Analysis of Helicobacter pylori vacA gene and serum antibodies to VacA in Japan.

Vacuolating cytotoxin, VacA, is one of the most important pathogenetic factors produced by Helicobacter pylori. However, it is not clear whether the diversity in disease outcome may be ascribed to variations in strain and/or to the host responses to virulence factors. In this study, we analyzed the vacA middle region sequence among 65 Japanese isolates to clarify the variation in strain and assayed antibody titer to VacA by ELISA using purified VacA to evaluate the host response to cytotoxin. The nucleotide sequence identities compared among Japanese isolates were 92.8 +/- 3.56%, and compared to 88.3 +/- 2.89% in tox+ strains reported in GenBank. Positive correlation was found between the antibody titers and the severity of atrophic change of the stomach. In Japan the nucleotide sequences of the vacA middle region were highly homologous and genetically closer to tox+ strains. Antibody titers and host response to cytotoxin may be associated with atrophy of the stomach.

Properties of a high-torularhodin-producing mutant of Rhodotorula glutinis cultivated under oxidative stress.

To characterize the properties of torularhodin, which is one of the carotenoid pigments produced by the yeast Rhodotorula sp., a mutant which produces large amounts of torularhodin was constructed and its tolerance against oxidative stress was investigated. The mutant we obtained was capable of producing large amounts of torularhodin in response to irradiation with blue light. The mutant, incubated under irradiation with white light that resulted in an increased production of torularhodin, exhibited resistance to growth inhibition induced by the addition of methylene blue as the generator of singlet oxygen. Leakage of lactate dehydrogenase to the growth medium from the mutant was not increased as compared to that from a parent strain and a high-beta-carotene-producing mutant. These results suggest that an increase in the production of torularhodin reduces the susceptibility to injury induced by an active oxygen species.

HLA-DQB1 locus and the development of atrophic gastritis with Helicobacter pylori infection.

Helicobacter pylori is known to be involved in digestive diseases such as peptic ulcer, atrophic gastritis, and gastric cancer. It is supposed that the incidence of these digestive diseases associated with H. pylori is influenced by the strain diversity of H. pylori, factors involving the host or environment, and the duration of infection. In this study, we directed our attention to HLA, a host factor, and investigated the relation between HLA-DQB1 genotype of H. pylori-infected patients and the development of atrophic gastritis. HLA-DQB1 genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism method on 122 H. pylori-infected patients with atrophic gastritis and 28 uninfected Japanese controls. Infected patients with developed atrophic gastritis were classified as the open type and those with undeveloped atrophic gastritis as the closed type. To estimate the grade of atrophic gastritis reliably, histological and serological evaluations were also undertaken. The allele frequency of DQB1*0401 was significantly higher in the open-type group compared to either the closed-type or the uninfected group. These results suggest that immunogenic factors play an important role in the development of atrophic gastritis in H. pylori-infected patients, and that DQB1*0401 is a useful marker for determining susceptibility to this disease.

The role of DNase and EDTA on DNA degradation in formaldehyde fixed tissues.

Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.

Prophylactic effect of Enterococcus faecalis FK-23 preparation on experimental candidiasis in mice.

The prophylactic effects of heat-killed cells of Enterococcus faecalis FK-23 (FK-23 preparation) on experimental candidiasis were investigated in normal and leukopenic mice. In cyclophosphamide-induced leukopenic mice, oral or intraperitoneal administration of the FK-23 preparation at a daily dose of 1.25 or 5 mg/mouse for 3 consecutive days prior to Candida albicans infection significantly prolonged survival periods of the infected mice, and decreased viable counts of C. albicans recovered from their kidneys. In normal mice, the FK-23 preparation administered at dosages ranging from 0.63 to 10 mg/mouse/day for 3 consecutive days was ineffective, while in leukopenic mice, the FK-23 administered orally caused a facilitated recovery in the number of white blood cells including neutrophils. Furthermore, intraperitoneal administration of the FK-23 preparation into mice augmented the anti-Candida activity of immunocompromised peritoneal exudate cells obtained from the animals. These results suggested the potential usefulness of the FK-23 preparation as a prophylactic agent for the management of patients with opportunistic fungal infections.

p53 mutations in flat- and polypoid-type colorectal tumors detected by temperature-gradient gel electrophoresis.

