Prevalence of symptomatic gastro-oesophageal reflux disease in Japanese patients with peptic ulcer disease after eradication of Helicobacter pylori infection.

BACKGROUND: The association between cure of Helicobacter pylori infection and the development of gastro-oesophageal reflux disease is controversial. AIM: To examine the prevalence of symptomatic GERD (sGERD) in Japanese patients with peptic ulcer disease after successful eradication and identify associated factors affecting sGERD development. METHODS: We retrospectively examined 72 patients (40 gastric ulcer and 32 duodenal ulcer) with successful eradication. Associated factors such as age, gender, drinking and smoking habits, body mass index, presence of gastric atrophy and hiatal hernia were analysed. RESULTS: Seven (9.7%) of 72 peptic ulcer patients newly developed sGERD. There were no differences in mean age, gender, smoking habit, drinking habit, body mass index, or presence of gastric atrophy and hiatal hernia between the sGERD and non-sGERD groups, while the proportion of subjects aged over 70 was significantly higher in the sGERD than the non-sGERD group. Six of 40 patients with gastric ulcer newly developed sGERD while only one of 32 patients with duodenal ulcer developed it. CONCLUSION: Approximately 10% of Japanese patients with peptic ulcer disease newly developed sGERD after cure of H. pylori infection. Age > 70 years was associated with development of sGERD. Eradication in patients in this age group should be carefully determined.

Donor selection in Japan: a trial of new criteria with predonation haemoglobin testing.

BACKGROUND AND OBJECTIVES: In Japan, eligibility for blood donation depends on blood specific gravity, which does not directly measure blood haemoglobin. Additionally, the criteria are not based on normal values. Therefore, we investigated the feasibility of predonation screening by using actual haemoglobin levels, and adopted a new criterion based on the normal range for men. MATERIALS AND METHODS: Using a portable device, we measured haemoglobin in 1032 prospective blood donors, then applied this method to all blood donations. The criterion for men was set at the 95th percentile of haemoglobin distribution, namely 13.0 g/dl and 13.5 g/dl, respectively, for 200-ml and 400-ml donations. That for women remained unchanged. RESULTS: The percentage of men ineligible by these criteria increased from 0.6 to 1.5%, while that of women decreased from 16.5 to 14.6%. Donors with abnormal haemoglobin levels were referred to hospitals. CONCLUSION: Predonation measurement of haemoglobin concentration, combined with the referral of those with abnormal values, provided a health benefit to that population.

Localization of CD36 and scavenger receptor class A in human coronary arteries--a possible difference in the contribution of both receptors to plaque formation.

CD36 and scavenger receptor class A types I and II (SR-AI/II) are major receptors for oxidized low density lipoproteins (OxLDL) expressed on macrophages. To elucidate the role of these two macrophage scavenger receptors in the development of coronary atherosclerosis, we examined the localization of CD36 and SR-AI/II in human coronary atherosclerotic lesions. Serial cryostat sections of 49 coronary arteries obtained from 43 autopsied cases were examined immunohistochemically. Regarding the relationship between the severity of atherosclerosis and immunoreactivities to CD36, there was almost no immunoreactivity to CD36 in regions with diffuse intimal thickening, while the expression of CD36 was accelerated in parallel with the progression of atherosclerosis. In contrast, SR-AI/II was expressed persistently from regions with diffuse intimal thickening to atherosclerotic plaques. We also clarified the differential distribution of CD36 and SR-AI/II in atheromatous plaques. Close to the luminal surface of the intima, macrophages were relatively small in size, contained lesser lipids, and expressed SR-AI/II more abundantly than CD36. In contrast, macrophages in the core region were larger in size, contained more lipids, were strongly positive for CD36 and showed a weaker immunoreactivity to SR-AI/II than those in the luminal surface of the intima. In conclusion, the expression of CD36 and SR-AI/II on macrophages may be regulated differently in the process of coronary atherogenesis.

Immunohistochemical localization of Betacellulin, a member of epidermal growth factor family, in atherosclerotic plaques of human aorta.

