Effects of antioxidative agents on apoptosis induced by ischaemia-reperfusion in rat intestinal mucosa.

BACKGROUND AND AIM: We have previously demonstrated that ischaemia-reperfusion induces apoptosis in the intestinal mucosa. To evaluate that reactive oxygen species enhanced intestinal apoptosis after ischaemia-reperfusion, we examined whether antioxidants reduced apoptosis. METHODS: Rats were infused through a duodenal tube with antioxidative agents, glutathione, rebamipide and dymethylsulfoxide during 2 h before an ischaemic insult. The superior mesenteric artery was occluded for 60 min, followed by 60 min reperfusion. Apoptosis was evaluated by percentage fragmented DNA (fragmented DNA/total DNA) and immunochemical staining. RESULTS: Increase in apoptosis in the intestinal mucosa after ischaemia-reperfusion was attenuated by intraduodenal infusion of antioxidative agents, but was not completely abolished. CONCLUSION: Scavenging effects of the antioxidative agents attenuated increases in intestinal apoptosis, indicating that oxidative stress after ischaemia-reperfusion plays an important role in induction of apoptosis in the intestinal mucosa.

Exogenous cysteine and cystine promote cell proliferation in CaCo-2 cells.

Previous studies have shown that intracellular glutathione, a ubiquitous intracellular thiol, is related to cell proliferation and that cysteine or its disulphide form, cystine, also induces cell proliferation. Cysteine is a thiol containing amino acid and a rate-limiting precursor of glutathione. Therefore, it is still unresolved as to whether the proliferative effect of cysteine or cystine is entirely mediated by a change in the intracellular glutathione status. The objective of this study was to delineate the relationship among cysteine/cystine (thereafter referred to as cyst(e)ine), intracellular glutathione and cell proliferation in the human colon cancer CaCo-2 cell line. CaCo-2 cells were cultured in cyst(e)ine-free Dulbecco's Modified Eagle Medium without serum, and treated with 200 microm cysteine and/or 200-400 microm cystine for 24 h. In the presence of DL-buthionine-[S, R]-sulfoximine (BSO), a glutathione synthesis inhibitor, exogenously administered cyst(e)ine did not change the intracellular glutathione content, but increased the intracellular cysteine as well as cystine level. Addition of exogenous cyst(e)ine following 5 mm BSO treatment significantly increased cell proliferation as measured by 3H-thymidine incorporation and protein content. Cell cycle analyses revealed that cyst(e)ine promoted cell progression from the G1 phase to the S phase. Correspondingly, cyst(e)ine treatment induced expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb). In conclusion, these data indicate that both cysteine and cystine have proliferative effects in CaCo-2 cells independent of an increase in intracellular glutathione. Induction of cyclin D1, phosphorylation of Rb, and subsequent facilitation of G1-to-S phase transition were involved in the proliferative effect of exogenous cyst(e)ine.

[A case of malignant lymphoma concomitant with multiple myeloma].

A 75-year-old woman was referred to us because of cough, high fever and skin erythema in April 1999. Malignant lymphoma (diffuse mixed cell type) was previously diagnosed in 1990 and she achieved complete remission after treatment with a series of CHOP regimen treatments. In 1998, multiple myeloma (IgG lambda type) was diagnosed and she was treated with a combination of melphalan and prednisolone. On physical examination, superficial lymphadenopathy and skin erythema were noted. Biclonal gammopathy (IgG kappa/lambda) was shown in serum, and Bence Jones protein in urine. Computed tomography showed pleural effusion and swelling of paraaortic lymph nodes. The bone marrow examination showed an increased number of abnormal plasma cells (19.2%) and no evidence of lymphoma. Left axillary lymph node biopsy revealed that she had non-Hodgkin's lymphoma (immunoblastic lymphadenopathy-like T cell lymphoma). She was treated with the CHOP regimen at reduced doses for both diseases. The lymphoadenopathy reduced after 6 courses of CHOP and 4 courses of CHOPE (CHOP + VP16), however, she had bone pain on November 1999 and received treatment with MCNU-VMP (MCNU + VDS + L-PAM + PSL). Her rib pain improved, but she died of systemic infection of herpes zoster virus. We report here a rare case of malignant lymphoma concomitant with multiple myeloma.

Induction of mild intracellular redox imbalance inhibits proliferation of CaCo-2 cells.

