The immunosuppressant leflunomide inhibits lymphocyte proliferation by inhibiting pyrimidine biosynthesis.

Leflunomide is a novel immunosuppressive compound that is effective in the treatment of animal models of autoimmune disease and human rheumatoid arthritis. The mechanism of action is unknown. Here we show that leflunomide blocked 1) increases in nucleolar size and number, 2) upregulation of the nuclear protein antigens (PCNA and Ki-67), 3) increases in uridine incorporation and total RNA and DNA content, 4) cell cycle progression and 5) proliferation in mitogen-stimulated rat spleen mononuclear cells and human peripheral blood mononuclear cells (HPBMC). Exogenous uridine reversed the leflunomide-dependent inhibition of the normal increase in total RNA and DNA content in mitogen-stimulated HPBMC and rat spleen cells. Uridine reversed the leflunomide-dependent inhibition of cell cycle progression in stimulated rat cell cultures. Either uridine or cytidine, which can be converted to uridine by cytidine deaminase, reversed the antiproliferative effect of leflunomide in HPBMC. Dihydroorotate accumulated in leflunomide-treated human T-lymphoblastoid cells, suggesting that the compound inhibited the fourth enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase. The results support the hypothesis that the in vitro effects of leflunomide on T-lymphocytes are due to inhibition of de novo pyrimidine synthesis.

Leflunomide interferes with pyrimidine nucleotide biosynthesis.

Leflunomide is an anti-inflammatory and immunosuppressive agent which blocks proliferation of transformed cells and mitogen stimulated normal lymphocytes but does not block T cell signaling mechanisms at antiproliferative concentrations. These properties are consistent with a mechanism involving interference with nucleotide metabolism. Leflunomide had anti-proliferative activity against all cells tested here. The anti-proliferative activities could be reversed by addition of uridine or cytidine to the cultures although some species and cellular differences were observed. Purine nucleotides had no effect. Measurements of nucleotide pools in a human T cell line and mitogen stimulated rat spleen cells treated with leflunomide showed that leflunomide preferentially reduces pyrimidine nucleotide levels. These results indicate that inhibition of pyrimidine biosynthesis is responsible for the anti-proliferative effects of leflunomide.

Vaccinating guinea pigs with recombinant glycoprotein D of herpes simplex virus in an efficacious adjuvant formulation elicits protection against vaginal infection.

Guinea pigs were immunized with glycoprotein gD-2t in SAF-m or saline, then challenged with herpes simplex virus, type 2 (HSV-2). Animals given gD-2t in SAF-m had higher anti-gD-2t antibodies, fewer and less severe vaginal lesions, and decreased ganglionic latency compared to animals given gD-2t in saline. Leucocytes from animals vaccinated with gD-2t in SAF-m had greater proliferative responses to gD-2t in vitro than cells from control animals. MHC II-restricted, gD-2t-specific cytotoxic T cells were induced in guinea pigs vaccinated with gD-2t in SAF-m. Thus, immunization of guinea pigs with gD-2t in SAF-m markedly reduced the incidence and severity of primary HSV-2 by eliciting both humoral and cell-mediated responses.

Improvement of hepatitis B vaccine by the use of a new adjuvant.

Humoral and cellular immune responses of mice and guinea-pigs to hepatitis B virus surface antigen when alum-precipitated or administered with Syntex Adjuvant Formulation (SAF) were compared. Two doses of HBsAg in SAF were sufficient to elicit antibody responses, and using SAF the dose of antigen could be reduced to one-tenth of that required to elicit antibody responses by alum-adjuvanted HBsAg. The use of SAF increased and made more consistent the antibody responses in young mice and in strains of mice with inherited low responses to HBsAg. Cellular responses to HBsAg were more consistently observed when SAF was used than when alum was used. SAF increased the formation of IgG2a antibodies in mice except in the B10.M strain; antibodies of this isotype activate complement and act synergistically with antibody-dependent effector cells more efficiently than antibodies of other isotypes. If SAF proves acceptable for human use it could improve vaccines against hepatitis B virus.

Long-term explant culture of human colon and a 3-step transformation model for rat colonic epithelium.

