positive "fibroblasts and myofibroblasts', mucosal haemoglobin content and PAS-positive mucus amount, while the red scar group could be discriminated from the white scar group based on the density of bFGF-positive "fibroblasts and myofibroblasts', density of bFGF-positive vascular endothelial cells, glandular index, haemoglobin content and PAS-positive mucus amount. The white scar group was difficult to discriminate from control. Our present results show that the recovery of glandular formation and mucus production continues throughout the repairing process, whereas the acceleration of angiogenesis and granulation formation is observed only in unhealed ulcers and at the red scar stage.

Enhanced levels of chemiluminescence and platelet activating factor in urease-positive gastric ulcers

Helicobacter pylori are believed to play an important role in the formation of gastric ulcer in a syndrome characterized by a high urease activity. On the other hand, the production of oxygen radicals and platelet activating factor (PAF) is enhanced in gastric ulcers. The present study is designed to investigate the relationship between the different aspects of gastric mucosal injury, urease activity, oxygen radical production, and PAF content in gastric specimens. Biopsy specimens taken from 35 gastric ulcer patients were studied. Urease activity was detected by a rapid urease test (CLO). Oxygen radical production was measured as a value of luminol-dependent chemiluminescence (ChL) and PAF content was determined by radioimmunoassay in the biopsy samples. The CLO-positive rate was significantly higher in the gastric ulcer group in comparison with that in controls. ChL values and PAF content were significantly increased in gastric ulcers, especially in CLO-positive specimens. The CLO-positive rate, ChL values, and PAF content were also found to be increased at a distant site beyond the ulcer lesions. During the course of macroscopic ulcer healing of CLO-positive cases, the CLO positive level and the ChL values were not significantly decreased, although PAF content was significantly lower. Enhanced oxygen radical and PAF production were observed not only in the ulcer region but also at a distant site from the ulcer in the urease-positive gastric mucosa. The persistent enhancement of ChL values during the healing stage of urease-positive gastric ulcers suggests its involvement in the recurrence of gastric ulcers.

Healing-Promoting Action of the Zinc-Cimetidine Complex on Acetic Acid-Induced Gastric Ulcers in Rats

We investigated the effect of the zinc-cimetidine complex on the healing of acetic acid-induced gastric ulcers in rats. When the effects of test drugs were assessed on the 15th day after acetic acid injection, the zinc-cimetidine complex at oral doses of 15.0 (11.4 mg as cimetidine), 30.0 and 60.0 mg/kg twice daily promoted the ulcer healing in a dose-dependent manner. Cimetidine was effective at oral doses of over 45.4 mg/kg twice daily. ZnCl2 was ineffective on all ulcer parameters. The effect of the combination of cimetidine and ZnCl2 was similar to that of cimetidine alone. The zinc-cimetidine complex had already inhibited the increase in thiobarbituric acid reactants in the ulcerated region before the ulcer-healing effect of this compound was recognized. A single oral administration of the complex at 15 and 30 mg/kg to normal rats was ineffective in inhibiting acid secretion and in increasing serum gastrin levels, although cimetidine was markedly effective on both parameters. These results indicate that the zinc-cimetidine complex at about 1 /4 the dose of cimetidine was as effective as cimetidine when the ulcer-healing effects of both compounds were compared with the same dose of cimetidine. In addition, the ulcer-healing effect of this complex may be due, at least in part, to the inhibition of lipid peroxidation but not due to the inhibition of acid secretion or the trophic effect of gastrin.

Effect of aspirin on ulcer site blood flow in cat stomachs.

Radioactive microspheres were used to measure blood flow in the cat stomach during exposure to saline, 0.075 M HCl, and then 15 and 30 min after exposure to 20 or 40 mM aspirin in HCl. At the end of the experiment, the stomach wall was divided into ulcerated regions and adjacent nonulcerated areas. When exposed to saline, both regions had similar blood flow: 27 +/- 5 and 25 +/- 5 ml.min-1.100 g-1 (means +/- SE). Addition of acid caused a significant increase in blood flow to 41 +/- 7 ml.min-1.100 g-1 only at those sites that eventually ulcerated in the presence of aspirin. In the adjacent nonulcerated regions, blood flow was 31 +/- 5 ml.min-1.100 g-1 and was not significantly greater than the flow recorded during saline exposure. Aspirin caused ulcer site blood flow to increase dramatically to 89 +/- 12 and 122 +/- 18 ml.min-1.100 g-1 after 15 and 30 min, whereas the adjacent nonulcerated tissue rose to 40 +/- 6 and 44 +/- 5 ml.min-1.100 g-1, respectively. The ulcer site hyperemia with acid alone suggests higher mucosal permeability in these regions allowing back-diffusion of acid and injurious agents. The present data obtained in the cat do not support the notion that ischemia plays a role in initiating nonsteroidal anti-inflammatory drug (NSAID)-induced ulcers, but rather that acute NSAID ulcers are associated initially with a hyperemia.