Intracellular redox status plays a critical role in cell function, such as proliferation. Oxidative stress, which elicits redox imbalance, also affects cell growth. Therefore, it is often difficult to distinguish the effects of redox imbalance from those of oxidative stress. The objective of this study was to determine the role of redox imbalance independent of reactive oxygen species (ROS) production, in proliferation of human colonic CaCo-2 cells. Low concentrations of diamide plus 1,3-bis(2 chloroethyl)-1-nitrosourea (BCNU) increased intracellular GSSG and decreased GSH and the GSH:GSSG ratio. These changes occurred within 30 min, which preceded a decrease in thymidine incorporation at 6 and 24 h. ROS formation was not detected under these conditions. This suppression of cell proliferative activity was attenuated by N-acetyl cysteine, in parallel with restoration of the intracellular GSH redox status. dl-buthionine-[S, R]-sulfoximine (BSO) decreased intracellular GSH level, but did not change the GSH:GSSG ratio. BSO alone had no effect on cell proliferation, but its presence exaggerated the suppressive effect of diamide plus BCNU. Flow cytometric analysis showed that cells were arrested at G1-to-S transition and G2/M phase. Collectively, this study shows that mild intracellular redox imbalance inhibited cell proliferation independent of ROS generation. Moreover, cells with compromised cellular GSH were susceptible to redox imbalance-induced inhibition of proliferation.

Endoscopic injection sclerotherapy for esophageal varices prolonged survival of patients with hepatocellular carcinoma complicating liver cirrhosis.

BACKGROUND: A prospective controlled study was performed between 1982 and 1991 to evaluate the efficacy of endoscopic injection sclerotherapy (EIS) in patients with esophageal varices complicated by hepatocellular carcinoma and liver cirrhosis. METHODS: The study included 83 patients with esophageal varices, hepatocellular carcinoma, and liver cirrhosis. Forty-three patients (group 1) underwent prophylactic EIS or emergent EIS for bleeding varices. EIS was performed weekly 4 to 6 times until the varices disappeared. The remaining 40 patients (group 2) underwent conservative therapy and did not undergo EIS. Survival rates were compared between the 2 groups. RESULTS: During the 5-year observational period, all patients who did not undergo EIS died. Sixteen in group 2 (40.0%) died of gastrointestinal bleeding including ruptured esophageal varices. In contrast, patients treated with EIS survived significantly longer (p<0.001). Nine patients (20.9%) treated with EIS experienced gastrointestinal bleeding as a result of which 5 (11.6%) died. EIS prolonged survival in patients classified as Child's A or B but did not affect survival in patients with Child's C hepatic function. EIS was effective in prolonging survival in patients with hepatocellular carcinomas smaller than 5 cm. However, EIS had no effect in patients with hepatocellular carcinomas that were larger than 5 cm. EIS prolonged survival only for patients with nodular hepatocellular carcinoma and had no effect in patients with massive and diffuse hepatocellular carcinoma. Further, EIS prolonged survival only for patients who did not have portal vein thrombosis. CONCLUSION: Based on this prospective study, we concluded that EIS was effective in prolonging the survival period of a select subset of patients with hepatocellular carcinoma.

Proportion of reflux esophagitis in 6010 Japanese adults: prospective evaluation by endoscopy.

Compared with findings in Western countries, the prevalence of reflux esophagitis in Oriental countries is estimated to be low. In this prospective study, we aimed to examine the proportion of reflux esophagitis in Japanese adults, as evaluated by endoscopy. Endoscopists were prospectively directed to grade esophageal mucosal breaks with esophagitis according to the Los Angeles Classification of Esophagitis in all subjects that underwent endoscopic examination. In total, 6010 subjects underwent endoscopic examination for evaluation of esophagitis grading from December 1996 to February 1998. The subjects included 4394 outpatients who were not receiving medication for gastrointestinal disease and 1616 subjects who visited the hospital for routine physical examinations. The overall proportion of esophagitis was 16.3%. Most of the subjects with esophagitis were classified as having grade A or B (proportion of grades A and B, 9.6% and 4.6%, respectively). The age-related proportion of esophagitis and of severe esophagitis (i.e., grades C and D) increased in females aged over 70 and in males aged over 80. Increased body mass index (partly due to decreased height caused by osteoporosis), and/or hiatal herniation, were related to the proportion of esophagitis in females aged over 70. These data indicated that reflux esophagitis is a common disease in Japan. However, severe esophagitis (grades C and D) is not common.

[Clinical characteristics of polycythemia vera in the elderly].