Our previous studies have suggested that colonic epithelium from rodents pretreated in vivo with suboptimal doses of carcinogen could be more easily maintained in explant culture. Transformation of colonic epithelium from these explants may be induced by subsequent exposure to additional genotropic agents. Therefore, we describe the development of a 3-step transformation model which uses (1) in vivo pretreatment with a suboptimal dose of 1,2-dimethylhydrazine (DMH) followed by (2) in vitro organ culture and exposure to xenotropic murine sarcoma virus (X-MSV), and finally (3) xenograft maintenance in nude mice to allow sufficient time for transformation. Male Wistar rats were injected intramuscularly with 20 mg DMH/kg 10 times per week followed by the removal and explant culture of the colon, which was then treated in vitro with X-MSV, and transplanted into nude mice after 1 week of culture. All the nude mice (n = 6) transplanted with rat colon explants contained viable xenograft explant epithelium and 1 of the 6 showed transformation. Our results demonstrate that the epithelium from animals pretreated with suboptimal doses of carcinogens can be easily transformed. We also demonstrate that human colonic epithelium is viable for an extended period of time in this model. Based on these results, we hypothesize that such a 3-step transformation model is applicable for carcinogenesis studies of various organs from different species, including human if one uses dysplastic or 'pre-neoplastic initiated' tissues obtained at surgery (e.g., ulcerative colitis; Barret's esophagus, etc.).

A pilot study on the usefulness of a new test for mass screening of colorectal cancer in Japan.

A new screening test (Shamsuddin and Elsayed, 1988) based on the enzymatic detection of the disaccharide beta-D-Gal(1----3)-D-GalNAc in the rectal mucus of patients with colorectal (CR) cancer and precancerous conditions such as inflammatory bowel disease (IBD) and polyp (precancerous lesions) was evaluated in 85 Japanese patients. Following a 15-minute reaction, a sensitivity of 80.0% (8/10) for CR cancer and 72.2% (8/11) for precancerous lesions was obtained. The overall specificity for combined CR cancers and precancerous lesions was 62.2% (28/45). Correlation with abnormal mucin production in the tissues of CR cancer and precancerous lesions was studied by high-iron diamine-Alcian blue and/or periodic acid-Schiff-Alcian blue (pH = 1.0). The agreement of the results with this test was 77.8% (7/9) for CR cancers and 75.0% (6/8) for precancerous conditions. Because of the simplicity of this test, low cost, stability of the sample and reagents and accuracy for CR cancer and precancerous lesions, the test may have potential use for mass screening of cancer and high risk individuals, particularly CR cancer in Japan.

Potential usefulness of a cultured glioma cell line induced by Rous sarcoma virus in B10.A mouse as an immunotherapy model.

A cultured glioma cell line, SR-B10.A, which was derived from a brain tumor induced in an adult female B10.A mouse by Rous sarcoma virus (RSV), has been established. The morphological appearance of the tumor produced by s.c. inoculating SR-B10.A cells was analogous to an astrocytoma of human glioma. Glial fibrillary acidic protein as well as S-100 protein was positive in these SR-B10.A tumor cells. A population doubling time of the cultured cells was 18.5 hours. Chromosomal analysis revealed a defect in one of the sex chromosomes. Integration of RSV genome was proven to be positive in SR-B10.A cells. It was possible to generate cytotoxic effector cells in the syngeneic B10.A mouse against SR-B10.A. The tumor-bearing syngeneic hosts harbored a suppressor activity in the splenocytes. Although recombinant human tumor necrosis factor (rH-TNF) had no growth inhibitory effect on the SR-B10.A cells in vitro, the s.c. implanted and growing tumor regressed when rH-TNF was administered intratumorally several times. In addition, this anti-tumor effect was completely abrogated when the host mice were treated with wholebody x-ray irradiation prior to the tumor cells inoculation. In contrast, neither rH-TNF (i.v.) nor cyclophosphamide (i.p.) induced the regression of SR-B10.A, indicating that efficacy of the locally administered rH-TNF is dependent on the host immune mechanism. These results suggest that SR-B10.A is a potentially useful tumor model in evaluating efficacy of immunomodulators.

Potential usefulness of a physical activity index in the assessment of quality of life of patients after gastrointestinal surgery.