Reports of cases of flat-type colorectal tumors are increasing in Japan, but almost nothing has been elucidated about the genetic abnormalities of these tumors. In this study, we have examined p53 mutations in six cases of colon cancer cell lines, 22 cases of flat-type colorectal tumors, and 27 cases of polypoid-type colorectal tumors using the polymerase chain reaction (PCR) and temperature-gradient gel electrophoresis (TGGE); the latter has recently been developed as a screening method for gene mutations. p53 mutations were observed in four colon cancer cell lines, six flat-type colorectal tumors, and three polypoid-type colorectal tumors, all of which were analyzed by direct sequencing. These mutations were observed only in adenomas with high-grade dysplasia and in colorectal cancers but not in adenomas with low-grade dysplasia. These observations suggest that p53 gene mutations are involved in flat-type as well as polypoid-type colorectal tumors at relatively later stages of carcinogenesis and that TGGE seems to be useful as one of the rapid screening methods.

Non-involvement of ras mutations in flat colorectal adenomas and carcinomas.

Ras gene mutations occur relatively early during colorectal tumor development and have been observed in 40-50% of malignant colorectal tumors. Advances in endoscopic techniques have made it possible to detect small, flat colorectal tumors that could not be detected by standard examinations. To determine whether ras gene mutations are also involved in the genesis of small, flat colorectal tumors, we examined ras point mutations in 34 cases of small polypoid or flat elevated colorectal tumors (32 adenomas, 2 carcinomas) and in 26 cases of small, flat colorectal tumors (13 adenomas, 13 carcinomas) by means of the polymerase chain reaction (PCR) and dot-blot hybridization. Ras gene point mutations were observed in 16 of the 34 tumors of the former type (47%), but in none of the 26 tumors of the latter type, even though the grade of dysplasia was severe in the flat tumors. Our results suggest that different genetic pathways for tumor progression may exist for polypoid and for flat colorectal carcinomas.

p53 gene abnormalities are closely related to hepatoviral infections and occur at a late stage of hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) accumulates a mutation of the p53 gene with a common substitution of nucleotide in a particular site. It is hypothesized that infection of hepatitis B virus (HBV) or exposure to aflatoxins could induce it. In Japan, the concentration of aflatoxins in the environment is low; however, infection of HBV and/or hepatitis C virus (HCV) is frequently seen in patients with HCC. The purpose of our studies was to determine whether these hepatoviral factors influence p53 alterations. In our results, p53 abnormalities, which were composed of loss of heterozygosity (LOH) and/or point mutation, were shown in 39% of patients. We postulated that they occurred at late stages in tumor growth based on the following two results. LOH analysis on p53 showed that most of the tumor nodule consisted of two phenotypes, LOH and non-LOH cancer cells. The p53 abnormalities correlated with the grade of cancer cell atypia which advanced with tumor growth. HBV and HCV infections were identified by polymerase chain reaction using DNA extracted from cancerous and noncancerous regions of the liver. By these methods, the patients who had been infected with either HBV or HCV showed an incidence of p53 abnormalities (45%) higher than those infected by neither (13%). However, the detection rate of these viruses was lower in the HCC region (33%) than that in the noncancerous region (56%) in cases with mutated p53. The low rate of HCV detection (22%) in the HCC region with altered p53 was attributable to these different viral detection rates. There was a difference in pattern of p53 mutational changes in patients depending upon whether they were infected by HBV or by HCV. Two of three HBV-infected patients had a transversional change of nucleotide at the G:C site to T:A. However, in cases with HCV, four of eight patients had a transitional change of nucleotide of p53. These results showed that HBV and HCV infections affect carcinogenic pathways causing p53 abnormalities independently.

