Solution structure of the IRF-2 DNA-binding domain: a novel subgroup of the winged helix-turn-helix family.

BACKGROUND: The transcription of interferon (IFN) and IFN-inducible genes is mainly regulated by the interferon regulatory factor (IRF) family of proteins, which recognize a unique AAGTGA hexamer repeat motif in the regulatory region of IFN genes. A DNA-binding domain of approximately 100 amino acids has been commonly found in the IRF family of proteins, but it has no sequence homology to known DNA-binding motifs. Elucidation of the structures of members of the IRF family is therefore useful to the understanding of the regulation and evolution of the immune system at the structural level. RESULTS: The solution structure of the DNA-binding domain of interferon regulatory factor-2 (IRF-2) has been determined by NMR spectroscopy. It is composed of a four-stranded antiparallel beta sheet and three alpha helices, and its global fold is similar to those of the winged helix-turn-helix (wHTH) family of proteins. A long loop (Pro37-Asp51) is found immediately before the HTH motif, which is not found in other wHTH proteins. The NMR signals of residues in this long loop, as well as the second helix of the HTH motif, are strongly affected upon the addition of the hexamer repeat DNA, suggesting that these structural elements participate in DNA recognition and binding. CONCLUSIONS: The structural similarity of the DNA-binding domain of IRF-2 with those of proteins in the wHTH family shows that the IRF proteins belong to the wHTH family, even though there is no apparent sequence homology among proteins of the two families. The sequential structure alignment program (SSAP) shows that IRF-2 has a slightly different structure from typical wHTH proteins, mainly in the orientation of helix 2. The IRF family of proteins should therefore be categorized into a subfamily of the wHTH family. The evidence here implies that the evolutional pathway of the IRF family is distinct from that of the other wHTH proteins, in other words, the immune system diverged from an evolutional stem at an early stage.

Role of carcinoembryonic antigen in the progression of colon cancer cells that express carbohydrate antigen.

Carcinoembryonic antigen (CEA) has been reported to promote the metastatic potential in some experimental tumors. Adhesion molecules are known to play an important role in the process of metastasis. Cytokines, including interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), which are produced by Kupffer cells, induce endothelial cells to express adhesion molecules. As a result, the present study was designed to investigate whether the interaction between CEA and Kupffer cells accelerated the metastatic potential of tumors in the liver. Kupffer cells isolated from the liver of male BALB/c mice were cultured with CEA, either with or without the addition of a cytokine inhibitor. The levels of IL-1beta and TNF-alpha were examined in a culture medium. An adhesion assay of colon cancer cell lines to human umbilical vein endothelial cells was also performed. When CEA was added to the Kupffer cell culture medium, cytokines were produced. Elevated levels of cytokines appeared to lead to increased rates of adhesion of cancer cells to endothelial cells. However, these phenomena were blocked by the addition of cytokine inhibitors. CEA stimulated Kupffer cells to produce cytokines. An elevated number of cytokines have been proven to promote the expression of adhesion molecules in endothelial cells. These processes are therefore considered to contribute to the metastasis of malignant cells to the liver. These results suggest that cytokine inhibitors may therefore play an important role in the inhibition of hepatic metastasis.

Quantitative assessment of severity of ventricular septal defect by three-dimensional reconstruction of color Doppler-imaged vena contracta and flow convergence region.

