The challenge of acute rejection in intestinal transplantation.

Early diagnosis and treatment of acute cellular rejection (ACR) after intestinal transplantation (ITx) is challenging. We report the outcome of three patients: two presented mild ACR improved with steroids. One presented steroid-resistant severe rejection, improved after rabbit anti-thymocyte globulin (r-ATG), but unfortunately died for encephalitis caused by opportunistic infections.

Role of macrophage colony-stimulating factor (M-CSF)-dependent macrophages in gastric ulcer healing in mice.

We examined the role of macrophage colony-stimulating factor (M-CSF)-dependent macrophages in the healing of gastric ulcers in mice. Male M-CSF-deficient (op/op) and M-CSF-expressing heterozygote (+/?) mice were used. Gastric ulcers were induced by thermal cauterization under ether anesthesia, and healing was observed for 14 days after ulceration. The numbers of macrophages and microvessels in the gastric mucosa were determined immunohistochemically with anti-CD68 and anti-CD31 antibodies, respectively. Expression of tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and vascular endothelial growth factor (VEGF) mRNA was determined via real-time reverse transcription-polymerase chain reaction (RT-PCR), and the mucosal content of prostaglandin (PG) E(2) was determined via enzyme immunoassay on day 10 after ulceration. The healing of gastric ulcers was significantly delayed in op/op mice compared with +/? mice. Further, significantly fewer macrophages were observed in the normal gastric mucosa of op/op mice than in +/? mice. Ulcer induction caused a marked accumulation of macrophages around the ulcer base in +/? mice, but this response was attenuated in op/op mice. The mucosal PGE(2) content as well as the expression of COX-2, VEGF, and TNF-α mRNA were all upregulated in the ulcerated area of +/? mice but significantly suppressed in op/op mice. The degree of vascularization in the ulcerated area was significantly lower in op/op mice than in +/? mice. Taken together, these results suggest that M-CSF-dependent macrophages play an important role in the healing of gastric ulcers, and that this action may be associated with angiogenesis promoted by upregulation of COX-2/PGE(2) production.

Overexpression of RegIV in peritoneal dissemination of gastric cancer and its potential as A novel marker for the detection of peritoneal micrometastasis.

BACKGROUND: Regenerating gene type IV (RegIV) is a candidate marker for cancer and inflammatory bowel disease. In this study, its potential as a novel marker for the detection of gastric cancer peritoneal micrometastases was examined. PATIENTS AND METHODS: RegIV mRNA levels in the peritoneal washes of 95 gastric cancer patients and 22 with benign disease were quantified by real-time RT-PCR. To examine whether expression of RegIV enhance tumorigenicity or not, thirty two mice were injected intraperitoneally or subcutaneously with RegIV transfectants of TMK-1 cells, parental TMK-1 cells, or neomycin control transfectants. RESULTS: RegIV expression was markedly higher in patients with peritoneal metastases compared to those without. The level of RegIV mRNA in gastric cancer patients was related to the extent of wall penetration. A cut-off value for RegIV-positive expression was based on an analysis of negative control patients with benign disease, and gastric cancer patients above the cut-off value constituted the micrometastasis (MM+) group. Based on this criteria, 3 out of 43 T1 or T2 cases were MM+ (93% specificity). Among 15 patients with peritoneal dissemination (7 out of 15 cases were positive by cytology), 14 cases were positive for RegIV expression (93% sensitivity), while analysis of carcinoembryonic antigen (CEA) mRNA failed to detect micrometastases in 4 cases (73% sensitivity). Combined analysis of CEA and RegIV improved the accuracy of diagnosis to 100%. The prognosis of RegIV-positive cases was significantly worse than that of RegIV-negative cases. Multivariate analysis using the Cox proportional hazards model suggested that RegIV may be an independent prognostic factor. Stable expression of RegIV significantly enhanced peritoneal metastasis in an animal model of gastric cancer. CONCLUSION: These findings suggest that RegIV mRNA expression has the potential to serve as a novel marker for detecting peritoneal dissemination in gastric cancer.

Frequent silencing of RUNX3 in esophageal squamous cell carcinomas is associated with radioresistance and poor prognosis.