Betacellulin (BTC), a new member of the EGF family, has been reported to be a potent mitogen for rat vascular smooth muscle cells (SMCs). BTC mRNA is known to be expressed in several human organs. However, the localization of BTC in human vascular tissues has not yet been clarified. We investigated whether or not BTC protein is involved in the pathogenesis of human atherosclerosis. Recombinant human BTC showed a mitogenic activity on cultured human aortic SMCs by measuring [3H]thymidine incorporation. The immunohistochemical localization of BTC, SMCs, macrophages, EGF receptors and ErbB4 was examined in autopsied human aortas. BTC was detected in both intimal and medial SMCs of the aortic wall. The percentage of BTC-positive medial SMCs in early types of atherosclerotic lesions decreased with age, but in adult, it was significantly higher in advanced types than in early types of atherosclerotic lesions. BTC-positive SMCs were predominantly localized in the medial side of the intima. Furthermore, numerous BTC-positive SMCs and macrophages were observed around the core lesion of atherosclerotic plaques. Receptors for BTC, EGF receptor and ErbB4, were expressed on SMCs, suggesting that BTC is associated with EGF receptor family-mediated signaling. BTC is produced in human aortic tissue and might play important roles in atherogenesis.

Roles of melatonin in gonadal maturation of underyearling precocious male masu salmon.

Testicular maturation of underyearling precocious male masu salmon (Oncorhynchus masou) is affected by photoperiod. It is accelerated by a short photoperiod (light-dark cycles of 8:16 h; LD 8:16) and delayed by a long photoperiod (LD 16:8). Circulating melatonin levels are high during the night and low during the day:the duration of the nocturnal elevation is longer under a short than under a long photoperiod, suggesting mediation of photoperiodic signals by melatonin. This study examined whether melatonin administration mimics short photoperiodic effects and whether it accelerates the testicular development of underyearling male masu salmon reared under a long photoperiod. Fish were randomly selected in June and were divided into two groups. They were reared under LD 16:8 (lights on 04:00-20:00 h) and fed pellets sprayed with melatonin (0.5 mg melatonin/kg body weight/day) or vehicle once a day at 11:00 h until October. The plasma melatonin profile of the melatonin-treated group was similar to that expected under a short photoperiod. Melatonin treatment had a stimulatory effect on the gonadosomatic index and pituitary gonadotropin (GTH) I contents. Plasma testosterone levels were significantly higher in the melatonin-treated group than in the control group in August. However, spermiation was observed in October in both groups and no significant differences were observed in GTH II contents in the pituitary in the two groups throughout the experiment. These results suggest that mimicking a short photoperiod by melatonin administration stimulated testicular development but did not completely activate the brain-pituitary-gonadal axis in precocious male masu salmon. Thus, melatonin is suggested to be one of the factors that mediates the transduction of photoperiodic information to the brain-pituitary-gonadal axis.

Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing.

Keratinocyte proliferation and migration are essential to cutaneous wound healing and are, in part, mediated in an autocrine fashion by epidermal growth factor receptor (EGFR)-ligand interactions. EGFR ligands are initially synthesized as membrane-anchored forms, but can be processed and shed as soluble forms. We provide evidence here that wound stimuli induce keratinocyte shedding of EGFR ligands in vitro, particularly the ligand heparin-binding EGF-like growth factor (HB-EGF). The resulting soluble ligands stimulated transient activation of EGFR. OSU8-1, an inhibitor of EGFR ligand shedding, abrogated the wound-induced activation of EGFR and caused suppression of keratinocyte migration in vitro. Soluble EGFR-immunoglobulin G-Fcgamma fusion protein, which is able to neutralize all EGFR ligands, also suppressed keratinocyte migration in vitro. The application of OSU8-1 to wound sites in mice greatly retarded reepithelialization as the result of a failure in keratinocyte migration, but this effect could be overcome if recombinant soluble HB-EGF was added along with OSU8-1. These findings indicate that the shedding of EGFR ligands represents a critical event in keratinocyte migration, and suggest their possible use as an effective clinical treatment in the early phases of wound healing.

Pulmonary stenosis in recipient twins in twin-to-twin transfusion syndrome: report on 3 cases and review of literature.