In an attempt to evaluate a quality of life (QL) grade of patients who underwent major gastrointestinal surgery, a physical activity index (PAI) representing their daily physical activity level was introduced in the present study. The relationship between the PAI and the QL grade, which was classified into four categories (excellent, good, fair, and poor) according to our diagnostic criteria, was investigated by using postoperative patients with total gastrectomy (n = 52) and other gastrointestinal major surgery (n = 54). The PAI value was derived from basal metabolic energy expenditure and whole day energy expenditure which was predicted by a 24 hours heart rate ratio method. The evaluation of the QL was based on clinical records and answers to a questionnaire submitted to the patient. The mean PAI of the gastrectomy patients (n = 15) was lowest during the postoperative 3 months (p less than 0.005), and was then gradually restored to nearly the preoperative value during the period between the 6th and 12th months. The four graded QL groups were compared with each other in terms of the mean PAI and energy expenditure. As a result, the QL grades were proportional to both of these values, indicating that the more favourable the QL grade, the higher the PAI and the energy expenditure (p less than 0.005). These results establish that if the evaluation of energy expenditure is valid the PAI could be potentially applicable to clinical use as one of the parameters that delineate the QL grade.

[Experimental study on combination chemotherapy of UFT and calcium antagonist in gastric cancer].

The antitumor effect of combined use of UFT and verapamil, a calcium antagonist, was examined in gastric cancer transplanted to BALB/c athymic nude mice. Four experimental groups included Group I (Control) which was administered 3% Gummi Alabicum (p.o.), Group II (UFT) which was administered 64.8 mg/kg UFT (p.o.), Group III (UFT + verapamil) which was administered UFT and 10 mg/kg verapamil (s.c.) and Group IV (verapamil) which administered same doses of verapamil. All mice were sacrificed at the day of 12 and pathological and flow cytometric studies were performed to those tumors. At the day of 8, significant retardation of tumor growth was observed in Group II and Group III compared with Group I (P less than 0.05). The ratio of tumor retardation of Group III was more predominant than that of Group II at the day of 9 (p less than 0.05). However, no difference in pathological and flow cytometric changes was observed between Group II and Group III. Weight loss and death, as side effects, were found in Group II and III. It was assumed that this may be depend on the over dose of UFT or methods of administration. These results suggested that combination chemotherapy of UFT and verapamil was effective in the retardation of gastric cancer, however the doses of UFT and verapamil have to be reexamined in order to control the side effect.

Ultrastructural study of two subtypes of gastric adenoma.

An ultrastructural study was performed to identify any differences in fine detail between type III and IV gastric polyps, which seem to differ from each other because they have different histological features and malignant potential. Clear ultrastructural differences were found, implicating microvilli, mucous granules, interdigitations, ribosomes, mitochondria and nuclei. Although these two subtypes have often been grouped together as borderline lesions or adenomas, the findings imply that they should be treated as essentially different.

Adenocarcinoma arising at the perineal wound after pull-through procedure for imperforate anus.

An extremely rare case is presented here, of an adenocarcinoma at the site of a long-standing perineal wound, the presence of which was a result of a pull-through procedure for imperforate anus. Wide local excision of a huge mass, including the perineal skin and the distal rectosigmoid segment, was carried out en-bloc on a 35 year old male. This tumor may have been caused by the repeated trauma and frequent ulceration around the perineal wound as a result of poor hygiene due to a fecal incontinence.

[Long-term maintenance of human gall-bladder epithelia as xenografts following explant organ culture].

