Biomedical subjects
T Toyota
Publications and source records attributed to T Toyota.
[A case of reflux esophagitis after eradication of Helicobacter pylori].
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Different susceptibility of mice to immune-mediated cholangitis induced by immunization with carbonic anhydrase II.
Carbonic anhydrase II (CA-II), an enzyme that catalyzes hydration of carbon dioxide to bicarbonate and hydrogen ions, is located exclusively in cholangiocytes in the liver. Recently, patients with autoimmune cholangitis have been reported to have serum antibodies to CA-II. Moreover, active immunization of susceptible mice with CA-II results in inflammation of submandibular glands, where CA-II is also expressed. In the present study, we attempted to produce cholangitis by immunization with CA-II using two strains of mice with different potential susceptibilities. Balb/c and DBA/1J mice were immunized with a dose of human CA-II (100 microg) intraperitoneally every other week on three occasions. One week after the final immunization, mice were killed and blood and tissue samples harvested. Light and electron microscopic evaluation for inflammation was performed under coded identification. After immunization of Balb/c mice, numerous mononuclear cells, mostly CD4-positive T cells, appeared around bile ducts; lymphocyte invasion between cholangiocytes was also seen. Inflammation was not observed outside the liver. Morphologic evidence of cholangitis was observed in 8 (53.3%) of 15 Balb/c mice and in 3 (20%) of 15 DBA/1 J mice. In the control mice immunized with bovine serum albumin (BSA), cholangitis was observed in only 1 (6.7%) of 15 Balb/c mice and none of 15 DBA/1J mice. Balb/c mice immunized with CA-II had statistically significant cholangitis compared with those immunized with BSA (p < 0.01), whereas DBA/1J did not show a significant difference from controls. Balb/c mice immunized with CA-II showed specific antibody production after immunization, whereas DBA/1J mice immunized with CA-II had anti-CA-II antibody even in preimmune sera. Adoptive transfer of splenocytes from CA-II-immunized Balb/c mice resulted in cholangitis in two (66.7%) of three Balb/c recipients. These data strongly suggest that the cholangitis can be induced by CA-II immunization in susceptible strains of mice.
[Immunohistochemical studies in depressed type adenomas of the stomach--comparison with protruded type adenomas and depressed type well differentiated mucosal gastric cancers].
In this study, we performed immunohistochemical studies using p53 and Ki-67 immunostaining in 48 depressed type adenomas, and compared with protruded type adenomas (n = 28) and depressed type well differentiated mucosal gastric cancers (mcancer: n = 46). Focal and diffuse p53 staining patterns were not noticed in depressed type adenomas and in protruded type adenomas, but were noticed in mcancers (focal: 4.3%, diffuse: 41.3%). Ki-67 positive cells were noticed in surface epithelium in 24.4% of depressed type adenomas, in 21.4% of protruded type adenomas and in 87.0% of mcancers. Ki-67 positive rate in foveolar epithelium (superior, middle, inferior) were noticed in (31.1%, 12.9%, 6.1%) of depressed type adenomas, in (33.4%, 15.4%, 6.9%) of protruded type adenomas and in (33.4%, 31.4%, 20.4%) of mcancers. These results suggest that from the point of immunohistochemistry, depressed type adenomas were similar to protruded type adenomas, but were different from mcancers.
[Kasabach-Merrit syndrome].
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Clinical studies of 13C-urea breath test in Japan.
The urea breath test (UBT) using isotope carbon is a non-invasive method for diagnosing H. pylori infection. In Japan, only 13C-UBT using a stable isotope is available, since the clinical use of radioactive isotope 14C-compounds is prohibited. Since there is no worldwide standard protocol for the 13C-UBT, it is therefore important to standardize this test to obtain a cheap, easy, reproducible method. Factors affecting the outcome of 13C-UBT values include sampling times and frequency, the amount of 13C-urea, the cut-off value, the avoidance of oral urease activity, and changes in gastric emptying times and 13C-urea distribution. Mouth rinsing reduces false-positive results due to oral urease activity, particularly within 20 min after ingestion of 13C-urea. A Japanese multicenter trial has shown that the diagnostic utility of 13C-UBT is superb, with the cut-off value 2.5/1000 in 20-min samples after ingestion of 100 mg 13C-urea. The 13C-UBT is a very useful method for diagnosing H. pylori infection.
Duct formation by immortalized mouse cholangiocytes: an in vitro model for cholangiopathies.
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Transgenic copper/zinc-superoxide dismutase ameliorates caerulein-induced pancreatitis in mice.