This report describes 3 cases of pulmonary stenosis in the recipient twin in twin-twin transfusion syndrome. Fetal echocardiography showed cardiomegaly, tricuspid valve regurgitation, and increased reverse flow in the inferior vena cava, as signs of congestive heart failure in all 3 cases. We diagnosed 2 cases of pulmonary stenosis by fetal echocardiography prenatally and confirmed our findings in all 3 cases postnatally. Two cases underwent postnatal balloon valvuloplasty to release the pulmonary valvular stenosis in neonatal period. The third one died soon after delivery and autopsy showed a slightly thickened pulmonary valve. One of the cases was diagnosed in the early second trimester (20 weeks of pregnancy), the earliest detection of fetal pulmonary stenosis reported in literature. The presence of high peak velocity of the pulmonary artery at 20 weeks of pregnancy preceded the development of pulmonary stenosis in this case. This supports the hypothesis that alterations in fetal hemodynamics may result in structural cardiac abnormality.

Localization of CD9, an enhancer protein for proheparin-binding epidermal growth factor-like growth factor, in human atherosclerotic plaques: possible involvement of juxtacrine growth mechanism on smooth muscle cell proliferation.

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), a member of the EGF family, has a potent mitogenic activity for vascular smooth muscle cells (SMCs). We previously reported that HB-EGF is involved in atherogenesis of human aorta and coronary arteries. ProHB-EGF (the membrane-anchored form of HB-EGF) has also been demonstrated to possess a mitogenic activity, which is approximately 30-fold increased when coexpressed with CD9 in mouse L cells. Thus, in the process of atherogenesis, CD9 may be involved in the proliferation of SMCs. We immunohistochemically investigated the localization of CD9 and proHB-EGF in the human aorta and coronary arteries. In normal aorta and coronary arteries, CD9 immunostaining was virtually negative, whereas proHB-EGF immunostaining was positive, especially in the arteries of babies. In contrast, in atherosclerotic lesions, some intimal SMCs were strongly positive for CD9 and proHB-EGF immunostaining. The juxtacrine growth activities of human aortic SMCs were inhibited in vitro by adding neutralization antibodies for CD9 or adding the specific inhibitor of HB-EGF. Besides, coexpressed CD9 and proHB-EGF cells markedly incorporated [(3)H]thymidine into the SMCs. CD9 is localized immunohistochemically in the SMCs of the atherosclerotic aorta and coronary arteries. CD9, when coexpressed with proHB-EGF, enhances proHB-EGF activities for SMC growth in a so-called juxtacrine manner in vitro and may be involved in atherogenesis.

Purification and some properties of a tetrodotoxin binding protein from the blood plasma of kusafugu, Takifugu niphobles.

A tetrodotoxin binding protein has been purified from the plasma of the puffer fish kusafugu, Takifugu niphobles, through DEAE-cellulose treatment, ammonium sulfate fractionation, Sephadex gelfiltrations and Sephacryl S-200 and Cellulofine A-500 column chromatography. Final purification by HPLC on a TSK G-3000 SL column yielded a protein which showed only a single protein peak. The molecular weight of the protein was estimated to be 116,000 and 91,000 by SDS-PAGE and mass spectrometry, respectively. A blast search on the amino-terminal amino acid sequence of the purified protein revealed that the protein had no homology to any other protein on data base.

Morphological and functional restoration of parietal cells in helicobacter pylori associated enlarged fold gastritis after eradication.