BACKGROUND: The aim of the present study was to investigate the feasibility and potential value of the computer-controlled, 3D, echocardiographic reconstruction of the color Doppler-imaged vena contracta (CDVC) and the flow convergence (FC) region as a means of accurately and quantitatively estimating the severity of a ventricular septal defect (VSD). METHODS AND RESULTS: We performed a 3D reconstruction of the CDVC and the FC region in 19 patients with an isolated VSD using an ultrasound system interfaced with a Tomtec computer. The variable asymmetric geometry of the CDVC and the FC region could be 3D-visualized in all patients. The 3D-measured areas of CDVC correlated well with volumetric measurements of the severity of VSD (r=0.97, P:<0.001). Regression analysis between the shunt flow rate (calculated from the product of the area of CDVC and the continuous Doppler-derived velocity time integral) and the corresponding reference results (calculated by cardiac catheterization) demonstrated a close correlation (r=0.95, P:<0.001). There was also a good correlation between shunt flow rates calculated using the conventional 2D, 1-axis measurement of the FC isovelocity surface area with the hemispheric assumption (r=0.95, P:<0.001); shunt flow rates calculated using 3D, 3-axis measurements of the FC region (r=0.97, P:<0.01); and reference results by cardiac catheterization. However, the 2D method substantially underestimated the actual shunt flow rate. CONCLUSIONS: The 3D reconstruction of the CDVC and the FC region may aid in quantifying the severity of VSD.

Inhibitory effects of safe and novel SOD derivatives, galactosylated-SOD, on hepatic warm ischemia/reperfusion injury in pigs.

BACKGROUND/AIMS: Oxygen-derived free radicals such as superoxide play an important role in ischemia/reperfusion (IR) injury during and after extensive liver surgery or liver transplantation. Superoxide dismutase (SOD) has protective effects against hepatic IR injury. The effect of native SOD is, however, limited because of rapid elimination from the blood circulation and poor affinity for liver cells. It was reported by our collaborators that a SOD derivative modified with galactose (Gal-SOD) was selectively delivered well to hepatocytes by direct attachment to galactose receptors. In the present study, the efficacy of this agent for attenuating hepatic warm IR injury was investigated using the pig model. METHODOLOGY: After 45-min clamping of the hepatic artery and portal vein, pigs were divided into 3 groups according to the following treatments. Ten milliliters of normal saline in Group 1 (n=5), 10,000 units/kg of native SOD in Group 2 (n=5) and 10,000 units/kg of Gal-SOD in Group 3 (n=5) were given just prior to hepatic reperfusion. Liver function including clearance of total bile acid (TBA) and hyaluronic acid (HA) was investigated. Lipid peroxidase of the liver tissue (LPO) and histological findings were examined. In addition, survival rates of the pigs in each group were evaluated. RESULTS: The survival rates at the 7th day after the operation were 60%, 80%, 100% in Groups 1, 2 and 3, respectively. Liver function tests, clearance of TBA and HA, and LPO levels were significantly improved in Groups 3 over findings in Groups 1 and 2. Congestion of hepatic tissues and vacuolization of hepatocytes in Group 3 were less than those in Groups 1 and 2. These results suggested that oxygen-derived free radicals were scavenged by Gal-SOD and IR injury was attenuated. CONCLUSIONS: A safe and novel agent, Gal-SOD has a protective effect against hepatic warm IR injury.

Sequential evaluation of left ventricular myocardial performance in children after anthracycline therapy.

This study prospectively assessed subclinical cardiotoxicity in patients undergoing chemotherapy by using the Tei index combining systolic and diastolic time intervals. A significant difference in the Tei index was observed between patients who received a low dose and those who received a moderate to high dose of anthracycline antibiotic drugs. The Tei index is a sensitive, accurate, and easy approach for detecting subclinical anthracycline cardiotoxicity.

Expression of p53 protein in gastric carcinomas is not independently prognostic.

BACKGROUND: Biologic significance of p53 protein expression in gastric carcinomas is unclear. The relationship between p53 expression and survival after curative operations for gastric carcinoma was evaluated. METHODS: Paraffin-embedded specimens from 135 patients who underwent curative resection of gastric carcinoma were analyzed immunohistochemically for p53 expression. RESULTS: Immunoreactivity of p53 was found in 37 of 135 gastric tumors. Six of 48 early gastric carcinomas expressed focally scattered p53. Pattern of p53 expression in the majority of advanced carcinomas was diffuse. In univariate analyses a significant relationship with survival was found for tumor size (p < 0.01), depth of invasion (p < 0.01), nodal involvement (p < 0.01), and p53 expression (p < 0.01). Significant relationships were found between p53 expression and nodal involvement (p < 0.01) and depth of invasion (p < 0.01). A multivariate analysis revealed that the independent predictors of recurrence were nodal involvement (p = 0.009) and depth of invasion (p = 0.009). In a logistic multiple regression analysis p53 expression was not independently related to the parameters, which suggests an aggressive tumor. CONCLUSIONS: Our results suggest that p53 expression did not correlate with the aggressiveness of gastric carcinomas and failed to be an independent predictor of outcome.

erbB-2 expression in well-differentiated adenocarcinoma of the stomach predicts shorter survival after curative resection.