[Antitumor activity of Enterococcus faecalis FK-23 preparation against murine syngeneic tumors].

The antitumor activity of a preparation of heat-killed cells of Enterococcus faecalis, FK-23. Intraperitoneal injection of the preparation prolonged the lifespan of C3H/He N mice which were intraperitoneally inoculated with MM46 mammary carcinoma. Not only intraperitoneal but also oral administration of the FK-23 preparation inhibited the growth of these carcinomas inoculated intradermally. The tumor-bearing mice produced tumor necrosis factor (TNF) in their sera 2 h after intravenous injection of OK432. This TNF level increased after the mice were fed with food supplemented with the FK-23 preparation. Additionally, the FK-23 preparation inhibited the growth of Meth A fibrosarcoma in cyclophosphamide-treated BALB/c mice.

Immunohistochemical study of epidermal growth factor and transforming growth factor-beta in the penetrating type of early gastric cancer.

We report that the penetrating type of early gastric cancer (PEN) is a specific type of early gastric cancer and that the poorly differentiated PEN type could be considered an initial lesion of linitis plastica-type cancer. We performed an immunohistochemical study to clarify the role of growth factors (epidermal growth factor [EGF] and transforming growth factor-beta [TGF-beta]) in the PEN type of early gastric cancer. The results indicated that the PEN type of early gastric cancer has a high growth capacity. Moreover, it was suggested that EGF was involved in its specific infiltrative growth and that both EGF and TGF-beta were involved in its specific scirrhous growth. From these findings, it was assumed that the immunohistochemical staining of EGF and TGF-beta in endoscopic biopsy specimens was useful for the diagnosis of the PEN type of gastric cancer and also for the diagnosis of the initial lesion of linitis plastica-type gastric cancer.

Multiple hormone secretion and gene expression in clones isolated from rat insulinoma cell lines.

Current evidence suggests that a multipotential endodermal cell may give rise to all islet cell phenotypes. Five (N1-N5) and twenty-one (m1-m21) pluripotent rat islet cell clones were isolated from two hormone-producing cell line (RIN-r, RINm5F) derived from a radiation-induced islet tumor. To investigate the characteristics of these clones, we analyzed the hormone expression and secretion by Northern blot, immunocytochemistry, and radioimmunoassay. Increased expression and secretion of insulin and glucagon were observed in these clones. The present examination might also be proof of the secretion of both insulin and glucagon in the single cell of the m21 clone isolated from RINm5F. These cell lines also overexpress the Ha-ras proto-oncogene. In order to determine whether or not the overexpression was caused by gene translocation, the insulin and Ha-ras gene loci in N3 isolated from RIN-r were assigned by in situ hybridization. Both of the genes were located on the long arm of chromosome 1, but no gene translocation was observed. These findings suggest that the expression of insulin and Ha-ras is not affected by chromosomal translocation, but it may be functionally linked in these clones. Overexpression of these three genes may indicate that these clones have the same characteristics as the embryonic immature islet cell.

Different origin of leiomyoblastoma by immunohistochemical study.

Leiomyoblastoma has been regarded as a neoplasm of smooth muscle origin. With recent progress in immunohistostaining techniques, many clinicopathological discrepancies have been pointed out about the origin of leiomyoblastoma. It has been claimed that gastrointestinal non-epithelial tumors should be regarded as stromal tumors in order to study their origin. In the present study, we performed various forms of immunohistostaining in seven cases of leiomyoblastoma to determine their origin. One case expressed desmine and muscle specific actin and was considered to be derived from smooth muscle. Four neoplasms expressed X-100 protein (two cases were also NSE positive) and were thought to be derived from the nerve. Two cases were of unknown derivation. These results suggest that the cells of leiomyoblastoma may arise from a primitive to totipotential cell of neural lineages that may anomalously express smooth muscle filaments.

Endoscopic mucosectomy for early gastric cancer using modified strip biopsy.