BACKGROUND/AIM: Helicobacter pylori infections are associated with hypochlorhydria in patients with pangastritis. It has previously been shown that eradication of H pylori leads to an increase in acid secretion in H pylori associated enlarged fold gastritis, suggesting that H pylori infection affects parietal cell function in the gastric body. The aim of this study was to evaluate the effects of H pylori infection on parietal cell morphology and function in hypochlorhydric patients. PATIENTS/METHODS: The presence of H pylori infection, mucosal length, and inflammatory infiltration were investigated in six patients with enlarged fold gastritis and 12 patients without enlarged folds. Parietal cell morphology was examined by immunohistochemistry using an antibody against the alpha subunit of H(+),K(+)-ATPase and electron microscopy. In addition, gastric acid secretion and fasting serum gastrin concentration were determined before and after the eradication of H pylori. RESULTS: In the H pylori positive patients with enlarged fold gastritis, fold width, foveolar length, and inflammatory infiltration were increased. In addition, the immunostaining pattern of H(+), K(+)-ATPase was less uniform, and the percentage of altered parietal cells showing dilated canaliculi with vacuole-like structures and few short microvilli was greatly increased compared with that in H pylori positive patients without enlarged folds. After eradication, fold width, foveolar length, and inflammatory infiltrates decreased and nearly all parietal cells were restored to normal morphology. On the other hand, altered parietal cells were negligible in H pylori negative patients. In addition, the basal acid output and tetragastrin stimulated maximal acid output increased significantly from 0.5 (0.5) to 4.1 (1.5) mmol/h and from 2.5 (1.2) to 13.8 (0.7) mmol/h (p<0.01), and fasting serum gastrin concentrations decreased significantly from 213.5 (31.6) to 70.2 (7.5) pg/ml (p<0.01) after eradication in patients with enlarged fold gastritis. CONCLUSION: The morphological changes in parietal cells associated with H pylori infection may be functionally associated with the inhibition of acid secretion seen in patients with enlarged fold gastritis.

Immunohistochemical localization of betacellulin, a new member of the EGF family, in normal human pancreas and islet tumor cells.

Betacellulin (BTC) purified from mouse beta cell tumor (betaTC-3) is a new member of the epidermal growth factor (EGF) family which can bind receptor tyrosine kinase, EGF receptor (erbB1) and erbB4. It has been demonstrated that proBTC mRNA was abundantly expressed in human pancreas tissue, and that BTC converted amylase-secreting rat acinar cell line (AR42J) into insulin-secreting cells, suggesting that BTC might be important for the growth and/or differentiation of islet cells. However, the cell type producing BTC in the pancreas has not been clarified. In this study, we examined the localization of BTC in human pancreas and islet cell tumors. Immunohistochemistry using specific antibodies to human BTC revealed that this protein was produced in alpha cells and duct cells, and probably in beta cells in normal adult pancreas. Furthermore, strong immunoreactivity to BTC was detected in primitive duct cells of the fetal pancreas, and both insulinoma and glucagonoma cells also showed positive immunoreactivity to BTC. EGF receptor (erbB1) and erbB4 were expressed mainly in islet and duct cells, and duct cells, respectively. These results demonstrate the localization of BTC and its receptors, and suggest that BTC may be one of the factors that have physiologically important roles such as growth and differentiation of islet cells in the human pancreas.

Demonstration of two different processes of beta-cell regeneration in a new diabetic mouse model induced by selective perfusion of alloxan.

To clarify the regeneration process of pancreatic beta-cells, we established a new mouse model of diabetes induced by selective perfusion of alloxan after clamping the superior mesenteric artery. In this model, diabetes could be induced by the destruction of beta-cells in alloxan-perfused segments, while beta-cells in nonperfused segments were spared. Intraperitoneal glucose tolerance tests showed glucose intolerance, which gradually ameliorated and was completely normalized in 1 year with a concomitant increase of insulin content in the pancreas. Histological examination showed neo-islet formation in the alloxan-perfused segment and the proliferation of spared beta-cells in the nonperfused segment. In the alloxan-perfused segment, despite a marked reduction of islets in size and number at an early stage, both the number of islets, including islet-like cell clusters (ICCs), and the relative islet area significantly increased at a later stage. Increased single beta-cells and ICCs were located in close contact with duct cell lining, suggesting that they differentiated from duct cells and that such extra-islet precursor cells may be important for beta-cell regeneration in beta-cell-depleted segment. In addition to beta-cells, some nonhormone cells in ICCs were positive for nuclear insulin promoter factor 1, which indicated that most, if not all, nonhormone cells positive for this factor were beta-cell precursors. In the nonperfused segment, the islet area increased significantly, and the highest 5-bromo-2-deoxyuridine-labeling index in beta-cells was observed at day 5, while the number of islets did not increase significantly. This indicated that the regeneration of islet endocrine cells occurs mostly through the proliferation of preexisting intra-islet beta-cells in the nonperfused segment. In conclusion, the regeneration process of beta-cells varied by circumstance. Our mouse model is useful for studying the mechanism of regeneration, since differentiation and proliferation could be analyzed separately in one pancreas.