BACKGROUND: Correlation between erbB-2 expression, histologic type of gastric carcinoma, and survival after curative resection was evaluated. METHODS: Paraffin-embedded specimens from 120 patients who underwent curative resection of gastric carcinoma were analyzed immunohistochemically for the expression of erbB-2. RESULTS: Enhanced erbB-2 expression correlated with tumor stage and depth of invasion. Well-differentiated adenocarcinomas had a higher incidence of erbB-2 expression than did poorly differentiated carcinomas. Survival rates of 33 patients with erbB-2-positive carcinomas were significantly lower than those of 87 with erbB-2-negative carcinomas (p < 0.001). Survival rates of patients with well-differentiated adenocarcinomas that were erbB-2 positive were significantly lower than those that were erbB-2 negative (p < 0.001). However, the presence of erbB-2 was not associated with altered survival in 46 patients with poorly differentiated carcinomas. Multivariate analysis of all 120 patients revealed that independent predictors of recurrent disease include nodal involvement (p = 0.003) and erbB-2 expression (p = 0.0051). CONCLUSIONS: Our results suggest that erbB-2 expression is a new marker associated with poor prognosis in well-differentiated gastric adenocarcinomas.

Sclerosing encapsulating peritonitis combined with peritoneal encapsulation.

The combined occurrence of idiopathic sclerosing encapsulating peritonitis and peritoneal encapsulation is described. A 52-year-old man presented with intestinal obstruction. The results of preoperative examinations were suggestive of sclerosing encapsulating peritonitis. Laparotomy revealed the concurrence of peritoneal encapsulation and sclerosing encapsulating peritonitis. The thick membrane of sclerosing encapsulating peritonitis was freed with multiple incisions. After operation, the patient reverted to the preoperative state. The condition, however, was alleviated with conservative therapy consisting of intravenous hyperalimentation and nasogastric suction. To our knowledge, the combined occurrence of sclerosing encapsulating peritonitis and peritoneal encapsulation has never before been reported.

Expression of Kirsten-ras p21 in gastric cancer correlates with tumor progression and is prognostic.

The purpose of this study was to determine the correlation between expression of ras oncoproteins and the tumor stage or outcome of patients with gastric carcinoma. After the specificity of each anti-ras monoclonal antibody was confirmed by protein immunoblot analysis, immunohistochemical assays for a common-ras antigen present in N-, Harvey- and Kirsten (K)-ras oncoproteins, as well as for K-ras specific antigen, were performed on paraffin-embedded carcinoma tissue from 110 patients who underwent curative resection. By Western blot analysis, there was more p21 in fresh cancer specimens than in normal specimens. K-ras expression distinguished advanced from early gastric carcinoma and correlated with depth of cancer invasion. Among the 110 patients, survival rates of those with carcinomas positive for the common-ras or K-ras antigens were significantly lower than of those with antigen-negative carcinomas (p < 0.05). In a multivariate analysis, nodal involvement (p = 0.002), serosal invasion (p = 0.012) and K-ras p21 expression (p = 0.044) were independently predictive of the recurrence. These results suggest that K-ras p21 is a useful marker of tumor progression and poor prognosis after curative resection.

MRI facilitated a diagnosis of endometriosis of the rectum.