Of 43 elderly patients who were suspected to have polycythemia between October 1990 and July 1998, 12 patients showed an increased red cell volume measured by 51Cr-labeled red blood cells. We analyzed the clinical characteristics of the 12 patients consisted of 7 men and 5 women, with a median age of 71 (range: 57-92). Chief complaints were headaches and dizziness (3 cases), symptoms of other conditions than polycythemia (4 cases). Five patients had no symptoms. Five of 6 patients over 70 years old had no symptoms due to polycythemia. Seven cases (58%) showed splenomegaly and three cases (25%) showed hepatomegaly. Laboratory findings were as follows: WBC 9.7 +/- 3.9 x 10(3)/microliter (mean +/- SD, p < 0.02 vs normal control), Hb 17.9 +/- 4.2 g/dl (p < 0.001), Plt 39.7 +/- 26.0 x 10(4)/microliter, EPO 13.8 +/- 5.2 mU/ml (p < 0.0001), NAP score 258 +/- 114, Vit. B12 1,686 +/- 2,156 pg/ml, arterial O2 saturation more than 92% in all cases. The diagnosis of all cases was polycythemia vera according to the diagnostic criteria of Polycythemia Vera Study Group. Associated conditions included 8 cases of thrombosis (cerebral thrombosis 4, thrombophrebitis 2, myocardial infarction 1, ischemic colitis 1) and 3 cases of malignancy (esophageal cancer 1, breast cancer 1, renal cancer 1), none of which was therapy-related cancer. Six patients (50%) had only phlebotomy, three (25%) only chemotherapy, and three (25%) both phlebotomy and chemotherapy. Patients over 80 years old needed neither intensive nor continuous treatment. Only one patient died due to esophageal cancer at age 89.

Minimal residual disease in acute myelogenous leukemia with PML/RAR alpha or AML1/ETO mRNA and phenotypic analysis of possible T and natural killer cells in bone marrow.

Here we studied minimal residual disease (MRD) of patients with acute myeloid leukemia (AML) who have PML/RAR alpha or AML1/ETO as well as the phenotypic analysis of lymphocyte subsets involved in antitumor immunity. Eight patients in long-term (LT; 3 to 15 years) and 15 patients in short-term (ST; up to 3 years) remission were studied. Using the reverse transcription-polymerase chain reaction (RT) assay, the limit of detection was 10(-5) to 10(-6) for PML/RAR alpha transcript and 10(-4) to 10(-5) for the AML1/ETO transcript. Simultaneously, T lymphocyte subsets and NK cells from the peripheral blood (PB) and bone marrow (BM) were investigated by flow cytometric analysis. Four of the eight patients in LT and 7 of the 15 patients in ST remission were MRD-positive. Although all MRD-positive patients in LT remission are still until now event-free, 3 of the 7 MRD-positive (MRD+) patients in ST remission soon relapsed. The total populations of CD4+, CD8+ and CD56+ [possible T-cell and natural killer (T/NK) populations] in the BM of ST patients and MRD+/LT patients were significantly (p < .01) low. The CD8+ CD28+ population showed the same tendency (p < .01-.02). The T/NK subsets in the BM of MRD-negative (MRD-) LT (MRD-/LT) patients showed similar numbers of cells as normal volunteers. Basically, the total percentage of the CD4+, CD8+ and CD56+ cell populations in the BM was increased and in the following order: MRD-/LT patients, normal volunteers, MRD+/LT patients and MRD+ or -/ST patients. The percentages of the T/NK-cell subsets in the PB were not significantly different among these groups. Thus, the difference of the possible T/NK-cell phenotype in the BM may strongly influence clinical and molecular remission. These results still remain to be confirmed by further studies of the functional anti-tumor immunity of T/NK cells of AML in remission.

Heterogenous expression of bcr-abl fusion mRNA in a patient with Philadelphia-chromosome-positive acute lymphoblastic leukaemia.

We performed molecular and cytogenetic analysis on a 56-year-old woman with Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukaemia (ALL) having two types of major and minor bcr-abl (M-bcr-abl, m-bcr-abl/fusion mRNA at diagnosis. In the course of her disease, unexpected heterogenous bcr-abl fusion mRNA was detected by sequential analysis using the reverse transcription and polymerase chain reaction (RT-PCR). The RT-PCR analysis showed both M-bcr-abl (b2-a2 type) and m-bcr-abl at diagnosis. Although b2-a2 type M-bcr-abl disappeared after complete remission (CR) was achieved with intensive induction chemotherapy, expression of both m-bcr-abl and a new type of M-bcr-abl mRNA (b1-a2 type), which may have been produced through splicing out of exon b2, was detected in the early stage of CR. The leukaemic cells contained these heterogenous bcr-abl mRNAs in both the CR and relapsed state, and showed more stable expression of the m-bcr-abl type mRNA than that of the b2-a2 type. These findings of heterogenous bcr-abl mRNA due to alternative or missplicing during the disease course in the present ALL case may provide important evidence of disease progression.

Evaluation of semiquantitative reverse transcriptase-polymerase chain reaction assay of PML/retinoic acid receptor alpha mRNA and in vitro differentiation assay as prognostic prediction in acute promyelocytic leukemia treated with all-trans retinoic acid.

