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.

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.

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.

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.

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.

Fluorometric study for the noninvasive determination of cellular viability in perfused rat liver.

Pyridine nucleotide fluorescence in perfused rat liver for the noninvasive determination of donor graft viability was investigated in relation to other metabolic indices, such as NAD concentration, adenine nucleotides, and mitochondrial phosphorylative activity. The amplitude between oxidation and reduction levels (RxA) in fluorometric trace, and the slope or the velocity of the trace curve from oxidation to reduction (RxV) were determined by the measurement of fluorescence from NAD(P)H, using a new fluorometric device, RxA and RxV decreased proportionally to the duration of preservation period (6, 12, 24, 48 hr) in simple cold storage. Other values of hepatic cell viability, such as total adenine nucleotides, energy charge, and mitochondrial phosphorylation rate, were simultaneously measured and also decreased proportionally to the duration of preservation period. There were close positive correlations between the percentage of RxA and NAD concentration (r = 0.724, p less than 0.01), between the percentage of RxA and total adenine nucleotides (r = 0.887, p less than 0.01), between the percentage of RxV and energy charge (r = 0.715, p less than 0.01), and between the percentage of RxV and phosphorylation rate/cytochrome a(+a3) (r = 0.837, p less than 0.01). These results suggest that this fluorometric method can provide an accurate noninvasive evaluation of donor graft viability--and, unlike the present indices of energy metabolism, it may be applied to evaluate the primary nonfunctioning graft prior to transplantation.

Pyridine nucleotide fluorometry in preserved porcine liver with fluorocarbon emulsion.

Oxidation-reduction changes in pyridine nucleotide fluorescence were investigated in perfused and preserved porcine liver. A fluorocarbon emulsion (FC-43) was administered to the perfusate as an oxygen carrier to obtain full oxidation level by portal perfusion at a physiological low flow rate. A satisfactory reading was obtained by portal perfusion with EuroCollins' solution containing 10% v/v FC-43 at a rate of 0.5 ml/min/g liver. The amplitude (R x A) and the changing velocity (R x V) from full oxidation to full reduction were determined in the resultant trace curve. Both R x A and R x V decreased in inverse proportion to the duration of preservation period (3, 6, 12 hr). Adenine nucleotide content, hepatic energy charge level, and ketone body ratio in the tissue were simultaneously measured, and they also decreased proportionally to the duration of preservation. There were close positive correlations between R x A and total adenine nucleotide concentration (r = 0.841, P less than 0.01), between R x V and energy charge (r = 0.787, P less than 0.01), and between R x V and tissue ketone body ratio (r = 0.881, P less than 0.01). These results suggest that pyridine nucleotide fluorometry can accurately follow the cellular function of isolated porcine liver by administration of FC-43 in perfusate. This fluorometry may also have potential application in evaluating viability of a large organ like the human liver graft.

Evaluation of cytoprotective drugs for liver preservation by pyridine nucleotide fluorometry.

The cytoprotective effects of membrane-stabilizing drugs, such as chlorpromazine, allopurinol, dibucaine, phenoxybenzamine, and OP41483 (prostacyclin analogue), administered to perfusate and preservation medium were studied in rat liver, after 24 hours' preservation, by assessment of pyridine nucleotide fluorescence. On the fluorometric trace curve, amplitude (RxA) and velocity (RxV) from oxidation to reduction were determined. Percent decrease of RxA (%RxA) and that of RxV (%RxV) after 24 hours' preservation were calculated. At the end of preservation, the concentration of total adenine nucleotides of the liver, hepatic adenylate energy charge, and prepared mitochondrial oxidative phosphorylative activity were also measured. In the groups given phenoxybenzamine, dibucaine, and allopurinol, there was no significant difference among these parameters. In the chlorpromazine group, energy charge and %RxV were higher than in the drug-free group (p less than 0.05). In the OP41483 group, both energy charge and phosphorylation rate were significantly higher (p less than 0.05) and %RxV was significantly high (p less than 0.01) at concentrations of more than 3 nmol/L, compared with the values for those without drugs. These results suggest that the Redoximeter can provide accurate information on the effectiveness of cytoprotective drugs. It is also suggested that OP41483 has potential application for maintaining graft viability for human liver transplantation.

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.

Bordetella heat-labile toxin causes release of radioactivity from smooth muscle cells labeled with [14C]arachidonic acid.

The effect of Bordetella heat-labile toxin (HLT) on cultured vascular or tracheal smooth cells labeled with [14C]arachidonic acid for 3 h was examined. At 37 degrees C, in the presence of Ca2+, HLT induced the release of radioactive substances from the cells in a dose-dependent manner but HLT had no effect on release of radioactivity from cells at 0 degrees C or in the absence of Ca2+. After cells were exposed to HLT, a 2-h lag period occurred before release of radioactivity was detected. The substances released from cells by HLT were identified as arachidonic acid and phosphatidylcholine.

