To evaluate correctly the proliferative activity of tumor cells, it is necessary to clarify not only S-phase fraction but also the growth fraction of tumor tissues. We used combined BrdU and DNA polymerase alpha (pol-α) immunohistochemistry to gastric biopsy specimens, and analyzed the proliferation of the neoplastic lesions of various degrees of malignancy. The results were as follows: The distribution of pol-α positive cells were almost the same as that of BrdU positive cells, but the percentage of pol-α positive cells was higher than that of BrdU positive cells irrespective of the mucosal specimens. In the adenomas, both BrdU and pol-α positive cells distributed generally superficially in the mucosal layer. In the well differentiated adenocarcinomas, both BrdU and pol-α positive cells distributed diffusely in the deeper layer of the mucosa. The ratio of the number of BrdU positive cells to that of pol-α positive cells, which means the S-phase fraction in the growth fraction, was higher in the tumor and that higher in the well differentiated adenocarcinomas than that of the adenomas. In conclusion, the combined BrdU and pol-α immunohistochemistry in gastric biopsy specimens are useful to evaluate the degree of malignancy.
A procedure for the detection and quantification of Helicobacter pylori in gastrointestinal tissue biopsy specimens by polymerase chain reaction (PCR) is presented. This method provides an accurate quantitative and sensitive measurement of the amount of H. pylori in the gastrointestinal tract without cultivation of this microorganism. We have used 30 cycles of PCR in the presence of 3.5mM Mg++ and demonstrated that the DNA content of one H. pylori cell is 0.0076pg. Using this approach, we analyzed samples of gastrointestinal tissue biopsies from 10 patients with various gastrointestinal disorder. Each of these patients had detectable H. pylori at levels ranging from 0.10 to 60.61 cells for each tissue cell. This new technique thus provides a useful way to detect H. pylori in gastrointestinal tissue biopsy specimens.
For the purpuse of studing pepsinogen secretion from gastric chief cells, we established a monolayer culture system of guinea pig chief cells and an enzyme immunoassay (ETA) system specific for guinea pig pepsinogen. Dispersed chief cells were obtained from gastric mucosa of a guinea pig using collagenase, GEDTA, and Percoll solution, suspended in DMEM/F-12 (1/1 containing 10%FCS) media, and cultured for 70hr. Then the monolayer culture system was established. Pepsinogen was purified from gastric mucosa of a guinea pig using DEAE-Sephacel and Sephacryl S-200 columns. Antibody to pepsinogen was raised by immunizing rabbit with the purified pepsinogen. A two-site EIA system was then established using β-galactosidase-labeled Fab' antibody. The EIA system showed sensitivity to measure above 1.5ng of guinea pig pepsinogen, and the monolayer culture system responded well to secretagogues. These systems are useful for studing pepsinogen secretion.
The effect of PGE1 and PGF2α in the ischemic intestinal tract were examined. In 40 mongrel dogs, we studied ischemic models of small intestine. PGE1 or PGF2α was injected into the anterior mesenteric artery after reperfusion according to each occulusion time, and the tissue blood flow was measured on both mucosal and serous sides of small intestinal loop by laser flowmeter to examine the relation to the extent of tissue damage. Tissue blood flow of the ischemic intestine after the injection of PGE1 increased by 148-208% in the 3-5hr occulusion group and by 86-110% in the 7-10hr occulusion group. Tissue blood flow after the injection of PGF2α decreased by 39-59% in the 3-5hr occulusion group and by 1-15% in the 7-10hr occulusion group. These results indicate that the effect of PGE1 and PGF2α in the ischemic intestine would be available up to 3-5hr of ischemia. Histological examination revealed that viability of the remaining crypt was high in the PGE1 injection group but low in the PGF2α injection group. These findings suggest that PGE1, if try at the early stage, would be effective for the treatment of ischemic lesion.
In this study SASP metabolite levels were measured in colonic mucosal specimens and plasma samples from 31 patients with ulcerative colitis (UC) under treatment with the drug. Colonic tissue specimens were obtained by endoscopically guided biopsy and plasma was isolated from peripheral blood. Measurements were performed by HPLC according to the procedure of Fischer et al. The levels of 5-ASA and SP in either of colonic tissue or plasma were significantly lower than those of Ac-5-ASA and Ac-SP, respectively. The tissue level of 5-ASA had a significant correlation to the dosage of 5-ASA. The tissue levels of 5-ASA and Ac-5-ASA were low in an active stage of UC than during a remission period and the difference observed with respect to the latter metabolite was significant. These findings suggest that acetylation of 5-ASA is inhibited in the colonic mucosa in an active stage of the disease.
To elucidate the long-term outcome of the ulcerative colitis we analyzed 124 patients suffering for more than 10 years concerning the long-term prognosis, cumulative rate of colectomy, cumulative risk of cancer development and cumulative survival rate. Long-term prognosis were divided to three categories as good, fair and poor, according to present disease activities, social availability and subjective judgement. One hundred and twenty four patients were divided to 66 patients with good prognosis, 27 fair and 31 poor (2:1:1). The rate of patients having active disease at each year were followed-up annually for 10 years according to final three categories of the prognosis. The serial curve of annual rate of having active disease of the patients with poor prognosis was the highest, fair prognosis the second, good prognosis the lowest. The factors affecting the final prognosis were the period before initiation of proper treatment, the severity of the first attack or the elder age (>40 year old) at the first attack. Twenty six patients were undergone colectomy in relation to ulcerative colitis. The cumulative colectomy rate was 16.5% at 10 years and 38.5% at 15 years after the onset. Malignant disease or severe dysplasia of the colon developed in 4 patients 10 to 14 years after the onset. Cumulative survival rate was equal to the expected survival curve during 16 years. The main causes of death were intracranial hemorrhage and colorectal malignant disease.
The Distribution pattern and proportion of the positive cells of proliferating cell nuclear antigen (PCNA) were examined immunohistochemically to study the proliferative activities of 33 cases of carcinoma found at the confluence of the main hepatic ducts. Expressions of CEA, CA19-9 and EGF receptor (R) were further examined with serial histological sections and comparatively analyzed with the result of PCNA staining and the clinico-pathological features of the carcinoma. PCNA positive cancer cells were observed in greatest abundance at the deeply infiltrated region of the carcinoma. CEA and CA19-9 were also expressed most strongly in this region and a stromal staining pattern was predominant. However, negative or apical staining patterns of CEA and CA19-9 were more frequent in the surface or the lateral spreading regions of the carcinoma, where the PCNA positive cancer cells were fewer in number. EGFR expression was rarely observed in the present study.
Plasma cholecystokinin (CCK) levels in rats with pancreatic insufficiency induced by a single injection of 50μl oleic acid into the pancreatic duct were determined by a sensitive and specific bioassay using the isolated rat pancreatic acini. Treatment with oleic acid significantly decreased pancreatic wet weight within 7 days, which lasted until the end of observation (56 days). Histologic examination revealed the destrution of acinar cells and the epithelium of intra- and interlobular ducts. Plasma CCK bioactivity was significantly increased from the pre-treatment values of 0.8±0.1pM to 5.1±1.4pM at 24h after oleic acid treatment. After this peak, plasma CCK levels gradually decreased. Even after 56 days, however, plasma CCK levels in oleic acid-treated rats were significantly high compared with those in control rats. In the present study, plasma CCK levels in rats with chronic pancreatitis did not correlate with the progress of pancreatic insufficiency.