Ring chromosome 20 and nonconvulsive status epilepticus. A new epileptic syndrome.

Six cases of epilepsy associated with ring chromosome 20 are presented. The study of these cases and 20 cases reported in the literature revealed that they constitute a distinct epileptic syndrome: frequent seizures consisting of a prolonged confusional state, with or without additional motor seizures, and an ictal EEG pattern of long-lasting bilateral paroxysmal high-voltage slow waves with occasional spikes. Neurological examination results were normal, and neuroimaging studies often failed to disclose a brain lesion. The seizures were resistant to antiepileptic drug therapy. Comparison of the electroclinical features of nonconvulsive status epilepticus in six patients with and four patients without ring chromosome 20 revealed that the group with the chromosomal anomaly had more frequent, comparatively brief episodes of confusion associated with a less prominent spike component on the EEG. We propose that epilepsy associated with ring chromosome 20 constitutes a new syndrome that may provide an opportunity to scrutinize a genetic mechanism of epilepsy.

Localization of heparin-binding epidermal growth factor-like growth factor in human coronary arteries. Possible roles of HB-EGF in the formation of coronary atherosclerosis.

BACKGROUND: Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a newly identified member of the EGF family. Our previous in vitro studies showed that HB-EGF is a potent mitogen and chemoattractant for vascular smooth muscle cells (SMCs), suggesting the role of HB-EGF in the pathogenesis of atherosclerosis. The purposes of the present study were to investigate the localization of HB-EGF in both normal and atherosclerotic human coronary arteries and to elucidate the possible roles of this growth factor in the formation of atherosclerotic lesions. METHODS AND RESULTS: The immunohistochemical localization of HB-EGF, SMCs, macrophages, and EGF receptors (EGFRs) was examined in human coronary arteries obtained at autopsy. The medial SMCs of coronary arteries in neonates, infants, and children consistently synthesized HB-EGF protein. In normal adults, however, the relative number of HB-EGF-positive medial SMCs decreased gradually with age after about 30 years of age. In nonatherosclerotic coronary arteries with diffuse intimal thickening, SMCs of the intima, especially those located in the area of the medial side of the intima, were strongly positive for HB-EGF protein. In atherosclerotic plaques of coronary arteries with eccentric intimal thickening, both SMCs and macrophages in and around the core lesions, in addition to the intimal and medial SMCs located adjacent to the plaque, produced HB-EGF protein. A strong immunostaining of EGFRs was observed in these SMCs, suggesting a close association of HB-EGF and EGFR expression. CONCLUSIONS: These data suggest that HB-EGF might play important roles in the migration of SMCs from the media to the intima, the proliferation of intimal SMCs, and the interaction between SMCs and macrophages in the process of coronary atherogenesis.

Expression of heparin-binding epidermal growth factor-like growth factor in neointimal cells induced by balloon injury in rat carotid arteries.