A 51-year-old pre-menopausal Japanese woman suffering from chronic lower abdominal pain was referred to our hospital. A barium enema showed a stenotic lesion in the recto-sigmoid region, and a pelvic computed axial tomography (CAT) scan revealed a thickened rectal wall. A colonoscopic examination showed the rectum to be constrictive, but the mucosa appeared to be intact. Magnetic resonance imaging (MRI) with T1 high-intensity revealed a cystic lesion in the thickened wall of the rectum, which led us to suspect possible bowel endometriosis. Part of the biopsy specimen showed endometrial epithelium within the interstitial layer of histologically normal mucosa; finally, endometriosis of the rectum was diagnosed. The patient became asymptomatic after the initiation of hormonal treatment and later experienced spontaneous menopause. MRI was effective for diagnosis and the patient did not undergo unnecessary laparotomy. Although bowel endometriosis is generally diagnosed by means of resected specimens, in our patient, diagnosis was made using MRI and biopsy, and hormonal therapy had an effective role as a bridge to menopause.

A second primary esophageal cancer developing 7 years after chemoradiotherapy for advanced esophageal cancer.

We report a rare case of advanced carcinoma and a second primary carcinoma of the esophagus, both of which were successfully cured by chemotherapy and operation at different times. In 1991, a 38-year-old Japanese man was diagnosed with advanced esophageal cancer, which was unresectable because of the bronchial invasion of the tumor. He was given chemotherapy with cisplatin (CDDP), combined with radiotherapy. During a 4-year follow-up, neither regrowth of the primary tumor nor distant metastasis occurred. In 1995, esophagoscopy demonstrated a lugol-unstained region located 3 cm distal from the area of radiation to the primary lesion shown by esophagography. Histological examination of a biopsy specimen showed the mucosa to be normal. Nevertheless, yearly surveillance by endoscopy and histological examinations showed that the mucosa of the esophagus gradually began to demonstrate mild dysplasia, followed by severe dysplasia; in 1998, a diagnosis of squamous cell carcinoma was made. Esophagectomy with lymph node dissection was performed. Microscopic examination revealed that there had been pathologic complete response for the original advanced esophageal cancer.

Human colon cancer produces a factor which induces the proliferation of venous endothelial cells.

This study was performed to investigate whether colorectal cancers produce proliferative factors for venous endothelial cells, and whether the proliferative activity is related to basic FGF and VEGF and also to the clinicopathological findings. Surgically resected specimens of 17 colorectal cancer patients were fragmented and cultured, and the supernatant was collected. A human umbilical endothelial cell line (EA-hy 926 cells) was incubated with the supernatant. The proliferative activity was examined and the levels of basic FGF and VEGF were measured. The activities were found to be significantly related to VEGF, the depth of tumor invasion and the tumor stage.

The low positive rate of serum alpha-fetoprotein levels in hepatitis C virus antibody-positive patients with hepatocellular carcinoma.

We analyzed the clinicopathological factors influencing the serum AFP levels at the time of diagnosis in 114 patients with resected HCC. The proportion of HCC patients with high serum AFP levels (> 100 ng/ml) has been steadily decreasing from a rate of 57.2% in 1980 to 33.3% in 1993. A significant relationship was noted between the serum AFP levels and the virus marker (p < 0.01) based on a multivariate analysis. The proportion of HCC patients with high serum AFP levels was significantly less in anti-HCV- positive HCC patients than in HBsAg-positive HCC patients (p < 0.01). The proportion of HBsAg-positive HCC patients has been steadily decreasing from a rate of 48% in 1980 to 15% in 1993. In contrast, the proportion of anti-HCV-positive HCC patients was 69% in 1993. It is thus assumed that the prevalence of anti-HCV-positive HCC patients is increasing recently, based on the fact that the incidence of HCC patients with high serum AFP levels is decreasing.

The detection of colorectal cancer at an asymptomatic stage by screening is useful.