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.

Color Doppler imaging of orbital venous flow in dysthyroid optic neuropathy.

Color Doppler imaging was performed to evaluate the venous stasis in 39 orbits, including 9 optic neuropathy orbits, of 20 patients with dysthyroid ophthalmopathy and 22 orbits of 11 healthy subjects. The superior ophthalmic vein (SOV) was detected in 26 dysthyroid ophthalmopathy orbits and in 13 control orbits. The blood flow in the SOV was in the anteroposterior direction in 20 dysthyroid ophthalmopathy orbits and in 13 control orbits. Reversed flow, ie, the flow in the posteroanterior direction, was seen in 6 dysthyroid ophthalmopathy orbits and in none of the control orbits. In dysthyroid ophthalmopathy orbits, the blood flow in the SOV was reversed in 5 (36%) of the 14 orbits with apical orbital crowding observed on computed tomography, which means there was compression of the optic nerve by enlarged extraocular muscles, as compared to in 1 (4%) of the 25 orbits without apical orbital crowding (P < 0.05). The percentage of the orbits having reversed flow in SOV was 44% of dysthyroid ophthalmopathy orbits with optic neuropathy as opposed to 7% of those without optic neuropathy (P < 0.05). Reversed blood flow in the SOV strongly supported the existence of severe venous stasis in the orbits, which may be related to the development of dysthyroid optic neuropathy.

Role of rectus muscle enlargement in clinical profile of dysthyroid ophthalmopathy.

Using logistic regression analysis, a correlation between enlargement patterns of the 4 rectus muscles and the occurrence of a variety of dysthyroid ophthalmopathy-associated ocular symptoms were analyzed in 698 eyes of 349 patients with this disorder. The condition of each rectus muscle was evaluated by computed tomography, and the various ocular symptoms were classified according to the Inoue classification system. The enlargement pattern, particularly when involving the inferior and medial recti, was found to have a strong statistical correlation with the occurrence of individual ocular symptoms. Additionally, the probabilities of the occurrence of most of the ocular symptoms were generally greater in those eyes with 2-, 3- or 4-muscle enlargement patterns than in those eyes with one or no enlarged rectus muscle.

Antigenic structure and relationship between serotypes 1 and 2 of Haemophilus paragallinarum.

Antigenic structure and relationship between serotypes 1 and 2 of Haemophilus paragallinarum were analyzed by the rapid plate agglutination and cross-absorption tests. Encapsulated strain forming iridescent colony type of both serotypes 1 and 2 had at least three antigens: heat-labile and trypsin-sensitive (L), heat-labile and trypsin-resistant (HL), and heat-stable and trypsin-resistant (HS). The L was a major antigen located in a surface and divided serologically into three parts: L1, L2, and L3. The L1 was specifici to serotype 1, the L2 was specific to serotype 2, and the L3 was a common antigen shared by serotypes 1 and 2. The HL and HS were common antigens between serotypes. By trypsinization or heating at 121 C, L antigen lost its agglutinability and agglutinin-producing ability. Nonencapsulated organisms forming noniridescent colony type lacked the L antigen. These results suggested that antigenic structure of H paragallinarum serotypes 1 was L1, L3, HL and HS, while serotype 2 was L2, L3, HL, and HS.

Biologic and serologic relationships between Page's and Sawata's serotypes of Haemophilus paragallinarum.

Biologic and serologic relationships between Page's serotypes A, B, and C and Sawata's serotypes 1 and 2 of Haemophilus paragallinarum were investigated. Of the 7 Page's serotype strains testes (which were biologically confirmed as H paragallinarum), serotype B strains Spross and 0222 (which produced a low-iridescent smooth-type colony) required V factor, but did not require chicken serum for growth. Serotype A strains 083, W, Georgia, and Germany and serotype C strain Modesto, as well as Sawata's serotype 1 strain 221 and serotype 2 strain H-18 (which produced a high-iridescent smooth-type colony) required V factor and chicken serum for growth. Strains 083, W, Georgia, and Germany of Page's serotype A and Modesto strain of serotype C, as well as strain 221 of Sawata's serotype 1 and H-18 of serotype 2, had a heat-labile, trypsin-sensitive L antigen. Because serotype A strains had serotype 1-specific antigen and common L antigen, which is shared by serotypes 1 and 2, these strains corresponded to Sawata's serotype 1. Serotype C strain Modesto, which had serotype 2-specific antigen and common L antigen, corresponded to serotype 2. However, serotype B strains Spross and 0222 lacked serotype B-specific L antigen, and were not typeable.