Radiotherapy is an effective treatment for some esophageal cancers, but the molecular mechanisms of radiosensitivity remain unknown. RUNX3, a novel tumor suppressor of gastric cancer, functions in transforming growth factor (TGF)-beta-dependent apoptosis. We obtained paired samples from 62 patients with advanced esophageal cancers diagnosed initially as T3 or T4 with image diagnosis; one sample was obtained from a biopsy before presurgical radiotherapy, and the other was resected in surgical specimens after radiotherapy. RUNX3 was repressed in 67.7% cases of the pretreatment biopsy samples and 96.7% cases of the irradiated, resected samples. The nuclear expression of RUNX3 was associated with radiosensitivity and a better prognosis than cytoplasmic or no RUNX3 expression (P<0.003); cytoplasmic RUNX3 expression was strictly associated with radioresistance. RUNX3 was downregulated and its promoter was hypermethylated in all radioresistant esophageal cancer cell lines examined. Stable transfection of esophageal cancer cells with RUNX3 slightly inhibited cell proliferation in vitro, enhanced the antiproliferative and apoptotic effects of TGF-beta and increased radiosensitivity in conjunction with Bim induction. In contrast, transfection of RUNX3-expressing cells with a RUNX3 antisense construct or a Bim-specific small interfering RNA induced radioresistance. Treatment with 5-aza-2'-deoxycytidine restored RUNX3 expression, increased radiosensitivity and induced Bim in both control and radioresistant cells. These results suggest that RUNX3 silencing promotes radioresistance in esophageal cancers. Examination of RUNX3 expression in pretreatment specimens may predict radiosensitivity, and induction of RUNX3 expression may increase tumor radiosensitivity.

In situ tissue engineering of the bile duct using polypropylene mesh-collagen tubes.

Multiple attempts have been made to replace biliary defects with a variety of materials. Recently, successful biliary reconstruction using the Gore-Tex vascular graft has been reported experimentally and clinically. We designed a new artificial bile duct consisting of collagen sponge and polypropylene mesh. We presently evaluated the feasibility of using this prosthesis as a scaffold for bile duct tissue regeneration in a canine model. Our prosthesis, a sponge made from porcine dermal collagen, is reinforced with a polypropylene mesh cylinder. We used the prosthesis to reconstruct the middle portion of the common bile duct in seven beagle dogs to evaluate its efficacy. While one dog died of biliary stricture 8 months after operation, six survived without problems to scheduled time points for tissue evaluation at 1 to 12 months. All prostheses had become completely incorporated into the host. A confluent epithelial lining was observed within 3 months. In cholangiograms the prosthesis displayed long-term patency in the six dogs and provided satisfactory bile drainage for up to 12 months. Our graft thus shows promise for repair of biliary defects and should lead to development of a new treatment for biliary reconstruction.

Frequent loss of RUNX3 gene expression in remnant stomach cancer and adjacent mucosa with special reference to topography.

Our previous studies suggest that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer. This study was conducted to determine whether alteration of RUNX3 gene expression could be detected in the normal-looking gastric remnant mucosa, and to ascertain any difference in the potential of gastric carcinogenesis between the anastomotic site and other areas in the remnant stomach after distal gastrectomy for peptic ulcer (RB group) or gastric cancer (RM group), by analysing RUNX3 expression with special reference to topography. A total of 89 patients underwent distal gastrectomy for gastric cancer from the intact stomach (GCI group) and 58 patients underwent resection of the remnant stomach for gastric cancer (RB group: 34 cases, RM group: 24 cases). We detected RUNX3 and gene promoter methylation by in situ hybridisation, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and methylation-specific PCR. The interval between the initial surgery and surgery for remnant gastric cancer (interval time) was 10.4 years in the RM group, and 27.5 years in the RB group. Cancers in the RB group were significantly more predominant in the anastomosis area (P<0.05). Within the tumour, downregulation of RUNX3 expression ranged from 74.7 to 85.7% in the three groups. The rate of downregulation of RUNX3 of adjacent mucosa was 39.2% (11 in 28 cases) in RB and 47.6% (10 in 21 cases) in RM, which are significantly higher than that of the GCI group (19.5%, 17 in 87 cases). In noncancerous mucosa of the remnant stomach in the RB group, RUNX3 expression decreased more near the anastomosis area. In the RM group, however, there were no significant differences in RUNX3 expression by sampling location. Based on RUNX3 downregulation and clinical features, residual stomach mucosa of the RM group would have a higher potential of gastric carcinogenesis compared to the RB or GCI group. Gastric stump mucosa of the RB group has higher potential especially than other areas of residual stomach mucosa. Measurement of RUNX3 expression and detection of RUNX3 methylation in remnant gastric mucosa may estimate the forward risk of carcinogenesis in the remnant stomach.