The role of oxidative stress in acute pancreatitis was investigated by comparing the pathological features of caerulein pancreatitis between transgenic mice that overexpress human Cu/Zn-superoxide dismutase (SOD) and nontransgenic littermates. Both the elevation of serum amylase and the formation of pancreatic edema during the pancreatitis were significantly reduced in the transgenic mice compared with the nontransgenic littermates. In the transgenic mice, the pancreatitis-associated reduction of Cu/Zn-SOD activity in the pancreatic tissues was significantly smaller than that in the nontransgenic mice. These results provide direct evidence that the elevation of intracellular oxygen radicals is an important factor for the progress of acute edematous pancreatitis.
Ultrastructural and confocal laser scanning microscopic examination of TUNEL-positive cells.
TdT-mediated dUTP-biotin nick end labelling (TUNEL) has been widely used for detecting cells with DNA fragmentation or apoptotic cells. However, since the concept of apoptosis is based on cellular ultrastructure, it is important to identify the morphological features of TUNEL-positive cells. In this study, we performed TUNEL and electron microscopic observation on serial semithin and ultrathin sections of pancreas from bilaterally adrenalectomized rats with caerulein-induced pancreatitis. TUNEL-positive cells were identified with two different ultrastructural patterns. One was characteristic of apoptosis, with condensed nuclei, intact mitochondria, and zymogen granules. The other pattern was one of marked cellular degeneration, possibly representing the end stage of cell death. Cells which did not demonstrate these ultrastructural patterns were not labelled by the TUNEL method. The three-dimensional structure of TUNEL-positive cells was also investigated by confocal laser scanning microscopy (CLSM), which showed the apoptotic nuclei exhibited various three-dimensional structures. These results confirm the utility of the TUNEL method in detecting apoptosis; application of the technique reported in this study will contribute to the further characterization of individual TUNEL-positive cells.
Effect of long-term treatment with complete Freund's adjuvant on KK-Ay mouse, a model of non-insulin-dependent diabetes mellitus.
Nonspecific stimulation with immunoadjuvants significantly improves glucose tolerance in animal models for non-insulin-dependent diabetes mellitus (NIDDM). In this study, we observed the effect of long-term treatment with complete Freund's adjuvant (CFA) on serum factors and histology of various organs in KK-Ay mice with NIDDM. The mice were injected with CFA weekly or every 2 weeks for 12 weeks. Glucose tolerance was significantly improved in the CFA-treated mice throughout the experiment. At the end of the experiment, hypertriglyceridemia was significantly reduced, but serum total protein, glutamic pyruvic transaminase, creatinine, and nonfasting insulin levels were not changed by the treatment. Fatty change of the liver and index of glomerular lesions of the kidney were significantly inhibited in the CFA-treated mice, whereas the pancreatic islet morphology remained unchanged. No toxic effect was observed by the CFA treatment. These results imply that the novel treatment with CFA could control NIDDM and inhibit the development of diabetic glomerular lesions in KK-Ay mice.
Relationship between glucocorticoid receptor and response to glucocorticoid therapy in ulcerative colitis.
PURPOSE: To clarify the relationship between the glucocorticoid receptor and the effectiveness of glucocorticoid therapy in patients with ulcerative colitis, we investigated the number and apparent dissociation constant of glucocorticoid receptor in peripheral blood mononuclear leukocytes of patients with ulcerative colitis. MATERIALS AND METHODS: Eleven patients with ulcerative colitis (5 who responded to intravenous glucocorticoids and 6 who did not) and ten control subjects were studied. The number and apparent dissociation constant of glucocorticoid receptor were measured using a whole-cell binding assay. Results were expressed as a median (interquartile range). RESULTS: The number of glucocorticoid receptors from the six nonresponders, five responders, and ten healthy controls were 4922 (range, 4484-5643), 3413 (range, 3183-4450), and 3610 (range, 2594-3979) binding sites/cell, respectively. The apparent dissociation constant of the glucocorticoid receptors from the nonresponders, responders, and healthy controls were 7.03 (range, 5.66-10), 4.27 (range, 4-5.13), and 6.18 (range, 5.86-6.74) nM, respectively. Nonresponders had a significant increase both in the number of binding sites and in the apparent dissociation constant compared with responders (P = 0.045; P = 0.029). CONCLUSIONS: The increased number and apparent dissociation constant of glucocorticoid receptor are closely associated with the effectiveness of glucocorticoid therapy. The measurement of the number and apparent dissociation constant of glucocorticoid receptor may be useful in predicting response to glucocorticoids.