BACKGROUND/AIMS: The validity of mass screening using fecal occult blood testing remains controversial. In addition, no controlled clinical study has yet been performed to show the usefulness of sigmoidoscopy. The purpose of the present study was to compare the surgical results achieved in asymptomatic patients with colorectal cancer detected by screening with those in symptomatic individuals. METHODOLOGY: A total of 285 patients underwent a surgical resection of colorectal cancer between 1991 and 1997 at our institution. Among them, 233 patients had complaints related to cancer at the time of diagnosis. In contrast, 52 were asymptomatic. In those 52 patients, colorectal cancer had been suspected based on routine screening including fecal occult blood testing, colonoscopy and/or elevated serum levels of carcinoembryonic antigen. RESULTS: Early stage of colorectal cancer was more frequently seen in asymptomatic patients than in symptomatic patients P < 0.01. The survival rates for asymptomatic patients was also superior to those of symptomatic patients P < 0.05. CONCLUSIONS: Screening using fecal occult blood testing, colonoscopy and tumor markers is thus considered to be beneficial for the early detection of colorectal carcinoma, which also tends to demonstrate good surgical results.

Transcatheter arterial embolization for advanced hepatocellular carcinoma resulting in a curative resection: report of two cases.

Transcatheter hepatic arterial embolization and lipiodolization have been reported to be effective palliative treatments for patients with unresectable hepatocellular carcinoma. We experienced 2 patients with advanced hepatocellular carcinoma which were initially considered to be unresectable due to the extreme extension of the primary lesions. Therefore, transcatheter hepatic arterial embolization with lipiodolization were selected as the treatments of choice. Thereafter, these tumors markedly decreased in size and, as a result, curative resections could subsequently be performed. The pathological examination of the resected specimens revealed necrosis and hyaline degeneration in the main tumors. Viable tumor cells, however, still remained adjacent to the main tumors. Such evidence indicated the limited efficacy of transcatheter hepatic arterial embolization with lipiodolization and the necessity of performing surgical treatment in combination with transcatheter hepatic arterial embolization with lipiodolization. Based on these findings, transcatheter hepatic arterial embolization with lipiodolization both appear to be a good mode of therapy for advanced hepatocellular carcinoma, and in selected patients, subsequent surgery can also be considered.

The effect of fenestration procedure on liver regeneration in a case of polycystic liver disease.

We encountered a case of polycystic liver disease for which unroofing and fenestration procedures were performed. The patient was a 55-year-old Japanese female with epigastralgia and abdominal fullness. On computed tomography, millions of low-density areas were seen, particularly in S6, 7, where huge cysts 15 cm in diameter were observed. Magnetic resonance imaging showed a T1 low T2 high-intensity lesion, which was compatible with simple cysts. Unroofing for the cysts in S6, 7 and fenestration of other cysts were performed. Histological examination revealed cuboidal and flat monolayer epithelia with no dysplasia in the wall of the cysts. The postoperative course was uneventful, and the patient's abdominal symptoms remarkably improved. The percentage of the liver volume which was increased in relation to standard liver volume was reduced from 241% (3386 mL: liver parenchyma 750 mL, cysts 2636 mL) to 180% (2525 mL, 1566 mL, 959 mL, respectively) after surgery. The potent mitogen, hepatocyte growth factor, was rapidly increased after the operation and stayed high during the observation period. In this patient, since no liver resection was performed, liver regenerative stimulus was considered to be the loss of space. This phenomenon represents a model of liver regeneration in response to loss of occupied space in an absence of shear stress.

Leiomyosarcoma occurring in the ligamentum teres of the liver: a case report and a review of seven reported cases.

We treated a 49-year-old male with leiomyosarcoma of the ligamentum teres of the liver. Preoperative hepatic imagings revealed a mass in the median segment of the liver. The patient underwent surgery, and the mass, measuring 6x5cm in size, was pathologically diagnosed as leiomyosarcoma arising from the ligamentum teres. To our knowledge, only eight cases of this rare tumor have been reported to date in the English literature.

Intraportal administration of low-dose recombinant human hepatocyte growth factor enhances effects of hepatocellular transplantation.