Balloon catheter injury of rat carotid arteries induces migration and proliferation of smooth muscle cells (SMCs), with subsequent neointimal formation. Several growth factors, such as platelet-derived growth factor and basic fibroblast growth factor, have been shown to be involved in this process, but the mechanisms that modulate the growth and/or migratory properties of SMCs remain unclear. In this study, we investigated whether heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is known to be a potent SMC stimulator from in vitro study, is associated with the proliferative response of SMCs to arterial injury. Northern blot analysis showed that the transcript levels of HB-EGF increased rapidly approximately 12-fold within 2 hours after injury and declined by 2 days but remained 3-fold at 14 days. In situ hybridization analysis demonstrated that the transcript of HB-EGF remained strongly expressed in the neointima, especially near the luminal surface, at 14 days after injury. Immunohistochemical staining showed that HB-EGF protein was positive in the endothelium and only faintly visible in medial SMCs in uninjured vessels. In contrast, 2 days after injury, positive HB-EGF immunostaining was detected in the medial SMCs along the luminal surface. At 7 days, the neointimal SMCs exhibited strong immunostaining for HB-EGF, and at 14 days, they exhibited a gradient of HB-EGF expression with strong immunoreactivity in the most luminal cells. SMCs labeled with 5-bromo-2'-deoxyuridine in their nuclei showed strong immunostaining for HB-EGF protein. Furthermore, the epidermal growth factor receptor to which HB-EGF can bind was also immunostained positively in neointimal SMCs. These data suggest that HB-EGF may play an important role of the proliferation and migration of SMCs in the process of neointimal accumulation induced by arterial injury, probably in an autocrine, paracrine, and/or juxtacrine manner.

Estimation of the degree of acclimatization to high altitude by a rapid and simple physiological examination.

The recent expansion in the geographical areas open to human activity has made it desirable to have an objective method to evaluate the degree of high-altitude acclimatization. In this study, we measured the arterial oxygen saturation value at rest and just after exercise in healthy high-altitude trekkers using a transportable pulse oximeter. During a 100-day stay at high altitude (around 4000 m), the degree of arterial hemoglobin saturation measured at rest was relatively stable. However, shortly after arrival at high altitude, even light exercise induced an acute reduction in the degree of arterial hemoglobin saturation; this reduction was ameliorated as the trekkers became acclimatized to the high altitude. Preliminary short trekking to high altitudes does not appear sufficient to induce this response. It is suggested that this rapid and simple physiological examination, the measurement of arterial oxygen saturation value after light exercise, could be a convenient means of estimating the level of high-altitude acclimatization among healthy subjects.

Detection of donor lymphocytes in the cerebrospinal fluid of a patient with metachromatic leukodystrophy following bone marrow transplantation.

An unrelated donor bone marrow transplant (UD-BMT) was carried out on a 10-year-old patient with metachromatic leukodystrophy (MLD). We collected cerebrospinal fluid (CSF) on day +168 and cultured it with recombinant IL-2 and PHA-P to examine the origin of cells in the CSF. Analysis on variable number of tandem repeat (VNTR) of lymphocytes in the CSF amplified by PCR revealed that lymphocytes in the CSF were of donor origin. These data support that BMT at an early stage may prevent deterioration in MLD. Although the patient developed grade III acute GVHD with rash and diarrhea, we successfully treated acute GVHD using rabbit anti-human thymocyte immunoglobulin (ATG). UD-BMT may be an alternative treatment for patients with MLD in the absence of an HLA matched family donor.

Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

Occurrence of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus).

The shore crab (Hemigrapsus sanguineus) is highly resistant to tetrodotoxin (TTX) although it contains no detectable amount of TTX (less than 5 MU/g, where 1 MU is defined as the amount of TTX killing a 20 g mouse in 30 min). Its body fluid was examined for neutralizing effects against the lethal activity of TTX. When the mixture of the body fluid and TTX was injected i.p. into mice, the lethal activity of TTX was significantly reduced; 1 ml of the body fluid was evaluated to neutralize 3.6-4.0 MU of TTX. Higher neutralizing activity (7.2-12.5 MU/ml of the body fluid) was exhibited by i.v. administration of the body fluid into mice before or after i.p. challenge of TTX. The lethal effect of paralytic shellfish poisons was not counteracted by the body fluid. Analysis by gel filtration on Sepharose 6B revealed that the body fluid contained TTX-binding high mol. wt substances (> 2,000,000) responsible for the neutralizing activity of the body fluid against TTX, which accounts for the high resistibility of the crab to TTX. When the crude toxin extracted from the liver of puffer (Takifugu niphobles) was mixed with the body fluid and chromatographed on Sepharose 6B, almost pure TTX was obtained from the fractions containing the TTX-binding high mol. wt substances, suggesting that the TTX-binding high mol. wt substances could be useful in purification of TTX from biological samples.