Gallbladder emptying and cholecystokinin and pancreatic polypeptide responses to a liquid meal in patients with diabetes mellitus.
To investigate gallbladder motility and its regulation in patients with diabetes mellitus (DM), we examined the gallbladder response to an intraduodenal test meal by measuring the temporal course of plasma cholecystokinin (CCK) and pancreatic polypeptide (PP) levels. Eighteen patients with type 2 DM and 7 healthy subjects (controls) were enrolled. The gallbladder volume was calculated by the sum-of-cylinders method from ultrasonographic images, and plasma CCK and PP were measured by radioimmunoassays. No significant difference was found in either the gallbladder response or in the CCK response between patients with DM and controls. However, the fasting plasma PP level of patients with DM was more than tenfold higher than that of controls. The integrated PP response (IPPR) of patients with DM to the test meal was 8.3-fold higher than that of controls. When patients with DM were grouped according to whether they had been treated with insulin or not, the fasting plasma PP of patients with DM without insulin treatment was significantly higher than the level in those treated with insulin. These results suggest that overproduction of PP-like immunoreactive substance(s) may occur in patients with DM, but the high plasma PP immunoreactivity does not appear to be related to the fasting gallbladder volume or to gallbladder emptying and filling.
Gastric mucosal blood flow response to stress in streptozotocin diabetic rats: regulatory role of nitric oxide.
To investigate cytoprotection against mucosal injuries of the stomach in patients with diabetes, we investigated gastric mucosal blood flow (GMBF), its response to a burn stress, and the involvement of nitric oxide (NO) in streptozotocin (STZ) diabetic rats. GMBF was measured by laser-Doppler velocimetry (LDV) and by the hydrogen gas clearance technique (HGC). The steady-state GMBF of STZ rats decreased according to the duration of diabetes, and insulin treatment blocked this decrease. Burn stress caused a rapid decrease in the GMBF. Reduction of the GMBF and gastric mucosal leakage of Evans blue (EB) after the burn stress were greater in the STZ rats than in the controls, but insulin treatment completely blocked this increase in EB leakage in the STZ rats. There was a significant negative correlation between the percent GMBF 3 h after the burn stress and EB leakage at the same time point. In the controls and the insulin-treated STZ rats, N-nitro-L-arginine (L-NNA), an NO synthase inhibitor, enhanced the decrease in postburn GMBF and EB leakage, but was without effect in the STZ rats. These results suggest that NO may be involved in the regulation of GMBF, and that persistent hyperglycemia may impair this regulation. These findings suggest that patients with diabetes have reduced cytoprotection against a variety of gastric mucosal injuries.
Angiotensin converting enzyme inhibitors suppress production of tumor necrosis factor-alpha in vitro and in vivo.
It has been reported that angiotensin converting enzyme (ACE) inhibitors have beneficial effects on insulin resistance and congestive heart failure, in which elevations of serum tumor necrosis factor-alpha (TNF-alpha) level have been indicated. Therefore, in this study, we examined effect of ACE inhibitors on TNF-alpha production both in vitro and in vivo by using human blood mononuclear cells and mice, respectively. LPS (20 micrograms/ml)-induced in vitro TNF-alpha production, measured by bioassay and enzyme-linked immunosorbent assay, was significantly inhibited with captopril, delapril and cilazapril in a concentration of 10(-3) mol/l. A single, oral administration of captopril, delapril and cilazapril at more than 10-fold doses of common clinical use in man significantly inhibited LPS (2 mg/kg)-induced serum TNF-alpha activity in Balb/c mice. These results indicate that ACE inhibitors such as captopril, delapril and cilazapril have an inhibitory effect on TNF-alpha production not only in vitro as previously reported, but also in vivo, although relatively high concentrations and large doses were required in this study.
Inhibitory effect of nicotinamide on in vitro and in vivo production of tumor necrosis factor-alpha.
Nicotinamide, a pellagra-preventive factor, has multiple functions such as inhibition of poly-ADP-ribose synthetase, inhibition of inducible nitric oxide synthase, free radical scavenging and suppression of major histocompatibility complex class II expression and ICAM-1 expression on endothelial cells. In addition to these, we have found an inhibitory effect of nicotinamide on production of tumor necrosis factor-alpha (TNF-alpha) in vitro and in vivo. Lipopolysaccharide (LPS)-induced in vitro TNF-alpha production by human peripheral blood mononuclear cells, measured by enzyme-linked immunosorbent assay (ELISA), was significantly inhibited with more than 1 x 10(-3) mol/l of nicotinamide, while interleukin-1-beta was not inhibited and interleukin-6 was slightly inhibited even with 10(-2) mol/l. Oral administration of nicotinamide with more than 62.5 mg/kg also significantly inhibited LPS-induced serum TNF-alpha production measured by ELISA and bioassay in Balb/c mice. Thus, nicotinamide has an inhibitory effect on TNF-alpha production that may be beneficial to TNF-alpha-mediated diseases.