Studies on the healing promoting action of Z-103 in chronic gastric ulcer models of rats

We studied the healing promoting action of Z-103 on the chronic gastric ulcer induced by acetic acid (AAU) or Fe-ascorbic acid (FAU) in rats. The area of the gastric ulcers, hydroxyproline (Hyp) and DNA contents in the ulcer region were measured as an index of ulcer healing. The area of gastric ulcers was the largest on day 4 and thereafter gradually decreased, but the ulcers still remained at the 14th day. Hyp contents in the ulcer region decreased until the 7th day in both models, and then this level increased. Significant decrease in DNA contents in the ulcer region was observed on the 7th day only in FAU. In AAU and FAU, administration of Z-103 (3 mg/kg/day x 2, p.o.) resulted in a significant decrease in the area of gastric ulcers on the 14th day and a significant increase in Hyp contents in the ulcer region on the 7th day as compared with the control group. Z-103 increased the DNA contents in the ulcer region on the 4th day in AAU and on the 7th day in FAU. These results suggest that tissue destruction surrounding the ulcer region in AAU and FAU models might occur until the 4th or 7th day after operation, and that the acceleration of ulcer healing by Z-103 on these models may be facilitated by the wound healing action of this drug.

Segmental Tuberculosis of the Colon Diagnosed by Colonoscopy

We describe the case of a woman with rectorrhagia and rectal tenesmus. Colonoscopy revealed a 3-4 cm-long ulcerated lesion with a reduced colon lumen 35 cm from the anus, and multiple biopsies were obtained from the ulcerated region, but histopathology was non-specific. Biopsy culture was positive for M. tuberculosis. Anti-tuberculous chemotherapy was initiated on the diagnosis of segmental tuberculosis of the colon. Subsequent evolution was favorable, both clinically and endoscopically. Emphasis is placed on the importance of colonoscopy and biopsy in the diagnosis of tuberculosis of the colon.

Determinants of the fluorescence emission spectrum of atheromatous plaques treated with haematoporphyrin in vitro

Studies were carried out on atheromatous plaques which had been incubated with the fluorescent dye haematoporphyrin. By varying the wavelength of excitation, it was possible to obtain fluorescence signals from different depths in plaque, since enhanced tissue penetration occurs when the wavelength of exciting light is increased. Moreover, the presence of ulcerated regions altered both excitation and emission spectra. These results suggest that corresponding measurements using laser/fibre-optic systems may be used to characterize plaques in vivo.

酢酸潰ようの治癒過程における胃粘膜アミン含量および関連酵素活性に対する抗潰よう薬の影響

In order to elucidate the action of an H2 blocker (cimetidine) and gastric mucosal protection agents (sucralfate and sofalcone) on the relapse and recurrence of gastric ulcer, the effects of cimetidine, sucralfate and sofalcone on the contents of histamine and serotonin and histidine decarboxylase (HDC) activity in the gastric mucosa were examined in the ulcer region and the intact region at the 10th day after the operation to produce acetic acid-induced gastric ulcer in rats. The following results were obtained: 1) HDC activity in the gastric mucosa of rats treated with cimetidine (100 mg/kg twice daily) tended to increase in the intact region, and it was significantly increased in the ulcer region. 2) Increased HDC activity due to cimetidine treatment was observed at the 10th day after interruption of cimetidine administration. 3) The HDC activity in the gastric mucosa was not changed by the treatment with sucralfate (500 mg/kg/day) and sofalcone (200 mg/kg/day). The results suggest that the increased HDC activity in the gastric mucosa might participate in the relapse and recurrence of gastric ulcer after discontinuation of cimetidine administration.

Investigations on guinea worm disease in anambra state, Nigeria. Treatment of patients, examination of drinking water and influence on school attendance.