The purpose of present study is to establish a method that should enable us to keep a human gall-bladder epithelial tissue alive for a period long enough to observe morphological change after an exposure to a certain carcinogenic agents. Human gall-bladder epithelia obtained from surgically resected specimen were kept as an explant organ culture in vitro for the first 2 weeks. They were, then, xenotransplanted to the subcutaneous (S.C.) space of the athymic nude mice. The gall-bladder tissues obtained from 7 cases of cholecystolithiasis and 1 case of cholecysto-choledocholithiasis were used as materials in the present study. And the longest survivor among them in the nude mice was extensively studied in the present paper. After an interval of 4 to 27 weeks, the xenografts were recovered from the recipient mice and studied by light microscopy and histochemistry. The morphological changes of the explant epithelia during the organ culture were also studied. Parts of the explants were implanted in 6 nude mice. Each of the mice received 3 pieces of explants on the s.c. space in both of their flanks. All xenografts showed subcutaneous cyst formation. The cyst obtained 4 weeks after the implantation was consisted of a monolayer of cuboidal epithelial cells and those recovered from the recipient mice 24 and 27 weeks after the implantation were consisted of well growing columnar and cuboidal epithelial cells. In addition, sinus-like structures were observed beneath the cystic epithelia. The viability of the cells was excellent.(ABSTRACT TRUNCATED AT 250 WORDS)

[Clinical significance of a physical activity index based on calorimetry in the assessment of quality of life after total gastrectomy].

In order to characterize the objective diagnostic criteria concerning quality of life (QL) of patients after total gastrectomy, a physical activity index (PAI) or a concept of daily physical activity was developed. Sixty patients of gastric cancer, of whom 38 patients underwent long loop Roux-en-Y gastrojejunostomy (LLRY) procedure after total gastrectomy, 13 patients gastroduodenostomy (Billroth I) and 9 patients gastrojejunostomy (Billroth II) after subtotal gastrectomy, respectively, were evaluated as part of this study. In addition, 3 cases of pancreatoduodenectomy (PD) and 5 cases of total esophagectomy were also evaluated. The evaluation of QL was based upon a clinical assessment and administration of patient questionnaire. The assessment of the PAI was performed by measuring the individual's whole day energy expenditure based upon 24 hour heart rate ratio (24h-HRR) method and the basal metabolic energy expenditure. The daily physical activity was graded into four categories according to the PAI value; light, moderate, moderately heavy and heavy. The results obtained were as follows: The value of the energy expenditure predicted by 24h-HRR method and that based on the results of bicycle ergometry (VO2/HR method) showed close correlation. There was no significant difference in the whole day energy expenditure among four operative procedure groups (Billroth I, Billroth II, LLRY and total esophagectomy). More than 80 per cent of LLRY patients, whose QL was evaluated as "excellent" or "good", showed no less than "moderate" PAI. In addition, one of the four patients whose QL was "fair" was categorized into "light" and the remaining three were "moderate".(ABSTRACT TRUNCATED AT 250 WORDS)

Verapamil enhancement of antitumor effect of cis-diamminedichloroplatinum(II) in nude mouse-grown human neuroblastoma.

The antitumor effect of combined use of cis-diamminedichloroplatinum(II) (CDDP) and verapamil, a calcium influx blocker, was examined in neuroblastoma transplanted to BALB/c athymic mice. The response of the tumor to CDDP was related to the dose administered. Regression of the tumor was observed when CDDP was administered at 4.2 mg/kg/injection. The retardation of tumor growth was observed in the group to which CDDP was administered at 2.1 mg/kg/injection. When verapamil was administered with CDDP, regression of the tumor was observed in the group treated with CDDP at 2.1 mg/kg/injection, and the retardation of tumor growth was observed in the group treated with CDDP at 1.4 mg/kg/injection. These results indicate that verapamil enhances the antitumor effect of CDDP against transplanted neuroblastoma in BALB/c athymic mice.

Histopathogenesis of intestinal metaplasia: minute lesions of intestinal metaplasia in ulcerated stomachs.

Minute lesions of intestinal metaplasia composed of a few metaplastic tubules were observed in the gastric mucosa during routine histological examination of gastrectomy specimens. The histological findings indicated that these lesions might be an initial stage of more advanced intestinal metaplasia. Accordingly, more than 18,000 serial sections in 10 stomachs with chronic ulcers were examined to clarify the histopathogenesis of the intestinal metaplasia. It was concluded from the three dimensional reconstruction of minute intestinal metaplasia lesions that these lesions originated during the regenerative process of healing of gastric erosions. The lesions were roughly globoid with a depression on the surface. It is thought that with continuous formation and healing of gastric erosions, more extensive intestinal metaplasia lesions would be formed by an increase in size and confluence of these focal minute intestinal metaplasia lesions.