Immunohistochemical studies on EGF family growth factors in normal and ulcerated human gastric mucosa.
Expression of members of the epidermal growth factor family, including epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), amphiregulin (AR), and Cripto, as well as their putative receptor, epidermal growth factor receptor (EGFR), was studied immunohistochemically in human gastric mucosa to evaluate their possible roles in cell proliferation of normal and regenerative gastric mucosa. We also examined the correlation betwen cell proliferation and EGFR by double immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and EGFR. In normal gastric mucosa, TGF-alpha, Cripto, and AR immunoreactivities were observed in the surface epithelial and parietal cells of gastric fundic glands, respectively. EGF immunoreactivity was not observed in any of normal mucosa examined. EGFR immunoreactivity was detected on foveolar cells in proliferative zones and in parietal cells. Double immunostaining revealed that EGFR immunoreactivity was distributed much more widely than PCNA immunoreactivity. PCNA positive epithelial cells adjacent to gastric ulcer margin expressed relatively intense EGFR but did not express any of the growth factors examined. On the other hand, relatively intense immunoreactivity of both TGF-alpha and Cripto was detected in PCNA-negative regenerative epithelium located distant from gastric ulcer margin. Relative immunoreactivity of AR in regenerative gastric epithelium associated with ulcer was not different from that in normal gastric mucosa. TGF-alpha, AR, and Cripto are considered to play important roles in normal gastric mucosal proliferation, and TGF-alpha and Cripto may be involved in ulcer healing, possibly via a paracrine mechanism.
Accessory cell function of a human colonic epithelial cell line HT-29 for bacterial superantigens.
The expression and up-regulation of cell adhesion molecules on a human colonic epithelial cell line HT-29, and the peripheral blood T lymphocyte proliferation responses to bacterial superantigens presented by this cell line were investigated, compared with peripheral blood monocytes. In HT-29 cells, there was constitutive expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3) at a low level, but no constitutive expression of HLA-DR, LFA-1, B7-1 and B7-2 molecules. After stimulation with the supernatants of staphylococcal enterotoxin B (SEB)-stimulated peripheral blood mononuclear cells for 48 h, there was significant up-regulation of HLA-DR and ICAM-1 molecules (both > 90% positive). However, this stimulation had no effect on the expression of LFA-1, B7-1, B7-2 and LFA-3 molecules. In the presence of all tested superantigens SEB, toxic shock syndrome toxin-1, and streptococcal pyogenic exotoxin A, stimulated HT-29 cells caused significant T cell proliferation. When monocytes were used as antigen-presenting cells (APC), the MoAbs against HLA-DR, B7-2 and LFA-3 showed a significant inhibition of SEB-induced T cell proliferation. Anti-ICAM-1 MoAb had no effect on this response. On the other hand, when stimulated HT-29 cells were used as APC, the MoAbs against HLA-DR and ICAM-1 significantly inhibited SEB-induced T cell proliferation. In contrast to monocytes, anti-B7-2 and anti-LFA-3 had no effect on this response. SEB could not induce HT-29 cells to produce IL-8 directly; however, SEB significantly induced the stimulated HT-29 cells to produce IL-8 in the presence of T cells. Thus these data demonstrate that the products of superantigen-stimulated T cell activation can increase the expression of HLA-DR and ICAM-1 molecules on HT-29 cells significantly. Stimulated HT-29 cells can serve as APC to bacterial superantigens. This response is an HLA-DR- and ICAM-1-dependent, but B7-2- and LFA-3-independent process, which was different from professional APC monocytes.
The lobular architecture of the normal human pancreas: a computer-assisted three-dimensional reconstruction study.
Tissue specimens of the normal pancreata from three adult patients were subjected to serial sectioning and computer-assisted three-dimensional reconstruction of the lobules, blood vessels, pancreatic ducts, and islets, to establish the anatomical relation among these structures. Most lobules, although more or less imperfectly demarcated by septa, were found to receive a single duct (lobular duct), justifying their definition as "glandular lobules." However, there were also lobules receiving not only a lobular duct of their own but a small accessory duct from a neighboring lobule. On the other hand, these lobules were shown not to correspond to the territories of arterial supply, leaving no ground for defining them as "vascular lobules."