Differential gene expression profiles of radioresistant oesophageal cancer cell lines established by continuous fractionated irradiation.

Radiation therapy is a powerful tool for the treatment of oesophageal cancer. We established radioresistant cell lines by applying fractionated irradiation in order to identify differentially expressed genes between parent and radioresistant cells. Six oesophageal cancer cell lines (TE-2, TE-5, TE-9, TE-13, KYSE170, and KYSE180) were treated with continuous 2 Gy fractionated irradiation (total dose 60 Gy). We compared expression profiles of each parent and radioresistant lines on a cDNA microarray consisting of 21168 genes. In the fractionated irradiation trial, four radioresistant sublines (TE-2R, TE-9R, TE-13R, KYSE170R) were established successfully, and we identified 19 upregulated and 28 downregulated genes common to radioresistant sublines. Upregulated genes were associated with apoptosis and inflammatory response (BIRC2 and COX-2), DNA metabolism (CD73), and cell growth (PLAU). Downregulated genes were associated with apoptosis (CASP6), cell adhesion (CDH1 and CDH3), transcription (MLL3), and cell cycle (CDK6). Some of these genes were known to be associated with radiation response, such as COX-2, but others were novel. Reverse transcription-polymerase chain reaction confirmed that genes selected by cDNA microarray were overexpressed in clinical specimens of radioresistant cases. Global gene analysis of radioresistant sublines may provide new insight into mechanisms of radioresistance and effective radiation therapy.

Overexpression of dopa decarboxylase in peritoneal dissemination of gastric cancer and its potential as a novel marker for the detection of peritoneal micrometastases with real-time RT-PCR.

We previously performed a global analysis of the gene expression of gastric cancer cell lines established from metastases to the peritoneal cavity with the cDNA microarray method, which made it possible to analyse the expression of approximately 21168 genes for the identification of novel markers for the detection of micrometastases in the peritoneal cavity. One of the upregulated genes is dopa decarboxylase (DDC), which is responsible for the synthesis of the key neurotransmitters dopamine and serotonine. We have examined its potential as a novel marker for the detection of peritoneal micrometastases of gastric cancer.DDC mRNA in the peritoneal wash from 112 gastric cancer patients was quantified for comparison of carcinoembryonic antigen (CEA) mRNA by means of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) with a fluorescently labelled probe to predict peritoneal recurrence. The quantity of DDC and CEA correlated with wall penetration. Real-time RT-PCR could quantitate 10-10(6) DDC-expressing gastric cancer cells per 10(7) mesothelial cells. The cutoff value was set at the upper limit of the quantitative value for noncancer patients, and those above this cutoff value constituted the micrometastasis (MM+) group. Of 15 cases with peritoneal dissemination, 13 were MM+DDC (87% sensitivity), and one of 48 t1 cases was MM+ (98% specificity). DDC levels in peritoneal washes from patients with synchronous peritoneal metastases were more than 50 times higher than in those from patients without metastasis (P<0.01). For 15 cases of peritoneal dissemination (seven cases were cytologically positive), DDC was positive in 13 cases (87% sensitivity), but CEA failed to detect micrometastases in four cases (73% sensitivity), indicating that DDC is in some cases superior to CEA for the detection of peritoneal micrometastases of gastric cancer in terms of sensitivity as well as specificity, especially for poorly differentiated adenocarcinomas. A combination of CEA and DDC improved the accuracy of diagnosis up to 94%. These results suggest that DDC is potentially a novel marker for peritoneal dissemination of gastric cancer and that quantitative RT-PCR of DDC is reliable and efficient for the selection of patients for adjuvant intraperitoneal chemotherapy to prevent peritoneal recurrence.