Adenomatosis of small intestine: case report.

A patient complained of severe diarrhoea for about four years. About 2.5 m of the jejunum was resected and duodenal polyps were also removed at laparotomy. Histologically, they were tubular or tubulovillus adenomas, which closely resembled colonic adenomas. No malignant change was detected in these adenomas. Severe inflammatory changes were seen in the jejunal mucosa and various gut hormones were seen in the same area. As far as we know no similar case with adenomatosis of the small intestine has been reported. We were unable to establish any association between adenomas and severe watery diarrhoea.

Response to 2'-deoxycoformycin after failure of interferon-alpha in nonsplenectomized patients with hairy cell leukemia.

Two patients with hairy cell leukemia with massive splenomegaly and severe pancytopenia were treated with recombinant alpha-A interferon (IFN-alpha-2a). There was no significant response to a trial of IFN-alpha-2a (11 and 20 weeks) with respect to blood counts or spleen size. Subsequent treatment with 2'-deoxycoformycin (dCF) for 8 consecutive weeks (4 mg/m2/wk) resulted in normalization of spleen size and a normalization of peripheral blood counts and bone marrow in one patient. The second patient demonstrated a reduction in spleen size and improved blood counts following 9 weeks of dCF therapy but eventually became refractory. This demonstrates that dCF is non-cross-resistant with interferon and confirms the efficacy of dCF in nonsplenectomized patients.

[Concentrations of cefmenoxime and cefotiam in the bile and gallbladder tissue following intravenous administration in patients with biliary tract diseases].

To test the effectiveness of cefmenoxime (CMX) and cefotiam (CTM) in patients with biliary tract diseases, concentrations of either antibiotic were measured after an intravenous bolus injection of 1.0 g of CMX or CTM, or simultaneous injection of both (1.0 g each). CMX or CTM was injected in 76 patients with biliary tract diseases (mostly cholelithiasis) prior to a cholecystectomy and concentrations of CMX or CTM were measured by the bioassay (agar well) method at 30 to 60 minutes after the injection. Average concentrations of both CMX and CTM in gallbladder bile and gallbladder tissue sufficiently exceeded the minimal inhibitory concentration (MIC) against main causative organisms of biliary tract infections. Concentrations of both antibiotics in gallbladder bile were significantly higher in patients with patent cystic ducts than with obstructed cystic ducts. Concentrations of both antibiotics in the gallbladder tissue reached at a similar high level regardless of the patency of the cystic ducts, but concentrations were lower in severely inflamed gallbladders. CMX and CTM were administered alternatively (cross-over fashion), or simultaneously (combined) to 13 patients with T-tube drainage or percutaneous transhepatic cholangio-drainage, and concentrations of both antibiotics in bile from the drainage tube were measured by high performance liquid chromatography at hourly intervals after the injection. Concentrations of both antibiotics were far greater than MICs against main attributable microorganisms in biliary tract infections. The concentration of CMX slightly exceeded that of CTM. Concentrations of both antibiotics were lower in bile of patients showing abnormally high serum GTP, A1-P, and total bilirubin levels than in bile of patients with normal values of these variables. It is speculated that the secretion of both antibiotics in the bile may decrease in cases with severe hepatic failure, but effective concentrations of both antibiotics in the gallbladder tissue should be maintained as long as the blood circulation in the gallbladder was maintained.

Appraisal of surgical treatment for distal ulcerative colitis.

Most patients with ulcerative colitis respond well to medical treatment, however surgical treatment may be required in cases with severe clinical symptoms. We treated eight patients with distal ulcerative colitis (DUC), limited to the rectum and distal sigmoid colon, who were treated at St. Mark's Hospital by excision of the rectum and sigmoid colon with permanent colostomy. Four who did not respond to medical treatment and had severe and intermittent symptoms of long-standing, three with no control of the distressing diarrhea with a shorter history, and those with a severe dysplasia evidenced by rectal biopsy were surgically treated. Two of eight had a recurrence at the proximal colon within 6 and 10 years respectively but responded well to conservative management. From these observations it concluded that the procedure for patients with DUC may be one of adequate operations. Histological features of the resected specimens did not relate to the postoperative outcome of these patients.