Investigation and treatment were performed on the cases with guinea worm disease (dracontiasis) in Abakariki district of Anambra State, Nigeria. In the classification of 63 patients under study by their professions, 76% were agricultural workers, 19% school boys and girls, and average age of the patients was 21 years old. For the treatment, albendazole was given to the patients, and the course up to the complete excretion of worm body was carefully observed. Whole worm body was excreted within 5 days in average, and the ulcerated region of the skin was healed thereafter. Bacterial colonies counts were examined on the drinking water by dividing water sources into five groups of ponds, shallow wells, deep wells, rivers and tap water. The results of the test revealed that the water in ponds, shallow wells and rivers was heavily contaminated and cyclops, the intermediary host of Dracunculus medinesis, was found in large quantity, suggesting frequent and easy infection of the disease. In elementary schools, many pupils (59.6% of all) were absent due to guinea worm disease, and this suggests that the disease exerts extensive social and economical influence such as the decrease of school attendance ratio or the decline of agricultural production. This disease is related with the hygienic standards of drinking water, and the prevention of the disease is extremely important. The construction of deep wells is urgently wanted, while it is also essential to provide good knowledge of hygiene to the inhabitants through education.

Gastric small lymphocytic proliferation with immunoglobulin gene rearrangement in pseudolymphoma versus lymphoma

The nature of gastric infiltrates consisting primarily of benign-appearing small lymphocytes is at present a controversial issue. Earlier reports of gastric lymphoma developing in gastric pseudolymphoma and more recent immunohistochemical studies demonstrating monoclonal B-cell populations in pseudolymphoma suggest that at least some cases represent low-grade lymphomas or clonal precursor lesions that may develop into lymphoma. Observations of a small lymphocytic infiltrate arising in the region of a gastric ulcer that lacked definitive morphologic evidence of malignancy (lymphoma) but was clearly a monoclonal B-cell proliferation by immunohistochemical and gene rearrangement studies support the notion that some gastric lymphoproliferative lesions that histologically have been called pseudolymphomas may include one or more clonal lymphoid expansions. A histopathologic/molecular model suggesting a potential pathway for the development of morphologically recognizable lymphoma from benign-appearing small lymphocytic infiltrates is presented, and the concept that for a variety of lymphoid proliferations clonality and malignancy may not be synonymous is discussed.

Changes in amine contents and regulating enzyme activity of gastric mucosa during the healing process of acetic acid induced ulcer in rats

Changes in the contents of various amines and histidine decarboxylase (HDC) activity in the gastric mucosa during the healing process of acetic acid induced gastric ulcer in rats were sequentially examined in the ulcer region and intact region at 2, 10, 40, 80, 180 and 365 days after the operation. The following results were obtained: 1) Histamine (HA) content in the ulcer region was decreased as compared with the intact region at 2 and 10 days and returned to the control level in 40 days. After 180 days, the contents in the ulcer region and intact region were also increased as compared with that of the normal control region. 2) Changes in serotonin (5-HT) content as well as HA content were observed. 3) Norepinephrine content in the ulcer region was decreased as compared with the intact region at 2, 10, 80 and 180 days. 4) HDC activity in the ulcer region was decreased as compared with the intact region at 2, 10 and 40 days, and a lower level was maintained still at 180 days. 5) In the relapse and recurrence of gastric ulcer at 365 days, HA and 5-HT contents in the intact region and ulcer region were not different from those in healing rats, but the contents of these amines were higher at 180 days. The results suggest that the change of HA, 5-HT contents and HDC activity in the gastric mucosa may be one of the factors involved in the relapse and recurrence of chronic ulcers.

Characterization of metalloproteinases in rat gastric tissues with acetic acid-induced ulcers.

To clarify the destruction of connective tissue in ulcerated regions, collagenase and gelatinolytic activities in homogenates of rat acetic acid-induced ulcers were examined. Gelatinolytic activity in the ulcerated regions was significantly higher than that in normal tissues. Collagenase, however, was not detectable. Gelatin-gel-electrophoresis showed that the gelatinolytic activity was due to several species, some of which crossreacted with a sheep anti-(rabbit prostromelysin) antibody. The H2-blocker, famotidine, significantly depressed the gelatinolytic activity in the ulcerated regions. Thus, both stromelysin and gelatinolytic enzymes may play important roles in the degradation of the basement membrane, especially type IV collagen.

Influence of Food-Intake during a Fixed Period on Hydrocortisone-Induced Relapse of Acetic Acid Ulcers in Rats

We studied the influence of food-intake during a fixed period on hydrocortisone-induced relapse of acetic acid ulcers in rats. The ulcer index, the defective area of the ulcerated region and the exposure index for the ulcer floor in the food-intake-time limited group markedly increased as compared to those in the control group. The index for the regenerated mucosa decreased in the food-intake-time limited group. Thus, hydrocortisone-induced relapsed ulcers were markedly increased by limiting the food-intake-time.