The Schizosaccharomyces pombe spo20(+) gene encoding a homologue of Saccharomyces cerevisiae Sec14 plays an important role in forespore membrane formation.

The Schizosaccharomyces pombe spo20-KC104 mutation was originally isolated in a screen for sporulation-deficient mutants, and the spo20-KC104 mutant exhibits temperature-sensitive growth. Herein, we report that S. pombe, spo20(+) is essential for fission yeast cell viability and is constitutively expressed throughout the life cycle. We also demonstrate that the spo20(+) gene product is structurally homologous to Saccharomyces cerevisiae Sec14, the major phosphatidylinositol transfer protein of budding yeast. This structural homology translates to a significant degree of functional relatedness because reciprocal complementation experiments demonstrate that each protein is able to fulfill the essential function of the other. Moreover, biochemical experiments show that, like Sec14, Spo20 is a phosphatidylinositol/phosphatidylcholine-transfer protein. That Spo20 is required for Golgi secretory function in vegetative cells is indicated by our demonstration that the spo20-KC104 mutant accumulates aberrant Golgi cisternae at restrictive temperatures. However, a second phenotype observed in Spo20-deficient fission yeast is arrest of cell division before completion of cell separation. Consistent with a direct role for Spo20 in controlling cell septation in vegetatively growing cells, localization experiments reveal that Spo20 preferentially localizes to the cell poles and to sites of septation of fission yeast cells. We also report that, when fission yeasts are challenged with nitrogen starvation, Spo20 translocates to the nucleus. This nuclear localization persists during conjugation and meiosis. On completion of meiosis, Spo20 translocates to forespore membranes, and it is the assembly of forespore membranes that is abnormal in spo20-KC104 cells. In such mutants, a considerable fraction of forming prespores fail to encapsulate the haploid nucleus. Our results indicate that Spo20 regulates the formation of specialized membrane structures in addition to its recognized role in regulating Golgi secretory function.

[A case of multiple liver metastases from breast cancer successfully treated with intra-arterial administration of paclitaxel].

A 57-year-old woman underwent modified radical mastectomy for left breast cancer (T4bN1M1: stage IV) in September 1999. Four-cycle CAF therapy had been administered as adjuvant therapy, but multiple recurrent tumors in the liver had grown bigger and the tumor marker (CEA) increased in value. Because CAF therapy was not effective, we tried to treat the patient with systemic and intra-arterial chemotherapy using paclitaxel. The side effects of this treatment were mild nausea and appetite loss, which required no treatments. This treatment reduced the multiple liver metastases on an abdominal CT and was thought to produce a partial response (PR). The time to response was the 101st day and PR has been continued.

Selective thermocoagulation of unresectable pancreatic cancers by using radiofrequency capacitive heating.

Recent investigations indicated that hyperthermia has antitumor effects. Several interstitial hyperthermic techniques were developed, and their clinical usefulness and safety were evaluated. However, few authors have attempted to study the use of interstitial hyperthermia for the treatment of pancreatic carcinomas. Therefore the efficacy of local selective thermocoagulation by radiofrequency was evaluated in 20 patients with unresectable carcinomas of the pancreas. A laparotomy and radiofrequency heating were performed in 20 patients with unresectable pancreatic carcinomas after informed consent. Local heat coagulation was induced by a 13.56-MHz radiofrequency pulse, produced by the heating apparatus. Four 2-cm needle electrodes were placed in the tumor, in a square array, at intervals of 2 cm. The heat was then administered for 15 min at a controlled temperature of 50 degrees C in the radiofrequency field (2x2x2 cc). All the patients were evaluated by computed tomographic scanning. Tumor markers in the blood also were assayed before and after the heating. Follow-up computed tomographic scans demonstrated that the tumor mass was enhanced heterogeneously, and after selective thermocoagulation, images revealed a change to a homogeneous low-density area. The blood levels of tumor markers decreased to below pretreatment values in 15 patients. Of the 20 cases treated with thermocoagulation, two had critical complications. One patient had septic shock, and another had gastrointestinal bleeding. The other 18 patients had no significant complications. These observations suggest that the selective thermocoagulation of tumor tissues using this equipment was relatively safe. These results justify further clinical trials for the treatment of patients with unresectable tumors without metastasis, or patients with benign pancreatic tumors such as insulinomas.