BACKGROUND/AIMS: Although recombinant human hepatocyte growth factor (rhHGF) is a potent mitogen, the dose used for patients is still not clear and must be low to avoid untoward effects. Firstly, the optimal strategy of the dose and route of rhHGF was investigated. Secondly, low-dose rhHGF, which would induce proliferation of transplanted hepatocytes, was explored using Nagase analbuminemic rats. METHODOLOGY: 1) Concentrations of rhHGF in the portal vein were measured after continuous administration of titrated rhHGF through the jugular vein or portal vein. 2) F344 rat hepatocytes (2 x 10(7) cells) were transplanted in the liver of Nagase analbuminemic rats. On the 7th day, the rats were subjected to a low-dose rhHGF treatment. RESULTS: When the rats were given rhHGF in a dose of 50 micrograms/kg/day, the mean concentration in the portal vein (0.8 +/- 0.1 ng/mL) was almost similar to the minimum concentration which stimulated hepatocyte proliferation in vitro. When low-dose rhHGF (50 micrograms/kg/day) was administered directly into the portal vein following hepatocyte transplantation in Nagase analbuminemic rats, the serum levels of albumin were significantly higher than in other groups. It was found that the concentration of rhHGF in the portal vein were 3.1 +/- 0.5 ng/mL with continuous intraportal infusion and 0.8 +/- 0.1 ng/mL with continuous systemic infusion. CONCLUSIONS: It was found that the minimal dose of rhHGF needed to stimulate hepatocyte proliferation was 50 micrograms/kg/day. With rhHGF (50 micrograms/kg/day), continuous intraportal infusion afforded a more favorable outcome in case of proliferation of hepatocytes.

Effects of recombinant human hepatocyte growth factor on the proliferation and function of porcine hepatocytes.

A porcine hepatocyte based bioartificial liver (BAL) is still insufficient to replace liver transplantation. In this experiment, to strengthen the performance of a BAL, the effect of human recombinant hepatocyte growth factor (rhHGF) on the proliferation and function of xenogeneic porcine hepatocytes was studied. Isolated porcine hepatocytes were seeded at various densities (5 x 10(3) to 8 x 10(4) cells/well) on a collagen coated 96 well plate in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS. After 4 hours, the medium was changed to DMEM with added insulin and dexamethasone. Subsequently, rhHGF was added at various concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, 40 ng/ml) and cultured for an additional 24, 48, and 72 hours, respectively. The proliferation of porcine hepatocytes in response to rhHGF reached a plateau at 2.5 ng/ml at 24 hours and subsequently decreased. The levels of porcine albumin vs protein present in the supernatant increased when cultured at high cell density. In conclusion, rhHGF was found to stimulate proliferation of porcine hepatocytes at low cell density and low concentration. rhHGF can also increase albumin synthesis at higher cell density, thus indicating its potential use in a more satisfactory porcine hepatocyte based BAL.

Effects of anticoagulants on porcine hepatocytes in vitro: implications in the porcine hepatocyte-based bioartificial liver.

For the clinical treatment with porcine hepatocyte-based bioartificial liver (BAL), the use of an anticoagulant in the extracorporeal system is essential. In this experiment, we studied the effect of various anticoagulants on cultured porcine hepatocytes. Porcine hepatocytes were isolated and seeded at a density of 2 x 10(5) cells on a collagen-coated plate in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS). Twenty-four hours later, the medium was changed to DMEM with various anticoagulants such as nafamostat mesilate (NM), sodium heparin (SH) and sodium citrate (SC) at concentration used clinically. As a control, the hepatocytes were cultured in only DMEM. After culturing for 6 hours, the viability of the porcine hepatocytes, lactate dehydrogenase (LDH) release, lidocaine clearance (cytochrome p450 function) and albumin synthesis were investigated. SC did not affect either the viability or the p450 function of the hepatocytes. In the NM group, the viability of porcine hepatocytes and lidocaine clearance were decreased significantly more than in the other groups. SH did not affect the viability of porcine hepatocytes, however, it seemed to reduce the p450 function. In conclusion, SC may therefore be the optimal anticoagulant available for hepatocyte-based BAL circuit in terms of its cell toxicity.