We studied the quantitative changes in PML/retinoic acid receptor alpha (PML/RAR alpha) fusion mRNA using the reverse transcriptase-polymerase chain reaction (RT-PCR) and the in vitro differentiation of leukemic cells from eight acute promyelocytic leukemia (APL) patients during treatment with all-trans retinoic acid (ATRA). In three patients, the intensity of the chimeric PML/RAR alpha bands decreased rapidly after the start of ATRA therapy. However, these three patients required variable periods of time to obtain complete remission (CR) (24, 29 and 100 days). In the five other patients, the chimeric bands decreased slowly, and the time until CR also varied (22, 28, 30, 39 and 67 days). As for the in vitro assay, leukemic cells from the three patients who achieved CR in a short period of time (22, 28 and 30 days) showed marked differentiation in response to 1 mumol/L ATRA, and leukemic cells from the four patients with slow or delayed clinical responses to ATRA did not show morphological differentiation in vitro. These findings suggest that the clinical response of APL patients to ATRA is predicted from the results of the in vitro differentiation assay, but not by RT-PCR analysis of the PML/RAR alpha fusion mRNA.

Gastric lesions in 76 patients with adult T-cell leukemia/lymphoma. Endoscopic evaluation.

BACKGROUND: Adult T-cell leukemia/lymphoma (ATLL) is caused by human T-lymphotropic virus type I. Gastric lesions in ATLL have not been described precisely, whereas the clinical features of ATLL have been well documented. The goal of the present study was to review gastric lesions, including gastric involvement, of patients with ATLL who were admitted to our hospital. METHODS: Endoscopic examination of the upper gastrointestinal tract was performed on 76 of 110 patients who were admitted to our hospital between 1981 and 1994. Gastric involvement was diagnosed by histologic examination of biopsy specimens of gastric lesions. Types of gastric lesions, histologic features, and survival periods in patients with ATLL were summarized. RESULTS: Of the 76 patients with ATLL who underwent an endoscopic examination, 23 had gastric involvement (30.3%). Twenty-seven patients had other gastric lesions: 10 with peptic ulcers (13.2%), 8 with gastric erosions (10.5%), 3 with submucosal tumors (3.9%), 2 with hyperplastic polyps (2.6%), 1 with gastric adenoma (1.3%), and 3 with gastric carcinomas (3.9%). The most frequent endoscopic configuration of gastric involvement with ATLL was the diffuse type with ulceration, and the most common histology was large cell type. Among those with the acute type ATLL, the survival period of those patients with gastric involvement was less than that of the patients without gastric involvement. In contrast, the survival period for lymphoma type ATLL did not differ among the groups regardless of gastric involvement. CONCLUSIONS: This study demonstrated that 30.3% of patients with ATLL had gastric involvement and 13.2% had peptic ulcers. Gastric involvement of ATLL was one of the prognostic factors in acute type ATLL, whereas it had no influence on the prognosis of lymphoma type ATLL.

Determinants of hydroperoxide detoxification in diabetic rat intestine: effect of insulin and fasting on the glutathione redox cycle.

The capacity for hydroperoxide detoxification in diabetic (DM) intestine was studied in streptozocin-induced DM rats by quantification of the intestinal glutathione (GSH) redox cycle, a key cellular pathway for peroxide elimination. A role for luminal glucose in regulation of redox cycle activity was examined in insulin-treated or 24-hour-fasted DM animals. Intestinal activities of the redox enzymes, GSH peroxidase, GSSG reductase, and glucose-6-phosphate dehydrogenase (G6PD), were significantly decreased by 17 hours' insulin treatment, whereas only G6PD was decreased by fasting. Mucosal GSH levels were also markedly decreased under these conditions. These results are consistent with an overall suppression of intestinal GSH redox cycle function by short-term administration of insulin. Insulin treatment for 7 consecutive days increased hepatic G6PD activity by fourfold but was without effect on intestinal G6PD, suggesting tissue specificity in insulin regulation of G6PD. The rate of metabolism of tert-butyl hydroperoxide (tBH) in isolated enterocytes was low in the absence of substrates (0.51 +/- 0.07 nmol/10(6) cells/min) but was increased fivefold by exogenous glucose (2.70 +/- 0.11 nmol/10(6) cells/min), indicating that glucose availability is an important contributor to intestinal detoxification of toxic hydroperoxides. Collectively, the current results show that GSH redox cycle enzymes in DM intestine are under coordinate insulin control, and that this control appears to be downregulated by short-term insulin treatment.

Hyperplastic polyps following treatment of acute gastric ulcers.

Although hyperplastic polyps are the most common polyps of the stomach, the etiology of these polyps is not completely understood. We report a 61-year-old woman who developed gastric hyperplastic polyps following acute gastric lesions. She was admitted for endoscopic injection sclerotherapy of esophageal varices. After the end of sclerotherapy, acute gastric lesions developed. For treatment of the lesions, omeprazole was used for 8 weeks followed by famotidine for 8 weeks. At the end of the treatment, she developed multiple gastric hyperplastic polyps, suggesting that acute gastric lesions and/or treatment of the gastric lesions are related to the development of hyperplastic polyps in the stomach.