We present a modification of strip biopsy (SB), one of the endoscopic treatment modalities for early gastric cancer. Using a side-viewing endoscope and a special electrocautery snare, we treated 6 patients with early carcinoma of the intestinal type with this modified strip biopsy (MSB). All tumors could be completely resected and 5 patients were not operated on: All 5 remained tumor-free at follow-up of at least one year. These results compared favorably with a historical control group of 17 patients with early gastric cancer treated with conventional strip biopsy. We therefore recommend applying our new method of MSB especially in the intestinal type of early gastric carcinoma; these preliminary data however, need confirmation in larger trials.

Fundamental study on the mechanism of DNA degradation in tissues fixed in formaldehyde.

The mechanism of DNA degradation and its clinical applications were examined. When purified lambda phage and extracted liver DNA were fixed in phosphate buffered formaldehyde, the DNA did not degrade, but there was incomplete digestion with endonuclease. Rat liver tissues were fixed under various conditions and DNA extracted. Immediate fixation with buffered formaldehyde at low temperature, or the addition of EDTA to buffered formaldehyde blocked the DNA degradation. Analysis of pulsed field gel electrophoresis also showed that DNA was degraded before extraction. These results suggest that tissue nuclease has an important role in DNA degradation in tissue. Furthermore, formaldehyde fixation at low temperature, which may take time and which decreases slightly the staining capacity, is useful for the extraction of intact DNA. For clinical application, the detection of provirus was examined. Genomic DNA was extracted from a necropsy sample of adult T cell leukaemia fixed in formaldehyde; human T cell leukaemia virus type-I (HTLV-I) provirus was successfully detected by Southern blotting.

[An autopsy case of extramural malignant leiomyoblastoma of the stomach with ovarian cancer: an immunohistochemical study].

The subject was an 85-year-old woman, who had been diagnosed as having an ovarian cancer and carcinomatous peritonitis and had been treated conservatively. She subsequently died from respiratory and renal insufficiency, and the autopsy that followed revealed that her pelvic cavity had been filled by a tumorous mass that size of a child's head. Histologically, the tumor was a serous cystadenocarcinoma of the ovary. Moreover another tumor, also the approximate size of a child's head, was found sited extramurally, beneath the posterior wall mucosa of the stomach body. Histological inspection of this tumor revealed a proliferation of round oval, and spindle-shaped tumor cells. A vacuolation of the cytoplasms and karyomitosis to the extent of 10/50 HPF also were observed. Based on the findings of being positive for Vimentin and a negative EMA, this tumor was diagnosed as being a malignant leiomyoblastoma of the stomach smooth muscle. The leioblastoma is a relatively uncommon neoplasm, and recent advances in immunohistochemical staining have indicated that some of these tumors are not only of smooth muscle derivation but also of nerve origin. Therefore, this tumor, given its morphological characteristics, had been generalized in this case as a gastric stromal tumor, and with negative findings for Desmin and S-100 protein, as well as positive for Vimentin.

[A case of vulva cancer responding to the recombinant human tumor necrosis factor (PT-950)--local injection therapy].

A case of vulva cancer responding to the recombinant human tumor necrosis factor (rHu-TNF: PT-050), which had been administered to double inguinal lymph nodes, is herein reported. A 60-year-old female underwent resection of a vulva cancer following a diagnosis of condyloma acuminatum. After surgery, however, exacerbation of a residual tumor, sited on the right of the vulva, and metastasis of the double inguinal lymph nodes were uncovered. For treatment, rHu-TNF was injected in the inguinal lymph nodes at a dose of 1 x 10(6)U per injection, 2 times a week. After 8 therapeutic sessions for 4 weeks, a marked response was observed not only in swelling of the lymph nodes, into which rHu-TNF had been injected, but also in the tumor of primary focus, which received no injections. On the 32 nd day after the final injection of rHu-TNF, the tumor of primary focus completely disappeared and the swelling of lymph nodes had diminished to 25% of the initial volume.