Immunologic relationship between Page's and Sawata's serotype strains of Haemophilus paragallinarum.

Immunologic relationship among Page's A, B, and C and Sawata's 1 and 2 serotype strains of Haemophilus paragallinarum was investigated. In cross-protection tests, two distinct type-specific immunities existed among the strains used. All Page's serotype A strains (083, W, Georgia, and Germany) were immunologically similar to Sawata's serotype 1 strain 221, although serotype C strain Modesto was similar to serotype 2 strain H-18. Cross-protection was not observed between these two serotypes. However, strains Spross and 0222 of serotype B lacked pathogenicity and protective antigenicity.

Hemagglutinin of Haemophilus paragallinarum serotype 2 organisms: occurrence and immunologic properties of hemagglutinin.

Hemagglutinating properties of Haemophilus paragallinarum serotype 2 and serotype C against freshly collected and glutaraldehyde (GA)- fixed chicken RBC were investigated. Different from serotype 1, the nontreated organisms of serotype 2 and serotype C lacked hemagglutinating activity. However, when the organisms were treated with potassium thiocyanate (KSCN) and/or sonication, activity occurred not only against GA-fixed chicken RBC, but also against GA-fixed RBC of various animal species. The maximum hemagglutination titer (1:64 to 1:256) was obtained against GA-fixed RBC with the KSCN-treated organisms that were also sonicated. The activity was inactivated by heating at 100 C or by treatments with formalin or trypsin, but not by treatments with hyaluronidase or neuraminidase. By using KSCN-treated and sonicated organisms and GA-fixed chicken RBC, a detection of hemagglutination-inhibition (HI) antibody was possible. The HI tests showed that serotype 2 hemagglutinin was immunologically distinct from serotype 1 and that the HI antibody correlated to protective activity against challenge exposure with serotype 2 and serotype C. Chicks having HI antibody greater than 1:5 were protected against challenge exposure with homologous type, but were not protected with serotype 1. Applicability of the HI test was also shown for evaluating protective potency in the serotype 2 vaccine, as well as in the serotype 1 vaccine.

Relationship between protective activity and antigen structure of Haemophilus paragallinarum serotypes 1 and 2.

The object in this investigation was to determine the relationship between protective activity and antigenic structure of Haemophilus paragallinarum, serotypes 1 and 2. A close relationship exists in both serotypes between protective activity and colonial phenotypic form (iridescent and noniridescent). Protective activities of both serotypes were related to a heat-labile, trypsin-sensitive (L) antigen of iridescent form that produced serotype-specific agglutinin to chickens. The chickens having the agglutinins were protected against challenge exposure with homologous strain, but not with heterologous strain. The chickens injected with unencapsulated organisms of noniridescent form that were derived from encapsulated organisms of iridescent form failed to produce both serotype-specific agglutinins and protection against challenge exposure with homologous strain. Most of the chickens injected with serotype 1 strain produced both hemagglutination-inhibition antibody and serotype 1-specific agglutinin, whereas those injected with serotype 2 produced serotype 2-specific agglutinin and protected against homologous challenge exposure. The protective activity was found in saline extract derived from encapsulated organisms of serotype 1, but was absent in those of serotype 2.

Mechanism of action of Bordetella heat-labile toxin on vascular smooth muscle strips and cells.

The mechanism of action of Bordetella heat-labile toxin (HLT) was analyzed in vitro using vascular smooth muscle strips (VSMS) and cultured cells (VSMC), from aortas of pig, guinea pig, rabbit, rat or mouse. HLT induced contractions to both VSMS and VSMC, but not other types of tissues and cells. HLT induced the increase of Ca2(+)-influx in parallel with the contraction. These HLT activities were not influenced by the addition of verapamil, diltiazem, or TMB-8, though a certain extension of the lag period was seen. The contractile action on VSMC was not influenced by a various blockers of cascades or systems. The HLT-induced contraction in VSMC was completely inhibited by H-7, but not by H-8. HLT did not induce specific activation of the protein kinases in VSMC. HLT induced the permeability changes in VSMS or VSMC membranes to cyclic nucleotides or trypan blue. Both HLT-induced contraction and permeability change were inhibited by dextran of M.W. 8,000. 125I-HLT bound immediately to VSMC depending on the HLT dose. At 37 degrees C, the binding ratio in maximum was approximately 0.8% of total HLT added at 60 min after exposure. At 4 degrees C, it was less than 20% of that at 37 degrees C. The labeled HLT binding to VSMC was released readily by washing. From these results, possible mechanism of HLT action was discussed.

[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.