Selective ablation of porcine and rabbit liver tissue using radiofrequency: preclinical study.

Temperature changes and their distribution induced by 13.56-MHz radiofrequency (RF) heating of agar phantom and porcine and rabbit liver were investigated. It was possible to produce selective local heating of approximately 50 degrees C in the RF field of 2 x 2 x 2 cm(3) of the pig or rabbit liver. Coagulation necrosis after heating became pronounced and the margin between the coagulated lesion and normal tissue became clearer with time. Within 1 week after RF heating at 50 degrees C for 20 min, the coagulated area was replaced selectively and totally by necrotic tissue.

[Selective thermocoagulation of unresectable malignant tumors using radiofrequency].

Based on our experimental findings on porcine liver, we have been conducting a clinical trial of selective hyperthermia by radiofrequency (RF) capacitive heating with laparotomy for patients with unresectable malignant tumors. In 10 patients with malignant tumors (8 carcinoma of the pancreas, 2 carcinoma of the gallbladder), laparotomy and RF heating were performed after informed consent. The local heat coagulation was produced by heating equipment using 13.56 MHz radiofrequency produced by Omron Corporation, Japan. Four 2-cm electrode needles were placed in the tumor in a square array at intervals of 2.0 cm. Hyperthermia was given for 30 min with a controlled temperature of 50 degrees C in the RF field (2 x 2 x 2 cm3). That of the surrounding area was maintained at less than 40 degrees C. The calculated volume treated by RF ranged between (2 x 2 x 2 cm3) x 1 and (2 x 2 x 2 cm3) x 6. We followed all patients by computed tomographic (CT) scan 2 weeks after coagulation. Tumor markers in the blood were assayed before and 14 days after heating. Follow-up CT scans demonstrated that after the tumor mass had been heterogeneously enhanced, it changed to a homogeneous low-density area in 6 of 10 patients. The levels of tumor markers decreased to lower than the pre-treatment values in 9 of 10 patients. In all patients, the changes in CT scans and/or decrease in the markers were confirmed. Complications such as bleeding or abscess formation were not observed. It was suggested that the selective hyperthermia was safely produced by this equipment. The encouraging results in these patients justify further clinical trials.

Extensive tissue oxygenation associated with mitochondrial DNA mutations.

Extensive tissue oxygenation in the mitochondrial myopathy patients caused by the mitochondrial DNA mutations was first demonstrated noninvasively by a tissue oxymeter measuring near infrared light. The extent of oxygenation of the tissue due to dysfunction of mitochondria correlated with the seriousness of mitochondrial DNA mutations resulting in defects in oxidative phosphorylation system, and causing suppressed oxygen utilization. Such oxygen stress furthers mitochondrial DNA mutations during the progressive course of the disease. This noninvasive diagnosis will find useful application in the diagnosis and management of patients of advanced age.

Purification and characterization of Bordetella calmodulin-like protein.

Bordetella calmodulin-like protein was purified from culture supernatant fluid of B. pertussis, B. parapertussis and B. bronchiseptica by successive chromatography on hydroxyapatite, Toyopearl HW-50F and QAE-Toyopearl 550C columns. The purified calmodulin-like protein appeared to be homogeneous by SDS-polyacrylamide gel electrophoresis. The apparent molecular mass of calmodulin-like protein on SDS-polyacrylamide gel electrophoresis was 10 kDa, which was smaller than bovine brain calmodulin (17 kDa). The purified calmodulin-like protein activated both Bordetella adenylate cyclase and mammalian phosphodiesterase in a Ca(2+)-dependent manner. This activation was inhibited by calmodulin antagonists. The calmodulin-like protein, like calmodulin, was retained by a hydrophobic resin in the presence of Ca2+ and eluted by the addition of EDTA. These results indicated that the Bordetella calmodulin-like protein is closely related to calmodulin. As a putative calmodulin the extracellular calmodulin may be involved in Bordetella pathogenesis.